No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Characterization of the manganese O2-evolving complex and the iron-quinone acceptor complex in photosystem II from a thermophilic cyanobacterium by electron paramagnetic resonance and X-ray absorption spectroscopy
Abstract
The Mn donor complex in the S1 and S2 states and the iron-quinone acceptor complex (Fe2+-Q) in O2-evolving photosystem II (PS II) preparations from a thermophilic cyanobacterium, Synechococcus sp., have been studied with X-ray absorption spectroscopy and electron paramagnetic resonance (EPR). Illumination of these preparations at 220-240 K results in formation of a multiline EPR signal very similar to that assigned to a Mn S2 species observed in spinach PS II, together with g = 1.8 and 1.9 EPR signals similar to the Fe2+-QA- acceptor signals seen in spinach PS II. Illumination at 110-160 K does not produce the g = 1.8 or 1.9 EPR signals, nor the multiline or g = 4.1 EPR signals associated with the S2 state of PS II in spinach; however, a signal which peaks at g = 1.6 appears. The most probable assignment of this signal is an altered configuration of the Fe2+-QA- complex. In addition, no donor signal was seen upon warming the 140 K illuminated sample to 215 K. Following continuous illumination at temperatures between 140 and 215 K, the average X-ray absorption Mn K-edge inflection energy changes from 6550 eV for a dark-adapted (S1) sample to 6551 eV for the illuminated (S2) sample. The shift in edge inflection energy indicates an oxidation of Mn, and the absolute edge inflection energies indicate an average Mn oxidation state higher than Mn(II). Upon illumination a significant change was observed in the shape of the features associated with 1s to 3d transitions. The S1 spectrum resembles those of Mn(III) complexes, and the S2 spectrum resembles those of Mn(IV) complexes. The extended X-ray absorption fine structure (EXAFS) spectrum of the Mn complex is similar in the S1 and S2 states. Simulations indicate O or N ligands at 1.75 +/- 0.05 A, transition metal neighbor(s) at 2.73 +/- 0.05 A, which are assumed to be Mn, and terminal ligands which are probably N and O at a range of distances around 2.2 A. The Mn-O bond length of 1.75 A and the transition metal at 2.7 A indicate the presence of a di-mu-oxo-bridged Mn structure. Simulations indicate that a symmetric tetranuclear cluster is unlikely to be present, while binuclear, trinuclear, or highly distorted tetranuclear structures are possible. The striking similarity of these results to those from spinach PS II suggests that the structure of the Mn complex is largely conserved across evolutionarily diverse O2-evolving photosynthetic species.
... However, the lightsaturated rate of oxygen evolution by PSII appears comparable between mesophilic and thermophilic cyanobacteria, ∼5,500 μmol of O 2 (mg of Chl) À1 h À1 [refer to Materials and Methods and Sugiura et al. (58) for oxygen evolution rates of PSII core complexes isolated from Synechocystis 6803 and T. elongatus, respectively]. This similarity in rates suggests identical mechanisms of O-O bond formation between species, which is consistent with various spectroscopic studies concluding that the OEC structures are nearly identical between mesophilic cyanobacteria, thermophilic cyanobacteria, and spinach (59)(60)(61)(62)(63)(64)(65)(66). Structural features in the map near the OEC are especially challenging to interpret due to the dynamic nature of the structure and challenges in modeling OEC atoms. ...
Significance
Photosystem II (PSII) is a photo-oxidoreductase that harnesses light energy to use water to make fuel. Water oxidation occurs at a metal cluster in the active site called the oxygen-evolving complex (OEC). Understanding PSII function has provided design principles for synthetic solar fuel catalysts; however, the details of water oxidation are obscured by the multiple states through which the mechanism proceeds, differences between species, and lability of the OEC. To better understand PSII function, we solved its structure from Synechocystis sp. PCC 6803. We observe significant differences compared with PSII from thermophilic cyanobacteria that highlight the need for reexamination of previous data using this structure for interpretation. The structure also provides a platform for studies of site-directed mutations of PSII.
The biological water oxidation takes place in Photosystem II (PSII), a multi-subunit protein located in thylakoid membranes of higher plant chloroplasts and cyanobacteria. The catalytic site of PSII is a Mn4Ca cluster and is known as the oxygen evolving complex (OEC) of PSII. Two tyrosine residues D1-Tyr161 (YZ) and D2-Tyr160 (YD) are symmetrically placed in the two core subunits D1 and D2 and participate in proton coupled electron transfer reactions. YZ of PSII is near the OEC and mediates electron coupled proton transfer from Mn4Ca to the photooxidizable chlorophyll species P680+. YD does not directly interact with OEC, but is crucial for modulating the various S oxidation states of the OEC. In PSII from higher plants the environment of YD• radical has been extensively characterized only in spinach (Spinacia oleracea) Mn- depleted non functional PSII membranes. Here, we present a 2D-HYSCORE investigation in functional PSII of spinach to determine the electronic structure of YD• radical. The hyperfine couplings of the protons that interact with the YD• radical are determined and the relevant assignment is provided. A discussion on the similarities and differences between the present results and the results from studies performed in non functional PSII membranes from higher plants and PSII preparations from other organisms is given.
A new synthesized and structurally characterized phenolato bridged di-nuclear manganese(II) complex, formulated as [Mn2L(OAc)2(DMF)2]PF6 (1) (where HL = 4-methyl-2,6-bis-[(2-morpholin-4-yl-ethylimino)-methyl]-phenol and OAc = acetate anion), can act as a turn-on Zn²⁺ sensing probe through the in situ formation of the corresponding dinuclear Zn(II) complex, [Zn2L(OAc)2]PF6 (2) via direct Mn²⁺ ion replacement in MeOH/DMF (1/14) medium. The selective and specific fluorescent change due to addition of Zn²⁺ ions to a solution of 1 enables the detection of Zn²⁺ ions in dimethylformamide (DMF). The fluorescent dinuclear zinc(II) complex 2 has also been produced through the direct reaction between Zn²⁺ ions and HL. Both complexes 1 and 2 have been characterized by using physico-chemical and spectroscopic tools, and finally by single crystal X-ray crystallography for a detailed structural determination. The crystallographic investigation indicates that the Mn(II) and Zn(II) ions in the complexes exhibit a distorted octahedral and distorted trigonal bipyramidal coordination geometry, respectively. Interestingly, complex 1 showed very weak fluorescence in solution and no emission in the solid state, whereas complex 2 gave a strong bluish-green coloured fluorescence in both solution and in the solid state, which may be due to the distinct packing of complex 2 compared to 1, as well as to intermolecular hydrogen bonding and C–H---π, C–H---N and C–H---O interactions in the crystal packing, likely playing crucial roles for the fluorescence behavior.
Chloride is a well-known activator of oxygen evolution activity in photosystem II. Its effects have been characterized over several decades of research, as methods have developed and improved. By replacing chloride with other small anions with a range of chemical properties, a picture of the requirements of a successful anion activator can be formulated. In this review, the results of experiments on the chloride effect using enzyme kinetics methods and electron paramagnetic resonance spectroscopy are described, with summaries for the major anion activators and inhibitors that have been studied.
In Cyanobakterien und Pflanzen erfolgt die Spaltung von Wasser in molekularen Sauerstoff, vier Protonen und vier Elektronen in einem Pigment-Protein Komplex, der Teil der Thylakoidmembran ist und als Photosystem II (PS II) bezeichnet wird. Die Wasserspaltung wird von einem funktionellen Teil des PS II katalysiert, der als Sauerstoff entwickelnder Komplex ( Oxygen Evolving Complex , OEC) bekannt ist und dessen katalytisches Zentrum von einem Mn4OxCa-Komplex gebildet wird. Der OEC durchläuft während der Wasseroxidation fünf Redoxzustände (S-Zustände). Die kürzlich veröffentlichten Kristallstrukturen von PS II aus den thermophilen Cyanobakterien Thermosynechococcus elongatus (T. elongatus) und T. vulcanus liefern Informationen über die komplexe Gesamtstruktur von PS II und die Anordnung vieler Kofaktoren. Bis heute existiert keine Kristallstruktur für PS II aus höherer Pflanzen. Da bisher die meisten funktionellen Untersuchungen an PS II Komplexen aus Spinat durchgeführt wurden, sind für eingehende Untersuchungen von Struktur-Funktionsbeziehungen vergleichende Funktionsstudien an PS II Komplexen aus Cyanobakterien und höheren Pflanzen erforderlich. Ziel dieser Arbeit ist die Untersuchung von Gemeinsamkeiten und Unterschieden beim Mechanismus der Sauerstoff-Entwicklung in T. elongatus und Spinat. Hierfür wurden insbesondere blitzinduzierte Sauerstoff-Oszillationsmuster ( Flash Induced Oxygen evolution Patterns , FIOPs) unter verschiedensten Bedingungen gemessen und im Rahmen eines erweiterten Kok-Modells analysiert. Es wurden folgende Ergebnisse erzielt: a) Die Temperatur-Abhängigkeiten der miss - und double hit -Wahrscheinlichkeiten, sowie die Lebensdauern der S-Zustände in beiden Organismen deuten auf strukturelle Unterschiede der Akzeptorseite von PS II und in der Umgebung des Tyrosin-Radikals YDox hin. b) Untersuchungen zu den Effekten von einem H/D Isotopen-Austausch auf die Reaktionen des OEC bei verschiedenen Temperaturen und pL-Werten (L = H oder D) zeigen, dass hierdurch die Reaktionen im PS II beider Organismen in vergleichbarer Weise beeinflusst werden. Im Gegensatz zu Spinat-Thylakoiden ist in Thylakoiden von T. elongatus aber YDox, das bei pH 7,0 stabil ist, bei pH 8,0 labil und wird im Dunkeln bei Raumtemperatur innerhalb einer Stunde reduziert. c) Durch Inkubation mit den exogenen Reduktionsmitteln NH2OH, N2H4 und NO wurde zum ersten Mal gezeigt, dass (i) Arrhenius-Diagramme für NH2OH induzierte S1 ? S0 and S0 ? S-1 Übergänge im OEC von Spinat-Thylakoiden einen Knickpunkt bei 29°C aufweisen. Unterhalb dieser Temperatur sind Aktivierungsenergie und prä-exonentielle Faktoren unabhängig von S-Zustand, wogegen oberhalb von 29°C beide Faktoren vom Redoxzustand abhängig sind. (ii) Das S-2 EPR Multiline -Signal wurde erstmals in monomeren und dimeren PS II core -Komplexen von T. elongatus durch NO -Inkubation erhalten. Kleine, reproduzierbare Verschiebungen einiger Tieffeld-Übergänge in den S-2 EPR- Multiline -Signalen von T. elongatus im Vergleich zu dem Spinat-Signal weisen auf leichte Unterschiede bei der Koordinations-Geometrie und/oder den Liganden des Mn4OxCa-Komplexes zwischen thermophilen Cyanobakterien und höheren Pflanzen hin. (iii) FIOPs von N2H4-reduzierten Thylakoiden von T. elongatus zeigen eine Besetzung des S-3 -Zustandes von bis zu ~70% an; die Stabilität dieses Redoxzustandes macht die Existenz eines Mn4(II4)-Komplexes für diesen Zustand unwahrscheinlich. Darüber hinaus weist die numerische Analyse der FIOPs von mit Hydrazin reduziertem PS II auf die Existenz von S-4- und S-5- Redoxzuständen hin. Diese Ergebnisse unterstützen die Zuordnung der Mangan-Oxidationszustände Mn4(III2,IV2) für den S1-Zustand.
Photosystem II (PSII) is a light driven, water-plastoquinone oxidoreductase which catalyses the most thermodynamically demanding reaction in biology. This highly endergonic reaction splits water into molecular oxygen, protons and electrons, thereby sustaining an aerobic atmosphere on earth and providing the reducing equivalents necessary to fix carbon dioxide to organic molecules, creating biomass, food and fuel.
The main electron transfer events in PS2 are:
Open image in new window
The pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) is used to measure the nuclear transition frequencies of paramagnetic nuclei magnetically coupled to unpaired electron spins.1 We have employed this technique to study the Mn center of the Photosystem II oxygen-evolving complex. ESEEM measurements were performed on the “multiline” Mn EPR signal associated with the S
2 state of the Kok cycle.
The great diversity of structural proposals for metal association in the OEC can be organized in three broad categories as single, dual or multiple centers. In the single center proposal, a tetranuclear assembly of manganese accumulates oxidizing equivalents and oxidizes water directly. The cubane proposal of Brudvig (1) and the double pivot mechanism of Christou (2) are two examples. These models have structural analogy to the Fe4S4 clusters. The multiple center idea (3) invokes a mononuclear Mn(IV) in an electron transfer equilibrium with a dinuclear center and a fourth, redox inactive manganese segregated from the other metal atoms. This is reminiscient of cytochrome oxidase where a dinuclear center reduces oxygen to water and two single site metal ions (copper and heme iron) contribute reducing equivalents. Two dual center possibilities are are two sets of dimers or, what we suggested (4), a monomer/trimer organization. The latter is consistent with Fe3S4 centers. A possible trinuclear structure is shown in figure 1. Three manganese are arranged in a distorted triangular orientation with the terminal manganese separated from the central manganese ion by 2.7 Å. The two terminal manganese ions are separated by either 3.3 Å or 4.3 Å. A mononuclear center is located at a distance >5 Å.
One of the outstanding problems facing current photosynthesis research is to work out the structure of the manganese-containing complex central to the water oxidising activity of photosystem 2 (PS2). This complex mediates the transfer of electrons from water to the reaction centre of PS2: ideally, one electron is transferred per quantum of light absorbed by the reaction centre. Four such steps advance the complex from the most reduced state, called S0, to the most oxidised, S4. Oxygen is evolved as S4 spontaneously converts to S0.
The nickel site in Thiocapsa roseopersicina hydrogenase was examined in the five redox forms defined by electron paramagnetic resonance spectroscopy at 77 K by use of X-ray absorption spectroscopy. These studies show that the nickel site is remarkably insensitive to changes in the redox state of the enzyme, a result that is inconsistent with nickel-centered redox chemistry. Model studies of a series of nickel complexes with ligands of the type RN(CH2CH2S)2 show that the products of one-, two-, and four-electron oxidations all reflect sulfur-centered chemistry. The role of the nickel site in binding hydrogen was explored by using a combination of electron nuclear double resonance and X-ray absorption spectroscopic techniques. These studies do not completely rule out a role for the nickel site, but they point to the possibility that nickel is not the hydrogen-binding site. These results are discussed within the context of biological and inorganic chemical literature pertaining to nickel thiolate complexes.
The structure of manganese cluster functioning to water cleavage is an urgent problem to be cleared. Is a water coordinated directly to one of the manganese ions? What is the structure of the manganese cluster? The EXAFS study[1] favors the two manganese cluster model, while the temperature dependence of EPR intensity could be illustrated by four manganese cluster models with different magnetic coupling among four manganese ions[2]. In this report we present the results of proton matrix ENDOR studies in the S2 state of photosystem II in deuterated buffer and in samples treated by ammonium chloride. Our preliminary result on an ordinary and deuterated PS II samples and its analysis has been reported[3]. Here we will try the analysis based on the dipolar interaction of a proton with the magnetic moment on the two separated positions of manganese ions and also will investigate the effect of NH4Cl treatment.
Involvement of residues of acidic amino acids in photoligation of manganese into the apo-water-oxidizing complex was investigated by use of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), a water-soluble carboxyl modifier. Treatment of Mn-depleted PSII membranes by EDC in the presence of nucleophiles induced a loss of photoactivation capability in the Mn complex and partial loss of capability of photooxidation of Mn2+, but little decrease in the DCIP photoreduction supported by diphenylcarbazide. The inhibition of diphenylcarbazide-photooxidation by submicromolar Mn2+, indicative of the intactness of high-affinity Mn-binding sites, was apparently abolished by EDC treatment. From amino acid quantitation analysis of D1 and D2 proteins and CP47 of the chemically-modified membranes, approximately three carboxyl groups of the D1 protein were found to be chemically-modified with EDC after removal of the functional Mn. These results suggest that acidic amino acids on the D1 protein are involved in photoactivation of the apo-water-oxidizing complex and probably in ligation of Mn to the water-oxidizing complex.
In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385-398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q400 component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.
This review describes the progress in our understanding of the structure of the Mn complex in Photosystem II over the last two decades. Emphasis is on the research from our laboratory, especially the results from X-ray absorption spectroscopy, low temperature electron paramagnetic resonance and electron spin echo envelope modulation studies. The importance of the interplay between electron paramagnetic resonance studies and X-ray absorption studies, which has led to a description of the oxidation states of manganese as the enzyme cycles through the Kok cycle, is described. Finally, the path, by which our group has utilized these two important methods to arrive at a working structural model for the manganese complex that catalyzes the oxidation of water to dioxygen in higher plants and cyanobacteria, is explained.
A comparative study of X-band EPR and ENDOR of the S2 state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S2 state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S2 state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. (1)H-ENDOR experiments performed at two different field positions of the S2 state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382-2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D2O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S2.
The electrochemical and spectroscopic characterization of the species formed upon interaction of iron(II) with 2-hydroxy-1,4-naphthoquinone (Lawsone) and its reduced form has been studied in dimethylsulphoxide. Neither the protonated nor the anionic form of the ligand interacts with iron(II) to form complexes of reasonable stability. The reduced species of the ligand (semiquinone) forms a very stable complex with iron(II), which has a 1 : 2 stoichiometry and presents a formation constant of 3.5 × 1025.8The magnetic susceptibility measurements of the iron(II)-semiquinone system indicate the existence of an intramolecular charge transfer between the metal ion and one of the radical ligands, so that the metal centre has the character of iron(III). The presence of the metal ion stabilizes the radical species formed upon reduction of the quinone, which may be relevant to understand the mechanisms involved in charge-transfer processes in biological systems.
Manganese was shown to be an essential trace metal for growth and thiosulphate oxidation by Thiobacillus versutus in chemostat culture. In the thiosulphate-oxidizing enzyme system of T. versutus, protein B was the only component found to contain manganese in significant amounts. When it was examined by electron spin resonance (ESR) spectroscopy, protein B gave a broad, complex spectrum, indicative of the presence of a dimeric manganese cluster, with manganese in the Mn(II) oxidation state.
The synthesis and characterization of several new catecholate derivatives of manganese and iron are described. The reaction of 3,5-di-tert-butylcatechol (DBCatHâ) with the amides M(N(SiMeâ)â)â (M = Mn, Fe) in the presence of pyridine (py) affords the title compounds in high yield. The dimers Mnâ(DBCat)â(py)â (1) and Feâ(DBCat)â(py){sub n} (n = 4 (4a), 6 (4b)) are obtained by treatment of catechol with the appropriate amide in pyridine. In hexane or toluene, this reaction gives the tetrametallic species Mâ(DBCat)â(py)â (M = Mn (2), Fe (5)) upon the addition of 2 equiv of pyridine or, in the case of 2, by recrystallization of the dimer from toluene. Mnâ(DBCat)â(py)â (3) is obtained by slow air oxidation of 2 or by addition of 1/3 equiv of 3,5-di-tert-butyl-o-benzoquinone to the reaction of Mn(N(SiMeâ)â)â with DBCatHâ in hexane with subsequent addition of pyridine. Compounds 1-5 were characterized by infrared, UV-visible, ¹H NMR, and EPR spectroscopy and X-ray crystallography.
Obwohl der Effekt, auf dem die Röntgenabsorptionsspektroskopie (EXAFS-Spektroskopie) beruht, seit etwa siebzig Jahren bekannt ist, fand sie bis vor zwanzig Jahren keine Anwendung. Gründe hierfür waren einerseits der Mangel an leistungsfähigen Röntgenquellen, andererseits das Fehlen einer geeigneten theoretischen Beschreibung. Mit dem Aufkommen der Synchrotronstrahlung hat sich die Situation von Grund auf geändert. Heute wird die EXAFS-Spektroskopie auf sehr unterschiedliche Probleme angewendet und immer häufiger bei chemischen Fragestellungen eingesetzt. Die außergewöhnliche Eigenschaft der EXAFS-Spektroskopie, in nahezu jedem beliebigem Gemisch, unabhängig vom Aggregatzustand sowie elementspezifisch mit hoher Empfindlichkeit lokale Strukturen bestimmen zu können, birgt jedoch die Gefahr, daß das Maß an erhaltbarer Strukturinformation überschätzt wird. Eine realistische Einschätzung der Leistungs-fähigkeit der EXAFS-Spektroskopie ist bei kristallinen Stoffen mittlerweile eingetreten, nicht aber bei amorphen Systemen. Hier reichen die Standpunkte von völliger Ablehnung bis zur Überinterpretation der EXAFS-Spektren. In diesem Artikel wird versucht aufzuzeigen, wie die EXAFS-Spektroskopie bei der Untersuchung nichtkristalliner Stoffe eingesetzt werden kann. Zunächst wer-den die Meßtechniken, Auswerteverfahren und die Simulation von EXAFS-Spektren erläutert. Mit den einfachsten Systemen — den amorphen Metallen und Nichtmetallen — beginnend, wird an repräsentativen, nicht unbedingt aktuellen Beispielen erläutert, welche Strukturinformationen die EXAFS-Spektroskopie liefert, wie sich bei binären Systemen die lokale Umgebung um beide Komponenten bestimmen läßt und wie sogar das Kristallisationsverhalten und Strukturänderungen bei chemischen Reaktionen untersucht werden können. Anschließend werden EXAFS-Untersuchungen an Lösungen, molekularen Flüssigkeiten und Schmelzen vorgestellt. Der abschließende Teil beschäftigt sich mit typischen Anwendungsgebieten der EXAFS-Spektroskopie, der Untersuchung von homogenen und heterogenen Katalysatoren sowie der Aufklärung von aktiven Zentren in Enzymen.
The Mn-4-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J, H. A., and Evans, M. C. W. (1997) Biochemistry, 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S, (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the So state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S-0 state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S-0 multiline signals detected in plant PSII and in S, elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S-2 state in S. elongatus is not affected by the addition of methanol in that (i) the S-2 multiline EPR signal is not modified by methanol and (ii) the spin state of the S-2 state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn-4 cluster.
The possibility of nitrogen ligation to the Mn in the oxygen-evolving complex from photosystem II was investigated with electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopies using N-14- and N-15-labeled preparations. Oxygen-evolving preparations were isolated from a thermophilic cyanobacterium, Synechococcus sp., grown on a medium containing either (NO3-)-N-15 as the sole source of nitrogen. The substructure on the "multiline" EPR signal, which arises from Mn in the S2 state of the enzyme, was measured with continous-wave EPR. No changes were detected in the substructure peak positions upon substitution of N-15 for N-14, indicating that this substructure is not due to superhyperfine coupling from nitrogen ligands. To detect potential nitrogen ligands with superhyperfine couplings of lesser magnitude than could be observed with conventional EPR methods, electron spin-echo envelope modulation experiments were also performed on the multiline EPR signal. The Fourier transform of the light-minus-dark time domain ESEEM data shows a peak at 4.8 MHz in N-14 samples which is absent upon substitution with N-15. This gives unambiguous evidence for weak hyperfine coupling of nitrogen to the Mn of the oxygen-evolving complex. Possible origins of this nitrogen interaction are discussed.
Manganese content and the amount of oxygen-evolving centres have been determined for purified PS II complexes from the cyanobacterium Synechococcus sp. by atomic absorption and measurements of flash-induced oxygen yields, respectively. A stoichiometry of six manganese per oxygen-evolving centre was observed. This was found not only for PS II preparations with different activities of oxygen evolution, but also when deactivating treatments by low-salt or NH2OH incubation were carried out. Our results call into question the current thinking that four Mn ions are located in the water-oxidizing complex. A hexanuclear manganese model is discussed, instead.
At hanging mercury-drop electrode (HMDE) Alizarin Red S (ARS) is electrochemically reduced in aqueous buffer solutions, giving rise to a prominent wave and a pre-wave. The prominent wave is diffusion-controlled and involves two electrons and two protons. The pre-wave is attributed to a strong reversible adsorption of the reduction product at the electrode. The reduction mechanism was proposed and discussed on the basis of the cyclic voltammetric and double potential-step chrono-amperometric & coulometric results. It was concluded that the reduction pathway is an ECEC mechanism in which the chemical reactions (C) are a dimerization of the formed radical anion and protonation of the dianion dimer, respectively. The rate-determining step is the protonation reaction. The adsorption process obeys a Langmuir isotherm with maximum surface excess of 1.4×10-10 mol cm-2 at a bulk concentration ≥4.6×10-5 mol dm-3, corresponding to 1.18 nm2/molecule. This indicates that ARS is horizontally oriented and resists reorientation upon increasing its bulk concentration.
Inversion recovery (T1) and microwave power saturation studies have been performed, between 4 and 25 K, on the EPR signal from the stable tyrosyl radical, YD˙, in photosystem II core complex preparations from higher plants. Measurements are reported from the dark stable S1 and first turnover S2 states of the photosystem catalytic Mn cluster and in two cryoprotectant regimes; sucrose and ethylene glycol/glycerol. The inversion recovery kinetics show a dominant, non exponential decay component which is well described by a through space dipolar relaxation model, with a weak exponential decay background (an order of magnitude less than the dipolar rate). The dipolar relaxation rate is only modestly temperature dependent and shows no consistent dependence on S state or cryoprotectant. In contrast, the background rate shows a S state dependence, consistent with an interaction between YD˙ and the Mn cluster in the multiline S2 state, over a distance of 30 Å. The fraction of centers exhibiting the dipolar relaxation component appears to be temperature dependent, but S state independent and consistent with the presence of a fast relaxing species interacting with YD˙. The present results and the possible nature of this interacting species are discussed in comparison with earlier YD˙ relaxation studies on photosystem II membrane samples.
Oxidation of water in photosynthetic organisms occurs in a chlorophyll containing enzyme, photosystem II. The catalytic manganese cluster of photosystem II cycles among five redox states called the Sn states. There are two forms of the S2 state, which give rise to different EPR signals and which differ in magnetic coupling among the manganese atoms. We have used difference infrared spectroscopy to obtain more information about the environment of the manganese cluster in these two forms of the S2 state. We present the 1600−1200 cm-1 region of the difference spectrum associated with the generation of the multiline state and the g = 4.1 S2 state. The difference spectrum associated with generation of the S2 multiline state from the dark stable S1 state shows broad spectral features in the 1500−1200 cm-1 region at 1490 (negative), 1331 (negative), 1393 (positive), and 1267 (positive) cm-1. Global 15N labeling has little impact on intensities or frequencies in this region of the spectrum. These vibrational features are not observed in manganese-depleted, EDTA-treated photosystem II. Also, these features are not observed in oxygen-evolving photosystem II upon illumination at 80 K, a temperature where the manganese cluster is not oxidized. We conclude that these lines arise from amino acid residues that are close to or ligating to the cluster. From the frequencies and the lack of sensitivity to 15N labeling, we favor the assignment of these lines to the asymmetric and symmetric stretch of one or more glutamate and/or aspartate residue(s). The spectral breadth of the lines is consistent with either an inhomogeneous or a homogeneous broadening mechanism. When illumination conditions are used that generate the g = 4.1 S2 EPR signal, these vibrational lines are not observed. These results are discussed in terms of current models for the catalytic site.
Although the effect on which X-ray absorption spectroscopy (EXAFS spectroscopy) is based has been known for about 70 years, up until about 20 years ago it had not found any application. The reasons for this were on the one hand the shortage of efficient X-ray sources, and on the other hand the lack of a suitable theoretical description. With the advent of synchrotron radiation the situation has changed completely. Today, EXAFS spectroscopy is applied to a variety of very different problems and used more and more frequently to answer chemical questions. The extraordinary behavior of EXAFS spectroscopy that allows the determination of local structures in almost any mixture, independent of the sample's physical state as well as element-specifically with high sensitivity, carries with it the danger that the amount of the obtainable structural information will be overestimated. A realistic estimate of the capability of EXAFS spectroscopy has in the meantime been achieved for crystalline materials, but not for amorphous materials. In this case the viewpoints range from complete rejection to overinterpretation of the EXAFS spectrum. In this article an attempt is made to show how EXAFS spectroscopy can be applied in the investigation of noncrystalline materials. First, the measurement techniques, data analysis procedures, and the simulation of EXAFS spectra are explained. Starting with the simplest systems, the amorphous metals and nonmetals, and using representative but not necessarily current examples, an explanation is given of the structural information provided by EXAFS spectroscopy, of how the local environment about the two components can be determined in binary systems, and of how even the crystallization process and structural changes can be investigated in chemical reactions. Thereafter, EXAFS studies on solutions, molecular liquids, and melts are presented. The final section is concerned with typical applications of EXAFS spectroscopy, the investigation of homogeneous and heterogeneous catalysts as well as the elucidation of active sites in enzymes.
We have investigated the EPR characteristics of native QB and QB analogues in higher plant PS II. We show that, as in cyanobacteria, an interaction between QA and QB iron-semiquinones (QA−-Fe2+-QB−) is observed which gives an EPR signal near g=1.6. Bicarbonate binding close to the non-haem iron is required to observe this interaction. The EPR signal of QB semiquinone is weak and difficult to distinguish from that of QA. The appearance of the g=1.6 signal from QA−-Fe2+-QB− after 77 K illumination is a better marker for the presence of QB semiquinone. The yield of QB semiquinone in plant PS II is lower than found in cyanobacteria. The brominated quinones DBMIB, TBTQ and bromanil were used as QB analogues to increase the yield of QA−-Fe2+-QB−. These analogues act by forming a stable semiquinone at the QB site and not by covalent binding.
We have studied the EPR signal in PS II from Phormidium laminosum with a g-value of 1.66, which we assign to an interaction between the semiquinones of Qa and Qb and the non-heme iron. 77 K illumination of samples from dark-poised redox titrations show the rise of the signal has a midpoint potential (Em) of about +60 mV, and it is lost with an Em of about −10 mV. Under the same conditions, the rise of the g = 1.9 signal from Q·−a-Fe2+ in the dark was found to be about +10-mV. The g = 1.66 signal can also be formed with a high yield by first illuminating dark-adapted PS II particles at 293 K, followed by a short period of darkness at 273 K and subsequent illumination at 77 K. We have measured the effect on signal yield of varying the period of darkness following 293 K illumination. Over 60% of the maximum signal size is seen after 1 min darkness, and increases further over 2 h. In these samples a signal attributed to Q·−b-Fe2+ is seen prior to 77 K illumination. Confirmation of the presence of Q·t-b was obtained by reductant-linked oxidation of the non-haem iron using phenyl-para-benzoquinone (PPBQ). Samples treated with the Qb-analogue tribromotoluquinone (TBTQ) give a modified EPR signal. We propose (i) that Qb is preserved in PS II preparations from P. laminosum; (ii) that Qb-semiquinone can be readily formed and trapped by freezing; and (iii) the g = 1.66 signal arises from a coupling between the primary and secondary plastosemiquinones and the non-haem iron.
The evaluation of Mn X-ray absorption fine structure (EXAFS) studies on the oxygen-evolving complex (OEC) from photosystem II is described for preparations from both spinach and the cyanobacterium Synechococcus sp. poised in the S[sub 1] and S[sub 2] states. In addition to reproducing previous results suggesting the presence of bis([mu]-oxo)-bridged Mn centers in the OEC, a Fourier transform peak due to scatterers at an average distance of > 3 [angstrom] is detected in both types of preparation. In addition, subtle but reproducible changes are found in the relative amplitudes of the Fourier transform peaks due to mainly O ([approximately]1.8 [angstrom]) and Mn ([approximately] 2.7 [angstrom]) neighbors upon cryogenic advance from the S[sub 1] to the S[sub 2] state. Analysis of the peak due to scatterers at [approximately] 3 [angstrom] favors assignment to (per 4 Mn in the OEC) 1-2 heavy atom (Mn, Ca) scatterers at an average distance of 3.3-3.4 [angstrom]. The EXAFS data of several multinuclear Mn model compounds containing such scattering interactions are analyzed and compared with the data for the OEC. Structural models for the OEC are evaluated on the basis of these results. 40 refs., 9 figs., 5 tabs.
The results of X-ray absorption spectroscopic studies of Thiocapsa roseopersicina hydrogenase poised in three forms exhibiting EPR signals due to the Ni center (A, B, and C) and two states that are epr silent with respect to the Ni center are reported. These spectra are used to examine the structural changes that occur during the reduction of the enzyme. Analyses of Ni K-edge spectra reveal the presence of weak features at ca. 8332 eV in the spectra obtained from forms A and B and the silent intermediate (SI) that are assigned to 1s --> 3d transitions. The lack of a significant pre-edge peak in the active form of the enzyme and low peak areas in other forms, coupled with the absence of edge features associated with planar four-coordinate Ni complexes, indicate that the Ni site in all states of the enzyme is five- or six-coordinate. No observable shift in edge energy occurs upon reduction of the enzyme to any level. This demonstrates that no significant change in the electron density of the Ni site occurs during reduction. Analyses of the EXAFS spectra obtained from scattering atoms in the first coordination sphere of Ni in all rive states of the enzyme that are defined by Ni EPR signals (or lack thereof) are consistent with a Ni site composed of 3 +/- 1 N(O)-donors at 2.00 +/- 0.06 angstrom and 2 +/- 1 S-donors at 2.23 +/- 0.03 angstrom. These results are discussed in light of various models for the structure and function of the Ni site in the enzyme. No evidence to support a redox role for Ni in hydrogenase is found in the XAS data.
The tetrameric dimer-of-dimers complex [(Mn2O2)2(tphpn)2]4+ possesses structural features and a parallel polarization EPR spectrum which are similar to those observed for the S1 state of the photosystem II water oxidation catalyst. We have probed the ground-state electronic structure of [(Mn2O2)2(tphpn)2]4+ by magnetic susceptibility and isothermal saturation magnetization measurements to understand the origin of the EPR spectrum in terms of the intra- and interdimer magnetic exchange interactions and the ground-state zero-field splitting. Both [MnIIIMnIV] dimers are treated as having an effective spin S'= 1/2 which are coupled (J' = +38.8 cm-1) to yield a zero-field split (D = +1.8 cm-1, E = -0.15 cm-1) triplet ground state. The fictitious J' has been related by vector algebra methods to the real J that describes the exchange through the alkoxide oxygen atoms of the tphpn ligands (J' = -4J(alkoxide)). This treatment shows that the effective ferromagnetic interaction between the two S' = 1/2 cores has its origin in an alkoxide mediated antiferromagnetic exchange interaction. The difference in the magnitude of the singlet-triplet splitting obtained via the effective model and that obtained using a model describing the full exchange symmetry of the tetramer is on the order of 1%. It has been determined that the zero-field splitting is not of single ion or dipolar origin but results from pseudodipolar coupling. We have shown that the electronic structure of [(Mn2O2)2(tphpn)2]4+ parallels the geometric structure of the complex, and these results are presented in light of their relevance to the manganese water oxidation catalyst.
The pKa value of [MnIV2(μ-O)3L′2] 2+ (L′ = 1,4,7-trimethyl-1,4,7-triazacyclononane) has been determined spectrophotometrically by carrying out titration experiments with concentrated sulfuric acid. The extremely low pKa value of -2.0 suggests that the electron density on the bridging oxygen atoms is very small. The asymmetric Mn-O-Mn vibration is observed at 670 cm-1, while the symmetric Mn-O-Mn vibration is present at 702 cm-1. The unusually high frequencies of these vibrations are due to the small Mn-O-Mn angle of 78°. Protonation of an oxygen bridge shifts both the asymmetric and symmetric vibrations to 683 cm-1. Electrochemical experiments in acetonitrile have shown that one-electron reduction of the complex is chemically irreversible. IR, EPR, and UV-vis studies of the reduced species suggest the presence of a MnIIIMnIV(μ-O)2(μ-OH) core. pH-dependent differential pulse voltammetry experiments in aqueous solutions have revealed an apparent pKa value of approximately 4.0 for the reduced mixed-valence species in various buffer systems. The reduction wave at pH > 4 is observed at around -0.10 V vs SCE. Cyclic voltammetry has revealed that the reduced species is prone to reaction with carboxylate groups. A bis(carboxylate)mono-oxo-bridged Mn(III)-Mn(III) species is formed in citric acid buffer which exhibits an anodic peak around +0.6 V vs SCE, and a UV-vis spectrum that is typical of such a species.
Two (mu-aqua)bis(mu-carboxylato)dimanganese(II) complexes with oxygen and nitrogen donor ligands have been synthesized. Crystal structure determinations reveal a distance of 3.6 angstrom between the two manganese atoms. Magnetic studies indicate weak antiferromagnetic exchange between the two Mn centers with a coupling constant of approximately -2 to -3 cm-1.
The effect of LaCl3 on the K3Fe(CN)6 (FeCy) reduction rate and the oxygen-evolving rate of PSII particles of spinach, and the spectral characterization of the D1/D2/Cytb559 of a PSII reaction center complex consisting of three polypeptides from spinach were studied. The experimental results showed that LaCl3 could significantly accelerate the transformation from light energy to electric energy, the electron transport, water photolysis and oxygen evolution of PSII of spinach, which was related to the spectral characterization of the D1/D2/Cytb559 complex. Soret band and Q band of Chl-a of UV-vis spectrum of D1/D2/Cytb559 complex were blue shifted, and the fluorescence emission peak was blue shifted in LaCl3 treated spinach compared with that in the control. The EXAFS (extended X-ray absorption fine structure spectroscopy) revealed that La3+ was coordinated with 8 nitrogen or oxygen atoms in the first coordination shell with LaN or LaO bond length of 0.254 nm, and with 6 nitrogen or oxygen atoms in the second coordination shell with LaN or LaO bond length of 0.321 nm in the D1/D2/Cytb559 complex. The CD suggested that the secondary structure of D1/D2/Cytb559 complex have been little affected by the treatment of LaCl3.
Proton matrix ENDOR of the manganese multiline in the S2, state of photosystem II membranes from spinach has been investigated. The spectral structure over a range of frequencies of ± 2 MHz centered on the position for a free proton was analyzed through employing a simulation method based on the dipolar interaction between electron and proton magnetic moments. The 6 pairs of lines resolved were attributed to the surrounding proton populations at distances varying within the range 2.7–6.0 Å from the putative manganese center. Two of the 6 pairs, namely those corresponding to protons at distances of 2.7 and 3.2 Å from the manganese center were eliminated on washing of the membranes with deuterated buffer. This suggests that these protons belong to the water molecules coordinating to the manganese cluster. The presence of ethylene glycol instead of glycerol and sucrose in the buffer broadened both EPR and ENDOR spectra. This suggests that ethylene glycol molecules are also accessible to the two-manganese cluster. A possible model of water association to the manganese ions in the photosynthetic water-oxidation system is proposed.
The activities of photosynthetic water oxidation and of the primary stable charge separation have been investigated as a function of the concentration of various salts by measuring flash-induced oxygen yields and absorption changes at 320 nm due to QA reduction. (1) At low salt concentrations, water oxidation is inhibited, whereas the charge separation is not affected. (2) The former can be reactivated by salts containing neithercalcium nor chloride (e.g., Na2SO4), indicating that these ions are not essential cofactors of water oxidation. (3) At higher concentrations, certain salts (e.g. CaCl2) reversibly inhibit water oxidation and in the range of molar concentration also the primary stable charge separation. (4) These activating and inhibiting effects are explained by equilibria between active and inactive conformational states of PS II. These equilibria depend on the concentrations and properties of the various salts. (5) Arguments are given that, also in higher plants, salt-dependent conformational states are responsible for the salt effects.
By studying the electron paramagnetic resonance (EPR) signals of QA/−-Fe2+TBTQ− and the oxidised non-haem iron we have found that detergent solubilisation of BBY Photosystem II (PS II) preparations using standard methods, involving either the detergents n-octyl β-d-glucopyranoside (OGP) or n-heptyl β-d-thioglucoside (HTG) at pH 6.0, results in loss of bicarbonate binding. New preparations including a dodecyl maltoside (DM) prepared CP47, CP43, D1, D2, cytochrome b-559 complex are described which at pH 7.5 retain native bicarbonate binding. These preparations provide a new system for studies on the ‘bicarbonate effect’ because bicarbonate depletion can now be achieved without displacement by another anion. They are also a more suitable starting material for the isolation of QA retaining D1/D2 reaction centres because the detrimental changes to the QA binding region are avoided.
IntroductionDuron Proteins that Interact with DioxygenThe Purple Acid PhosphatasesOther (μ-Oxo)Diiron Centers in ProteinsDimanganese Centers in ProteinsPerspectives
The light-induced oxidation of water to O2 is catalyzed by a four-manganese atom cluster associated with Photosystem II (PS II). This chapter summarizes ongoing investigations
of the oxidation state, the structure and the associated cofactors calcium and chloride of the catalytic Mn cluster using
X-ray and electron paramagnetic resonance (EPR) spectroscopy. Manganese K-edge X-ray spectroscopy, Kβ X-ray emission spectroscopy
(XES), and extended X-ray absorption fine structure (EXAFS) studies have not only determined the oxidation states and structural
features, but also changes that occur in oxidation state of the Mn cluster and in its structural organization during the accumulation
of oxidizing equivalents leading to O2 formation. Combining X-ray spectroscopy information with X-ray diffraction studies, and consistent with the available EPR
data, we have succeeded in limiting the range of likely structures of the Mn cluster. EXAFS studies at the strontium and calcium
K-edges have provided evidence that the catalytic center is a Mn/Ca heteronuclear complex. Based on the X-ray spectroscopy
data, models for the structure and a mechanism for O2 evolution are presented.
Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.
We present an overview of anionic interactions with the oxidation-reduction reactions of photosystem II (PSII) acceptors. In section 1, a framework is laid for the electron acceptor side of PSII: the overview begins with a current scheme of the electron transport pathway and of the localization of components in the thylakoid membrane, which is followed by a brief description of the electron acceptor Q or QA and the various heterogeneities associated with it. In section 2, we review briefly the nature of the active species of the bicarbonate (HCO
3−) effect, the location of the site of action of HCO
3−, and its relationship to interactions with other anions. In section 3, we review data on the anion effects on the reoxidation of Q
A− and on the various reactions involved in the two-electron gate mechanism of PSII, and provide a hypothesis as to the action of HCO
3− on the protonation reactions. New data obtained by one of us (G) in collaboration with J.J.S. van Rensen, J.F.H Snel and W. Tonk for HCOg
3−-depleted thylakoids, demonstrating the abolition of the binary oscillations contained within the periodicity of 4 observed for proton release, are also reviewed. In section 4, we comment on the measured binding constant of HCO
3− at the anion binding site. And, in section 5, we review our current concept of the mechanism of the HCO
3− effect on the electron acceptor side of PSII, and comment on the possible physiological roles for HCO
3−. Measurements of HCO
3− reversible anionic inhibition in intact cells of a green alga Scenedesmus are also reviewed.
The linear, four-step oxidation of water to molecular oxygen by photosystem II requires cooperation between redox reactions driven by light and a set of redox reactions involving the S-states within the oxygen-evolving complex. The oxygenevolving complex is a highly ordered structure in which a number of polypeptides interact with one another to provide the appropriate environment for productive binding of cofactors such as manganese, chloride and calcium, as well as for productive electron transfer within the photoact. A number of recent advances in the knowledge of the polypeptide structure of photosystem II has revealed a correlation between primary photochemical events and a ‘core’ complex of five hydrophobic polypeptides which provide binding sites for chlorophyll a, pheophytin a, the reaction center chlorophyll (P680), and its immediate donor, denoted Z. Although the ‘core’ complex of photosystem II is photochemically active, it does not possess the capacity to evolve oxygen. A second set of polypeptides, which are water-soluble, have been discovered to be associated with photosystem II; these polypeptides are now proposed to be the structural elements of a special domain which promotes the activities of the loosely-bound cofactors (manganese, chloride, calcium) that participate in oxygen evolution activity. Two of these proteins (whose molecular weights are 23 and 17 kDa) can be released from photosystem II without concurrent loss of functional manganese; studies on these proteins and on the membranes from which they have been removed indicate that the 23 and 17 kDa species from part of the structure which promotes retention of chloride and calcium within the oxygen-evolving complex. A third water-soluble polypeptide of molecular weight 33 kDa is held to the photosystem II ‘core’ complex by a series of forces which in some circumstances may include ligation to manganese. The 33 kDa protein has been studied in some detail and appears to promote the formation of the environment which is required for optimal participation by manganese in the oxygen evolving reaction. This minireview describes the polypeptides of photosystem II, places an emphasis on the current state of knowledge concerning these species, and discusses current areas of uncertainty concerning these important polypeptides. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/43541/1/11120_2004_Article_BF00037001.pdf
A genetic system has been developed for site-specific mutagenesis of the D2 polypeptide of Photosystem II in the cyanobacterium Synechocystis 6803. In chloroplasts, this polypeptide is encoded by the psbD gene (1). The discovery of significant amino acid homologies between the D2 polypeptide and the M-subunit protein of the bacterial photosynthetic reaction center has led to the proposal that certain amino acid residues in D2 might bind the reaction center chlorophyll, the quinone QA, and the ferrous non-heme iron.
The cyanobacterium Anabaena contains at least three copies of DNA sequences related to the unique gene encoding the 32 kd thylakoid membrane protein in spinach chloroplast DNA, based on hybridization with the cloned spinach probe. Two of the identified Anabaena DNA fragments were isolated from a recombinant lambda library and the complete nucleotide sequences of the coding regions were determined. Both fragments contain open reading frames coding for proteins of MW 39 950. The predicted amino acid sequences are 94% identical; 87% of the positions are identical to those of the corresponding spinach protein. The nucleotide sequences of the 5' and 3' flanking regions of the two Anabaena genes differ considerably. Based on S1 nuclease protection, primer extension, and Northern hybridization experiments it is concluded that only one of the two cloned genes is transcribed in Anabaena cells growing on complete medium (containing ammonia). Under these conditions it appears that none of the other related sequences, not yet cloned, is transcribed. Transcription of only one member of the multigene family provides a possible explanation for the ability to isolate mutants resistant to the herbicide DCMU, whose target is believed to be the 32 kd protein.
The paramagnetic resonance absorptions in several trichelate complexes of Cr+3 have been investigated at room temperature and at 1.2‐ and 3.0‐cm wavelengths. From single crystal measurements on chromic acetylacetonate the orientations of the electric axes were determined and the parameters in the spin‐Hamiltonian found to be g = 1.983±0.002, ∣D∣ = 0.592±0.002 cm—1, and ∣E∣ = 0.052±0.002 cm—1. The asymmetric single absorption peak with g = 4 found in several of the powders and in their liquid solutions is explained on the basis of the relatively large value of D associated with the individual trichelate molecules. The apparent inadequacy of the ``ionic'' model for several chromic compounds is also discussed.
The first MnIV Schiff base complex to be structurally characterized by X-ray crystallography, MnIV(saladhp)2(1)(saladhp = 2-salicylideniminato-1,3-dihydroxy-2-methylpropane) has been synthesized and its physical properties reported (EPC=–460 mV vs. Ag/AgCl and a small axial zero field splitting parameter); the reported data are of potential importance to the understanding of manganoenzymes.
Electron paramagnetic resonance (EPR) spectroscopy and O2 evolution assays were performed on photosystem II (PSII) membranes which had been treated with 1 M CaCl2 to release the 17, 23 and 33 kilodalton (kDa) extrinsic polypeptides. Manganese was not released from PSII membranes by this treatment as long as a high concentration of chloride was maintained. We have quantitated the EPR signals of the several electron donors and acceptors of PSII that are photooxidized or reduced in a single stable charge separation over the temperature range of 77 to 240 K. The behavior of the samples was qualitatively similar to that observed in samples depleted of only the 17 and 23 kDa polypeptides (de Paula et al. (1986) Biochemistry25, 6487–6494). In both cases, the S2 state multiline EPR signal was observed in high yield and its formation required bound Ca2+. The lineshape of the S2 state multiline EPR signal and the magnetic properties of the manganese site were virtually identical to those of untreated PSII membranes. These results suggest that the structure of the manganese site is unaffected by removal of the 33 kDa polypeptide. Nevertheless, in samples lacking the 33 kDa polypeptide a stable charge separation could only be produced in about one half of the reaction centers below 160 K, in contrast to the result obtained in untreated or 17 and 23 kDa polypeptide-depleted PSII membranes. This suggests that one function of the 33 kDa polypeptide is to stabilize conformations of PSII that are active in secondary electron transfer events.
Photosystem II (PS II) particles retaining a high rate of O2 evolution were prepared from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland, and the composition and properties of their peripheral proteins were investigated. The following results were obtained. (1) The O2-evolving PS II particles of S. vulcanus contained only one peripheral protein with a molecular mass of 34000 which corresponded to the 33 kDa protein in higher plant PS II particles, but no other peripheral proteins corresponding to the 24 and 16 kDa proteins of higher plant PS II particles. (2) The cyanobacterial peripheral 34 kDa protein was removed from the particles by 1 M CaCl2-washing concomitant with total inactivation of O2 evolution, and the inactivated O2 evolution was reconstituted to 75% of the original activity by rebinding of this protein back to the washed particles. (3) The cyanobacterial peripheral 34 kDa protein rebound to CaCl2-washed spinach PS II particles and restored O2 evolution to an appreciable extent (28%). (4) The spinach peripheral 33 kDa protein rebound to CaCl2-washed PS II particles of S. vulcanus and partially restored O2 evolution (60%). These results suggested that the peripheral 34 kDa protein of S. vulcanus possesses the determinants for both binding and activity reconstitution identical with those of the peripheral 33 kDa protein of spinach.
In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S1 and S2 states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S2 state. Low-temperature illumination of samples prepared in the S2 state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH2OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl− with F− exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.
An oxygen-evolving, Photosystem II particle was isolated from the thermophilic, blue-green alga, Phormidium laminosum, according to the procedure of Stewart and Bendall (Stewart, A.C. and Bendall, D. (1979) FEBS Lett. 107, 308–312). Our particle has an oxygen-evolution activity of 1500–1600 μmol O2/mg chlorophyll per h. The oxygen-evolution activity has a pH optimum at 5–6, and is abolished at pH 9. Maximum oxygen evolution occurs at approx. 47°C in whole cells, but at 29°C in the particles. The activity decreases to 50% when the cells are heated for 30 min at 55°C; with the particles, 50% inactivation occurred at 47°C for the same heating time of 30 min. Flash excitation of the particle at 100 K produced absorbance changes whose difference spectrum in the ultraviolet-to-near infrared region shows photochemical charge separation and recombination of P-680+ and Q− in the dark with of 1.75 ms. An EPR spectrum for the P-680+ free radical, with g 2.0027 and ΔHpp = 8 G, was constructed from flash-induced EPR changes under conditions identical to those used for obtaining P-680 absorbance changes. The actinic light-induced variable fluorescence yield is 5-fold that induced by the weak probing beam alone. Addition of dithionite to the particle brings the fluorescence to the same maximum level. Under the reducing condition, strong actinic light caused the fluorescence to decrease. This observation is consistent with the notion that variable fluorescence yield in Photosystem II originates, as in green-plant chloroplasts, from recombination luminescence, the attenuation of which corresponds to photoaccumulation of reduced pheophytin under these conditions. Broad segments (300 nm) of the difference spectrum for pheophytin photoreduction were recorded by an intensified photodiode array in conjunction with a phosphoroscopic photometer. Kinetic spectrophotometric assays together with chemical analysis showed a rather clean and simple stoichiometry in these particles, namely, 1 P-680:1 Ph:1 Q:4 Mn:44 Chl. Initial investigation failed to reveal the doublet EPR spectrum previously observed for Ph−·Q− Fe in spinach subchloroplast particles (Klimov, V.V., Dolan, E. and Ke, B. (1980). FEBS Lett. 118, 97–100). A hyperfine EPR spectrum consisting of 16–20 lines and presumably associated with the manganese clusters in the oxygen-evolving protein has been confirmed in these particles. Tris washing but not washing with EDTA eliminates this signal. Active oxygen-evolving particles also yield the IIvf signal with a of approx. 800 μs. Upon Tris washing, the IIf signal appears which decays in 23.5 ms.
The LF-1 mutant of the green alga Scenedesmus obliquus is completely blocked on the oxidizing (water-splitting) side of photosystem II (PS II) while the reaction center and reducing side remain functional. A 34-kDa protein found in the PS II reaction center core complex of wild-type cells is replaced by a 36-kDa protein in the mutant cells. Both of these proteins are labeled by azido[14C]atrazine and are recognized by polyclonal antibodies raised against the herbicide-binding, D1 protein of Amaranthus hybridus. The data provide a new perspective on the role of the D1 protein by implying that it affects the oxidizing side of PS II in addition to performing its well established function on the reducing side.
An oxygen-evolving complex has been highly purified from the thermophilic cyanobacterium Synechococcus sp. The complex, which reproducibly showed 5 major polypeptide bands of 47, 40, 35, 30 and 9 kDa on SDS-polyacrylamide gel electrophoresis and contained 3.2 Mn per QA, had an oxygen-evolving activity of 300–400 chl per h in the presence of 5 mM MnCl2; or CaCl2. The complex most likely represents a minimum functional unit of the photosynthetic oxygen evolution.
Sharp-line fluorescence, paramagnetic resonance in the ground state, and optical absorption due to Mn4+ in alpha-Al2O3 have been observed (analog of Cr3+). The Mn4+ valence state is obtained by charge compensation with Mg2+. The increased charge of Mn4+ compared to Cr3+ results in a stronger crystal field and greater covalency whose effects are clearly seen in both the optical and paramagnetic resonance results. The ground state splitting is 0.39 cm-1, almost the same as in ruby, while the metastable 2E state splitting is 80 cm-1. When the crystals are irradiated with ultraviolet (
Incubation of PS II membranes with herbicides results in changes in EPR signals arising from reaction centre components. Dinoseb, a phenolic herbicide which binds to the reaction centre polypeptide, changes the width and form of the EPR signal arising from photoreduced Q−AFe. o-Phenanthroline slightly broadens the Q−AFe signal. These effects are attributed to changes in the interaction between the semi-quinone and the iron. DCMU, which binds to the 32 kDa protein, has virtually no effect on the width of the Q−AFe signal but does give rise to an increase in its amplitude. This could result from a change in redox state of an interacting component. Herbicide effects can also be seen when Q−AFe is chemically reduced and these seen to be reflected by changes in splitting and amplitude of the split pheophytin− signal. Dinoseb also results in the loss of ‘Signal II dark’, the conversion of reduced high-potential cytochrome b559 to its oxidized low-potential form and the presence of transiently photooxidized carotenoid after a flash at 25°C; these effects indicate that dinoseb may also act as an ADRY reagent.
— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O2 in weak light and light flashes to analyze the interactions between light induced O2 precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O2. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O2 evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, (S) successively accumulates four + charges. The S4+ state produces O2 and returns to the ground state S0. 5. Besides S0 also the first oxidized state S+ is stable in the dark, the two higher states, S2+ and S3+ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S*1←S2, has a (first) half time ≤ 200 μsec. The S*2 state and probably also the S*0 state are processed somewhat more slowly (˜ 300–400 μsec).
Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum. Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria (Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl2 or MgCl2 inhibited O2 evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O2 evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O2 evolution in parallel.
Freezing of spinach or barley chloroplasts during continuous illumination results in the trapping of a paramagnetic state or a mixture of such states characterized by a multiline EPR spectrum. Added Photosystem II electron acceptor enhances the signal intensity considerably. Treatments which abolish the ability of the chloroplasts to evolve oxygen, by extraction of the bound manganese, prevent the formation of the paramagnetic species. Restoration of Photosystem II electron transport in inhibited chloroplasts with an artificial electron donor (1,5-diphenylcarbazide) does not restore the multiline EPR spectrum. The presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in a modified signal which may represent a second paramagnetic state. The paramagnetic forms appear to originate on the donor side in Photosystem II and are dependent on a functional oxygenevolving site and bound, intact manganese. It is suggested that magnetically interacting manganese ions in the oxygen-evolving site may be responsible for the EPR signals. This suggestion is supported by calculations.
Photosystem 2 preparations with very high rates of oxygen evolution from the thermophilic cyanobacterium Phormidium laminosum have been studied by EPR spectrometry. In the presence of DCMU the g = 1.82 signal of the iron—quinone electron acceptor (Q) can be observed. It is proposed that DCMU is necessary to disrupt a magnetic interaction, between the semiquinone forms of Q and the secondary acceptor B, which otherwise prevents detection of the Q−Fe signal. A doublet EPR signal arising from magnetic interaction between Q−Fe and the reduced intermediary electron acceptor pheophytin (I−), and a spin-polarized triplet signal assumed to arise from the back reaction between I− and P680+ can also be seen. Preliminary redox titrations of Q reduction have been carried out, indicating Em ⋍ 0 mV.
Durch Oxidation einer methanolischen Lösung von Mn(II)-perchlorat, Sorbit und [NMe4]OH bei Raumtemp. wurde der Titelkomplex (I) dargestellt und als NMe4-Salz isoliert.
Extended X-ray absorption fine structure (EXAFS) amplitude and phase functions have been calculated from first principle for K and L edges of nearly half of the elements in the periodic table. It is shown that for L II,III edges, the transition from the initial state to the d final state is favored by almost a factor of 50 over the transition to the s final state such that the L II,III EXAFS can be analyzed in the same way as K and L I edges with the use of the l = 2 phase shifts. These theoretical EXAFS functions exhibit significant new structures (as a function of electron wave vector) which are in accord with experiments. Chemically interesting trends are observed for these functions as a function of atomic number Z. It is believed that these ab initio EXAFS functions can be used in EXAFS data analysis to provide accurate structural (interatomic distances) and chemical (type and number of neighboring atoms, Debye-Waller factors) information.
EPR examination of the class II (deeply trapped) mixed valence complexes ((bpy)/sub 2/MnO/sub 2/Mn(bpy)/sub 2/)/sup 3 +/ (the bipyridyl(III,IV) dimer) and its phenanthroline analogue in acetonitrile solution verifies that these complexes possess inequivalent Mn ions at room temperature. Isotropic hyperfine structure for two Mn ions is resolved with A/sub 1/ = 167 +- 3G and A/sub 2/ = 79 +- 3G for both complexes. The hyperfine pattern with parallel A/sub 1/ parallel approx. = 2 parallel A/sub 2/ parallel and the small g anisotropy are consistent with high-spin Mn(III) antiferromagnetically coupled to Mn(IV), producing an S = 1/2 ground state. At room temperature a rate of less than 10/sup 8/ s/sup -1/ is estimated for the thermally activated intramolecular electron transfer, consistent with an upper limit of 10/sup 6/ s/sup -1/ calculated from Hush's theory. The magnetic susceptibility of the (III,IV) complexes is characteristic of a strongly antiferromagnetically coupled S = (2, 3/2) pair. The temperature dependence of the data was in good agreement with the isotropic Heisenberg exchange Hamiltonian H = -2JS/sub 1/S/sub 2/, yielding J= -150 +- 7 cm/sup -1/ for the bipyridyl(III,IV) dimer and J = -134 +- 5 cm/sup -1/ for the phenanthroline analogue. 3 figures, 2 tables.
X-ray absorption spectra at the manganese K edge are presented for spinach chloroplasts and chloroplasts that were Tris treated and hence unable to evolve oxygen. A significant change in the electronic environment of manganese is observed and is attributed to the release of manganese from the thylakoid membranes with a concomitant change in oxidation state. A correlation of the K-edge energy, defined as the energy at the first inflection point, with coordination charge has been established for a number of manganese compounds of known structure and oxidation state. Comparison of the manganese K-edge energies of the chloroplast samples with the reference compounds places the average oxidation state of the chloroplasts between 2+ and 3+. With use of the edge spectra for Tris-treated membranes which were osmotically shocked to remove the released manganese, difference edge spectra were synthesized to approximate the active pool of manganese. Coordination charge predictions for this fraction are consistent with an average resting oxidation state higher than 2+. The shape at the edge is also indicative of heterogeneity of the manganese site, of low symmetry, or both.
Synthetic procedures for preparing the green Mn4(3,5-DBSQ)8 tetramer are reported. Spectral and structural characterization of the complex shows that the metal ions are in the Mn(II) form and that the quinone ligands are bonded as semiquinones. Weak magnetic exchange between the high-spin d5 metal ions and the paramagnetic ligands results in a magnetic moment of 5.1 μB per Mn(3,5-DBSQ)2 unit at room temperature. Treatment of the tetramer with pyridine results in formation of the Mn(3,5-DBCat)2(py)2 monomeric addition product. A structural study has shown that the metal ion of this molecule is Mn(IV) and that the quinone ligands are bonded in their catecholate form. EPR spectra verify the d3 configuration of the metal ion. In toluene solution at room temperature purple crystals of the complex give a green solution. At temperatures below 230 K the solution assumes the purple color of the crystals. Spectral evidence suggests an equilibrium between MnII(3,4-DBSQ)2(py)2 (green) and MnIV(3,4-DBCat)2(py)2 (purple) forms of the complex over the temperature range between 230 and 300 K in toluene. In pyridine the equilibrium temperature range is increased by approximately 100 K. The physical and structural properties of the manganese complexes are compared with the properties of the manganese protein associated with photosynthetic water oxidation.
The synthesis of several binuclear, oxobis(carboxylato)-bridged dimanganese(III) complexes, [Mn2O(O2CR)2(HB(pz)3)2] where R = CH3 (1), C2H5 (2), or H (3), and HB(pz)3- is the hydrotris(1-pyrazolyl)borate ligand, is described. X-ray structural studies of 1·CH3CN and 1·4CH3CN reveal two six-coordinate manganese atoms bridged by μ-oxo [av Mn-O,1.78 Å; Mn-O-Mn, 125.1°] and two μ-acetato (av Mn-O, 2.07 Å) groups and capped by two tridentate HB(pz)3- ligands. Each high spin, d4 Mn(III) center has its empty d-orbital directed toward the short Mn-Ooxo bond axis, the consequences of which are a shortening of Mn-N bonds trans to the μ-oxo group and markedly reduced antiferromagnetic coupling of the two Mn(III) centers compared to the two high spin, d5 Fe(III) centers in the [Fe2O(O2CCH3)2(HB(pz) 3)2] analogue. In the latter, the spin exchange coupling constant J = -121 cm-1, whereas χM vs. T measurements over the range 4.2 < T < 300 K for 1 reveal a J value of ∼ -0.5 cm-1. The greater paramagnetism and rapid spin relaxation of the manganese complexes leads to large isotropic shifts and narrow lines in their proton NMR spectra. All protons were observed in the 67 to -56 ppm region, and most could be assigned on the basis of deuterium substitution. The methyl resonance of the bridging acetate ligands in 1 occurs at +65.6 ppm, which should be useful for identifying the {Mn2O(O2CCH2R)2}2+ core in biology. These results suggest that substitution of Mn(III) for Fe(III) in metalloproteins such as hemerythrin or ribonucleotide reductase, that are known or believed to contain such cores, would provide a powerful NMR structural probe. The results of UV-vis, Raman, and infrared spectral studies are reported, including work on isotopically substituted 1, from which the symmetric and asymmetric Mn-O-Mn bridge bond stretching frequencies are assigned at 558 and 717 cm-1, respectively. Electrochemical studies of 1 reveal a quasi-reversible one-electron oxidation at 0.51 V vs. the Fc+/Fc couple to form the mixed valence Mn2(III,IV) complex. The ESR spectrum of a species, which was chemically generated from 1, exhibits a 16-line 55Mn hyperfine pattern that is typical of the Mn2(III,IV) trapped valence state. This spectrum closely matches that observed for the 300 K form of the S2 state of the manganese complex involved in photosynthetic water oxidation in green plants. Further oxidation of the mixed valence species reveals a second, quasi-reversible wave at 1.22 V vs. Fc+/Fc, tentatively assigned as the Mn2(IV,IV) complex.
Tris(3,5-di-tert-butylcatechol) anion (DTBC) complexes of manganese(II), -(III), and -(IV) have been studied in aprotic solvents by electrochemical, spectroscopic, and magnetic methods. The blue [MnIV(DTBC2-)3]2- complex reversibly binds oxygen at room temperature to form a red-brown complex [MnIV(DTBC2-)2(SQ-·)(O 2-·)]2- (SQ = semiquinone) with an apparent formation constant of 2.9 atm-1 at 25°C.
Hydroxyl-rich Schiff base ligands react with Mn(II) and Mn(III) salts in basic methanolic solution, generating monomeric Mn(IV) complexes. The general stoichiometry is MnL2, where L represents a dianionic, tridentate Schiff base ligand that uses one imine nitrogen, one phenolate oxygen, and one alkoxide oxygen atom to form a neutral octahedral complex. The molecular structure of Mn(SALADHP)2 (where H2SALADHP = 1,3-dihydroxy-2-methyl-2-(salicylideneamino)propane) has been determined by X-ray crystallography. The compound crystallizes from DMF/ether in the monoclinic space group P21/a (Z = 4, a = 10.676 (5) Å, b = 16.473 (10) Å, c = 17.541 (7) Å, β = 102.82 (4)°, V = 3008 (3) Å3), and the structure has been refined by using full-matrix least-squares methods to a final R = 0.076, Rw = 0.072 with 2186 data greater than 3σ(I). Those complexes that form one five-and one six-membered ring (where L can be H2SALAHE = 2-(salicylideneamino)-1l-ethanol, H2SALAPDH = 1,3-dihydroxy-3-phenyl-2-(salicylideneamino)propane, H2SALATHM = tris(hydroxymethyl)(salicylideneamino)methane, and H2NO2SALAPDH = 1,3-dihydroxy-3-(4-nitrophenyl)-2-(salicylideneamino)propane) are believed to be isostructural to Mn(SALADHP)2. The X-band EPR spectra, obtained at 90 K in DMF/methanol, show low-field features ranging between g = 4.32 and g = 5.45. The EPR spectrum for Mn(SALADHP)2 arises from a rhombically distorted S = 3/2 spin system with E/D = 0.22. The SALAHP ligand also forms mononuclear Mn(IV) complexes; however, it contains two six-membered chelate rings. All complexes exhibit room-temperature solid-state and solution magnetic moments in the range 3.80-4.3 μB, further substantiating the Mn(IV) formulation. The Epc values for the complexes in Me2SO range between -320 and -480 mV vs. Ag/AgCl. The stabilization of the Mn(IV) oxidation state by alkoxide oxygen ligation is demonstrated by the nearly 1 V more negative reduction potential of these compounds compared to that of another compound, bis(salicylato)(bipyridine)manganese(IV), with an N2O4 coordination environment.
The complexes (Et3NH)2[Mn(Cat)3] and K2[Mn(3,5-(t-Bu)2Cat)3]·6CH 3CN (Cat = catecholate) have been synthesized, and the latter has been characterized by X-ray diffraction (trigonal system, space group R3, a = 14.760 (9) Å, c = 50.752 (32) Å, Z = 6, final R = 6.9%, final Rw = 7.1%). The [Mn(3,5-(t-Bu)2Cat)3]2- anion has cristallographic threefold symmetry with short Mn-O bond lengths (1.922 (3) and 1.891 (3) Å) and no evidence of either dynamic or static Jahn-Teller distortion. The electron paramagnetic resonance spectrum of the [Mn(Cat)3]2- ion at 77 K is characteristic of a d3 system with large zero-field splitting. The magnetic behavior of K2[Mn(3,5-(t-Bu)2Cat)3]·6CH 3CN is fully consistent with that expected for a simple d3 system. The present structural, magnetic susceptibility, and magnetic resonance results establish that these essentially identical chelates are tris(catecholate)manganese(IV) rather than (semiquinone)bis(catecholate)manganese(III) complexes. The present structural results also show that the "bite distance" of catechol (2.58 Å) is too short to offer a regular octahedral environment to Mn(IV). This fact has a pervasive influence on the chemistry of the system. It causes trigonal compression of the Mn(IV) ion and thereby gives rise to the large zero-field splitting observed. In the Mn(III)-catechol system, it actually determines the solution chemistry. Because Mn(III) is even larger than Mn(IV) and has either two or four elongated bonds (by the Jahn-Teller theorem), three catechol ligands cannot span all six coordination sites in a chelating fashion. The short bite distance of catechol therefore effectively prevents formation of [MnIII(Cat)3]3-. Moreover, the difficulty of forming a tris complex of Mn(III) with a catechol-like ligand may contribute significantly to stabilization of [Mn(3,5-(t-Bu)2Cat)3]2- as tris(catecholate)manganese(IV) instead of (semiquinone)bis(catecholate)manganese(III).
A Mn-containing enzyme complex is involved in the oxidation of H/sub 2/O to O/sub 2/ in algae and higher plants. X-ray absorption spectroscopy is well suited for studying the structure and function of Mn in this enzyme complex. Results of X-ray K-edge and extended X-ray absorption fine structure (EXAFS) studies of Mn in the S/sub 1/ and S/sub 2/ states of the photosynthetic O/sub 2/-evolving complex in photosystem II preparations from spinach are presented in this paper. The S/sub 2/ state was prepared by illumination at 190 K or by illumination at 277 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); these are protocols that limit the photosystem II reaction center to one turnover. Both methods produce an S/sub 2/ state characterized by a multiline electron paramagnetic resonance (EPR) signal. An additional protocol, illumination at 140 K, produces a state characterized by the g = 4.1 EPR signal. The authors have previously observed a shift to higher energy in the X-ray absorption K-edge energy of Mn upon advancement from the dark-adapted S/sub 1/ state to the S/sub 2/ state produced by illumination at 190 K. The Mn K-edge spectrum of the 277 K illuminated sample is similar to that produced atmore » 190 K, indicating that the S/sub 2/ state is similar when produced at 190 or 277 K. A similar edge shape and an edge shift of the same magnitude are seen for the 140 K illuminated sample. These results indicate that the g = 4.1 signal arises from oxidation of the Mn complex and that the structural differences between the species responsible for the g = 4.1 signal and the multiline EPR signal are subtle. They conclude from the edge and EXAFS studies that the light-induced S/sub 1/ to S/sub 2/ transition at 190 K or at 277 K involves a change in the oxidation state of Mn with no EXAFS-detectable change in the coordination of Mn in the O/sub 2/-evolving complex.« less
Electron paramagnetic resonance (EPR) signals arising from components in photosystem II have been studied in membranes isolated from spinach chloroplasts. A broad EPR signal at g = 4.1 can be photoinduced by a single laser flash at room temperature. When a series of flashes is given, the amplitude of the g = 4.1 signal oscillates with a period of 4, showing maxima on the first and fifth flashes. Similar oscillations occur in the amplitude of a multiline signal centered at g ≃ 2. Such an oscillation pattern is characteristic of the S2 charge accumulation state in the oxygen-evolving complex. Accordingly, both EPR signals are attributed to the S2 state. Earlier data from which the g = 4.1 signal was attributed to a component different from the S2 state [Zimmermann, J.-L., & Rutherford, A. W. (1984) Biochim. Biophys. Acta 767, 160-167; Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] are explained by the effects of cryoprotectants and solvents, which are shown to inhibit the formation of the g = 4.1 signal under some conditions. The g = 4.1 signal is less stable than the multiline signal when both signals are generated together at low temperature. This indicates that the two signals arise from different populations of centers. The differences in structure responsible for the two different EPR signals are probably minor since both kinds of centers are functional in cyclic charge accumulation and seem to be interconvertible. The difference between the two EPR signals, which arise from the same redox state of the same component (a mixed-valence manganese cluster), is proposed to be due to a spin-state change, where the g = 4.1 signal reflects an S = 3/2 state and the multiline signal an S = 1/2 state within the framework of the model of de Paula and Brudvig [de Paula, J. C., & Brudvig, G. W. (1985) J. Am. Chem. Soc. 107, 2643-2648]. The spin-state change induced by cryoprotectants is compared to that seen in the iron protein of nitrogenase.
The prospects are shrinking rapidly for a future for society based on liquid hydrocarbons as a major source of energy. Among the wide array of alternative sources that are currently undergoing scrutiny, much attention is attracted to the photolysis of water to produce hydrogen and oxygen gases. Water, the starting material, does not suffer from lack of abundance, and there is every likelihood that the environmental consequences of water splitting will be negligible. Solar radiation is the obvious candiate for the ultimate energy source, but of course water cannot be photolyzed directly by the relatively low-energy wave-lengths, greater than 300 nm, that penetrate the earth's atmosphere. Nevertheless, the photolysis of water to produce O and reduced substances, with reduction potentials equivalent to that of H, is accomplished efficiently using sunlight by higher plant photosynthesis. There are even organisms that, under special conditions, will evolve H gas photosynthetically, but not efficiently when coupled with O production. To produce a molecule of O from water requires the removal of four electrons from two HO molecules.
The room-temperature EPR characteristics of Photosystem II reaction center preparations from spinach, pokeweed and Chlamydomonas reinhardii have been investigated. In all preparations a light-induced increase in EPR Signal II, which arises from the oxidized form of a donor to P-680+, is observed. Spin quantitation, with potassium nitrosodisulfonate as a spin standard, demonstrates that the Signal II species, Z⨥, is present in approx. 60% of the reaction centers. In response to a flash, the increase in Signal II spin concentration is complete within the 98 μs response time of our instrument. The decay of Z⨥ is dependent on the composition of the particle suspension medium and is accelerated by addition of either reducing agents or lipophilic anions in a process which is first order in these reagents. Comparison of these results with optical data reported previously (Diner, B.A. and Bowes, J.M. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoyunoglou, G., ed.), Vol. 3, pp. 875–883, Balaban, Philadelphia), supports the identification of Z with the P-680+ donor, D1. From the polypeptide composition of the particles used in this study, we conclude that Z is an integral component of the reaction center and use this conclusion to construct a model for the organization of Photosystem II.
The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z+ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl2, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P+Q− recombination and a slow Z+ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl2. These results are interpreted as a light-induced release of strongly bound Ca2+ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca2+.
Fluorescence detection, in principle, permits the detection of the extended x‐ray absorption fine structure (EXAFS) of more dilute atoms than can be obtained in absorption. To take advantage of this it is necessary, in practice, to eliminate the background that normally accompanies the fluorescence signal. We describe an x‐ray filter assembly that accomplishes this purpose. The unique characteristic of the assembly is a slit system that minimizes the fluorescence background from the filter. The theory of the slit assembly is presented and is found to agree with measurements made on the Fe EXAFS of a dilute sample. The filter assembly has a better effective counting rate in this case than that of a crystal monochromator design.
Incubation of highly active, O2-evolving PS II preparations at alkaline pH inhibits donor side electron-transfer reactions in two distinct fashions, one reversible the other irreversible. In both cases, O2 evolution is inhibited, with concomitant loss of the light-induced multiline and g = 4.1 EPR signals and an increased steady-state level of EPR Signal II induced by continuous illumination. However, the inhibition that is observed between pH 7.0 and 8.0 is readily reversible by resuspension at low pH, while above pH 8.0 the effect is irreversible. In addition, under repetitive flash conditions the ms decay kinetics remains largely unchanged at pH ≤ 8.0 but shows about a 2-fold increase in amplitude and is slowed at pH above 8.0. The irreversible component of inhibition most likely can be attributed to the loss of Mn and the 16, 24 and 33 kDa proteins. The reversible component may be mediated by displacement of Cl− from an anion-binding site by OH− or by titration of ionizable groups on the protein(s) associated with water-splitting. We propose that the reversible inhibition blocks electron transfer between the O2-evolving complex and an intermediate which serves as the direct donor to Signal II, while the irreversible inhibition blocks the reduction of Signal II by this intermediate donor species.
The structure of the Mn complex in the oxygen-evolving system and its mechanistic relation to photosynthetic oxygen evolution are poorly understood, though many studies have established that membrane-bound Mn plays an active role. Recently established procedures for isolating oxygen-evolving subchloroplast Photosystem II (PS II) preparations and the discovery of a light-induced multiline EPR signal attributable to the S2 state of the O2-evolving complex have facilitated the preparation of samples well characterized in the S1 and S2 states. We have used extended X-ray absorption fine structure (EXAFS) spectroscopy to probe the ligand environment of Mn in PS II particles from spinach, and in this report we present our results. The essential feature of the EXAFS results are that at least two Mn atoms per PS II reaction center occur as a binuclear species with a metal-metal distance of approx. 2.7 Å, with low Z atoms, N or O, at a distance of approx. 1.75 Å and at approx. 1.98 Å, which are characteristic of bridging and terminal ligands. These results agree well with those derived from whole chloroplasts that provided the first evidence for a binuclear manganese complex (Kirby, J.A., Robertson, A.S., Smith, J.P., Thompson, A.C., Cooper, S.R. and Klein, M.P. (1981) J. Am. Chem. Soc. 103, 5529–5537).
The decay kinetics of EPR Signal II has been used to monitor accumulation of positive charge in dark-adapted O2-evolving and inhibited PS II preparations from spinach. In fully active samples the Signal II transients following four saturating flashes exhibit periodic oscillations in amplitude and decay time. These oscillations result because electron transfer from the O2-evolving complex to Z+, the species giving rise to Signal II, becomes slower in the higher S-states. A small signal is induced following the first flash because of rapid rereduction of Z+ in the S1 state, but transients are resolved following the second and third flash, corresponding primarily to the S2 and S3 states, respectively, with half-times of 0.6 and 1.3 ms. The oscillatory pattern and decay times are similar to those previously reported in whole chloroplasts (Babcock, G.T., Blankenship, R.E. and Sauer, K. (1976) FEBS Lett. 61, 286–289). The period-four behavior is confirmed by extension of our observations to eight flashes. Upon partial inhibition of O2-evolution by NaCl washing, which extracts the 16 and 24 kDa peptides, or upon incubation at pH 7.75, a large transient is observed following the first flash, and subsequent flashes induce signals of similar amplitude and decay time. Extraction of the 16, 24 and 33 kDa peptides by CaCl2 washing also eliminates the oscillations and markedly slows the decay kinetics. The absence of period-four oscillations in these inhibited samples indicates that turnover of the O2-evolving complex is blocked. Furthermore, the large signals induced following the first flash indicate slow rereduction of Z+ in the lower S-states, suggesting that even partial advancement is inhibited. Addition of Ca2+ to NaCl-washed preparations restored the period-four oscillations. Thus, the 16 and 24 kDa peptides are not necessary for turnover of the O2-evolving complex, but in their absence Ca2+ is required for electron transfer from the O2-evolving complex to Z.
A study of signals, light-induced at 77 K in O2-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q−AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin− signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin− signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.
Electron paramagnetic resonance (EPR) spectroscopy of the iron-semiquinone complex in photosynthetic bacterial cells and chromatophores of Rhodopseudomonas viridis is reported. Magnetic fields are used to orient the prolate ellipsoidal-shaped cells which possess a highly ordered internal structure, consisting of concentric, nearly cylindrical membranes. The field-oriented suspension of cells exhibits a highly dichroic EPR signal for the iron-semiquinone complex, showing that the iron possesses a low-symmetry ligand field and exists in a preferred orientation within the native reaction-center membrane complex. The EPR spectrum is analyzed utilizing a spin hamiltonian formalism to extract physical information describing the electronic structure of the iron and the nature of its interaction with the semiquinones. Exact numerical solutions and analytical expressions for the transition frequencies and intensities derived from a perturbation theory expansion are presented, and a computer-simulated spectrum is given. It has been found that, for a model which assumes no preferred orientation within the plane of the membranes, the orientation of the Fe2+ ligand axis of largest zero-field splitting (Z, the principal magnetic axis) is titled 64±6° from the membrane normal. The ligand field for Fe2+ has low symmetry, with zero-field splitting parameters of |D1|=7.0±1.3 cm−1 and |E1|=1.7±0.5 cm−1 and for the redox state Q1−Fe2+Q2−. The rhombic character of the ligand field is increased in the redox state Q1Fe2+Q−2, where . This indicates that the redox state of the quinones can influence the ligand field symmetry and splitting of the Fe2+. There exists an electron-spin exchange interaction between Fe2+ and Q−1 and Q−2, having magnitudes |J1|=0.12±0.03 cm−1 and , respectively. Such weak interactions indicate that a proper electronic picture of the complex is as a pair of immobilized semiquinone radicals having very little orbital overlap (probably fostered by superexchange) with the Fe2+ orbitals. The exchange interaction is analyzed by comparison with model systems of paramagnetic metals and free radicals to indicate an absence of direct coordination between Fe2+ and Q−1 and Q−2. Selective line-broadening of some of the EPR transitions, involving Q− coupling to the magnetic sublevels of the Fe2+ ground state, is interpreted as arising from an electron-electron dipolar interaction. Analysis of this line-broadening indicates a distance of 6.2–7.8 Ȧ between Fe2+ and Q−1, thus placing Q1 outside the immediate coordination shell of Fe2+.
The molecular structure of the photosynthetic reaction centre from Rhodopseudomonas viridis has been elucidated using X-ray crystallographic analysis. The central part of the complex consists of two subunits, L and M, each of which forms five membrane-spanning helices. We present the first description of the high-resolution structure of an integral membrane protein.
CO2 depletion leads to an approximately 10-fold increase in the light-induced EPR signal at g = 1.82, attributed to the QA− · Fe2+ complex, in Photosystem II-enriched thylakoid membrane fragments. Upon reconstitution with HCO3−the signal decreases to the size in control samples. The split pheophytin− signal is broader in control or reconstituted than in CO2-depleted samples. It is concluded that HCO2− strongly influences the localization and conformation of the QA− · Fe+ complex. The QA− · Fe2+ and split pheophytirr− EPR signals from triazine-resistant Brassica napus were virtually identical to those from triazine-susceptible samples, indicating that the change in the 32-kDa azidoatrazine-binding protein does not lead to a confonnational change of the Qa− · Fe2+ complex.
A method is reported for the isolation of a highly resolved oxygen-evolving photosystem II reaction center preparation. This preparation can be separated from the more complex photosystem II membranes isolated by the procedure of Berthold et al. [(1981) FEBS Lett. 134, 231-234] by use of octylglucopyranoside at elevated ionic strengths; the oxygen-evolving material can be collected by centrifugation at relatively low g values (40000 x g) in yields estimated to be more than 80%. This new preparation lacks the 17 and 23 kDa extrinsic polypeptides; addition of calcium and chloride produces activities approaching 1000 [mu]mol O2/h per mg chlorophyll. Although activity is maximal in the presence of 2,5-dichloro-[beta]-benzoquinone, the response of activity to ferricyanide and 3-(3,4-dichlorophenyl)-1,1-dimethylurea indicates that the reducing side of photosystem II has been modified in this new oxygen-evolving reaction center preparation. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/26236/1/0000316.pdf
Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S(2) state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at approximately 30 cm(-1), inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S(2) state and that the g = 4.1 signal arises from monomeric Mn(IV).
The structure of the Mn complex of photosystem II (PSII) was studied by X-ray absorption spectroscopy. Oxygen-evolving spinach PSII membranes containing 4-5 Mn/PSII were treated with 0.8 M CaCl2 to extract the 33-, 24-, and 16-kilodalton (kDa) extrinsic membrane proteins. Mn was not released by this treatment, but subsequent incubation at low Cl- concentration generated preparations containing 2 Mn/PSII. The Mn X-ray absorption K-edge spectrum of the CaCl2-washed preparation containing 4 Mn/PSII is very similar to spectrum of native PSII, indicating that the oxidation states and ligand symmetry of the Mn complex in these preparations are not significantly different. The Mn extended X-ray absorption fine structure (EXAFS) of CaCl2-washed PSII fits to a Mn neighbor at approximately 2.75 A and two shells of N or O at approximately 1.78 and approximately 1.92 A. These distances are similar to those we have previously reported for native PSII preparations [Yachandra, V. K., Guiles, R. D., McDermott, A. E., Cole, J. L., Britt, R. D., Dexheimer, S. L., Sauer, K., & Klein, M. P. (1987) Biochemistry (following paper in this issue)] and are indicative of an oxo-bridged Mn complex. Our results demonstrate that the structure of the Mn complex is largely unaffected by removal of 33-, 24-, and 16-kDa extrinsic proteins, do not provide ligands to Mn. The Mn K-edge spectrum of the CaCl2-washed sample containing 2 Mn/PSII has a dramatically altered shape, and the edge inflection point is shifted to lower energy. The position of the edge is consistent with a Mn oxidation state of +3.(ABSTRACT TRUNCATED AT 250 WORDS)
The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)