No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The S1YZ. metalloradical intermediate in photosystem II: An X- and W-band EPR study
Abstract
Visible light illumination at liquid He temperatures of photosystem II (PSII) membranes poised in the S1-state, results in the production of a metalloradical signal with resonances at g
= 2.035 and g
2.0 at X-band (J. H. A. Nugent, I. P. Muhiuddin, and M. C. W. Evans, Biochemistry, 2002, 41, 4117–4126). A similar signal has been obtained by near IR excitation of samples poised in the S2 state (D. Koulougliotis, J.-R. Shen, N. Ioannidis, and V. Petrouleas, Biochemistry, 2003, 42, 3045–3053). The signal has been attributed to the magnetic interaction of the tyrosyl Z radical with the Mn cluster in the S1 state. In an effort to obtain further information about the interactions of tyrosine Z with the Mn cluster, and about the integer-spin S1 state we have employed EPR spectroscopy at two frequencies, X and W-band. The spectrum at W band is characterized by novel resonances at g
= 2.019, g
2.00 and g
= 1.987. For the analysis of the spectra at the two microwave frequency bands a spin Hamiltonian has been applied under the following basic assumptions: The S1 state of the Mn cluster is characterized by two low lying spin states Sa
= 0 and 1. The major features of the spectra are attributed to the interaction of the Sa
= 1 state with the spin Sb
= 1/2 of the tyrosyl radical. Potential contributions from the Sa
= 0 state are suppressed under the present experimental conditions. A satisfactory fit reproducing all features of the spectra is achieved with the same set of fitting parameters for the signals at both bands. An anisotropic ferromagnetic exchange interaction results from the fit with the coupling value being of the same order of magnitude with the value of the zero field splitting term of the Mn cluster (S
= 1).
... ) and the CaMn 4 cluster in PSII have been described from each of the S 1 , S 2 , S 3 and S 0 states when samples are illuminated with visible and/or NIR light at liquid helium temperatures27282930313233343536373839. The split S 1 and split S 3 signals are asymmetric: the former is characterised by a low-field peak at g = 2.035 and the latter has a double trough at the high-field side of g = 2 together with a weak broad peak at the low-field side. ...
... Both signals were quickly induced and reached the maximum amplitude after 4 min illumination with visible light. The split S 3 signal can be induced with both visible and NIR light (Fig. 1B) [35]. With visible light, 60% of the maximum amplitude was formed after illumination for 4 min and the maximum was slowly reached after 60 min. ...
EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn(4) cluster are known in all S states except S(4). Many signals are insufficiently understood and the S(0), S(1), and S(3) states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S(0), S(1), and S(3) states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S(0), split S(1), split S(3) and S(2) state multiline EPR signals. We demonstrate that quantification of the S(1), S(3) and S(0) states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S(1) --> S(2) transition proceeds without misses following a saturating flash at 1 degrees C, whilst substantial misses occur in the S(2) --> S(3) transition following the second flash.
... Dexheimer and Klein suggested that the state giving rise to the S 1 g = 4.8 signal converts to the S 2 g = 2 multiline state but does not correlate with the S 2 g = 4.1 signal, [53] while Gregor and Britt showed that the S 2 g = 4.1 signal preferentially decays to the S 1 multiline centered at g % 12. [57] Sioros et al. supported that the S 1 Y Z C component with the characteristic EPR signal at g = 2.035 advances to the S 2 multiline, while a 26 G wide signal advances to the g = 4.1 conformation. [58] EPR investigations of the g = 2.035 component of the S 1 Y Z C at X-band and W-band frequencies revealed that the major features of the spectra are due to the interaction of the S = 1 state of the Mn 4 cluster with the S = 1/2 spin of Y Z C. [59] These observations are consistent with the idea that the S 1 state consists of two isomers that are the predecessors of the g = 2 or g = 4.1 forms of the S 2 state. The two EPR signals described above remain without explicit atomistic explanation and even their common origin has been questioned. ...
The dark-stable resting state of the biological oxygen-evolving complex is shown to accommodate a rare type of functionally important MnIII orientational Jahn–Teller isomerism that is identified as the electronic origin of subsequent valence isomerism in the catalytic cycle of water oxidation.
Abstract
The tetramanganese–calcium cluster of the oxygen-evolving complex of photosystem II adopts electronically and magnetically distinct but interconvertible valence isomeric forms in its first light-driven oxidized catalytic state, S2. This bistability is implicated in gating the final catalytic states preceding O−O bond formation, but it is unknown how the biological system enables its emergence and controls its effect. Here we show that the Mn4CaO5 cluster in the resting (dark-stable) S1 state adopts orientational Jahn–Teller isomeric forms arising from a directional change in electronic configuration of the “dangler” MnIII ion. The isomers are consistent with available structural data and explain previously unresolved electron paramagnetic resonance spectroscopic observations on the S1 state. This unique isomerism in the resting state is shown to be the electronic origin of valence isomerism in the S2 state, establishing a functional role of orientational Jahn–Teller isomerism unprecedented in biological or artificial catalysis.
... [55] This signal could also be induced in spinach by removal of two extrinsic PSII proteins, [56] showing that the spectroscopic response of the OEC in the S 1 state is sensitive to small structural perturbations.D exheimer and Klein suggested that the state giving rise to the S 1 g = 4.8 signal converts to the S 2 g = 2multiline state but does not correlate with the S 2 g = 4.1 signal, [53] while Gregor and Britt showed that the S 2 g = 4.1 signal preferentially decays to the S 1 multiline centered at g % 12. [57] Sioros et al. supported that the S 1 Y Z C component with the characteristic EPR signal at g = 2.035 advances to the S 2 multiline,w hile a2 6G wide signal advances to the g = 4.1 conformation. [58] EPR investigations of the g = 2.035 component of the S 1 Y Z C at X-band and Wband frequencies revealed that the major features of the spectra are due to the interaction of the S = 1state of the Mn 4 cluster with the S = 1/2 spin of Y Z C. [59] These observations are consistent with the idea that the S 1 state consists of two isomers that are the predecessors of the g = 2org = 4.1 forms of the S 2 state.T he two EPR signals described above remain without explicit atomistic explanation and even their common origin has been questioned. [55] Here we show that they are both attributable to the S 1 state and have ao ne-to-one correspondence with the Jahn-Teller isomers described above. ...
The tetramanganese–calcium cluster of the oxygen‐evolving complex of photosystem II adopts electronically and magnetically distinct but interconvertible valence isomeric forms in its first light‐driven oxidized catalytic state, S2. This bistability is implicated in gating the final catalytic states preceding O−O bond formation, but it is unknown how the biological system enables its emergence and controls its effect. Here we show that the Mn4CaO5 cluster in the resting (dark‐stable) S1 state adopts orientational Jahn–Teller isomeric forms arising from a directional change in electronic configuration of the “dangler” MnIII ion. The isomers are consistent with available structural data and explain previously unresolved electron paramagnetic resonance spectroscopic observations on the S1 state. This unique isomerism in the resting state is shown to be the electronic origin of valence isomerism in the S2 state, establishing a functional role of orientational Jahn–Teller isomerism unprecedented in biological or artificial catalysis.
... Such a difference could be caused e.g. by changes in the bridging of the two spin centers that would modify the strength of the exchange coupling, or by differences in the localization of the spin density that would modify the dipolar coupling. Elaborate theoretical simulations in conjunction with multifrequency EPR investigations have been carried in the past of the S 2 Y Å z split EPR signals in preparations inhibited in the S 2 to S 3 step by acetate-treatment or calcium depletion [26][27][28][29] as well as of the S 1 Y Å z in untreated preparations [30]. The simulations took into account the isotropic exchange interaction between the Mn cluster and the Y z radical, the point-dipolar interaction between the two spin centers, the hyperfine interaction of the four Mn ions, and the g-anisotropy, especially for the Mn cluster. ...
... Y Z @BULLET is not trivial to detect by conventional EPR techniques. However, at low temperatures, where the S state transitions are blocked, any formed Y Z @BULLET can only decay by recombination with the acceptor side of PSII (with Q A ˉ) which makes the live time of Y Z @BULLET much longer and thus, possible for measurement101102103. Moreover, at cryogenic temperatures, the Y Z radical interacts magnetically with the CaMn 4 O x -cluster. ...
Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q(A)- Fe²(+) and Phe- Q(A)- Fe²(+) as well as from the free Phe- radical were observed. The multiline EPR signals from the S₀- and S₂-states of CaMn₄O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn₄O(x)-cluster in the S₀-, S₁-, S₂-, and the S₃-state were induced by illumination of the PSII membranes at 5K and characterized. In addition, EPR signals from auxiliary donors Y(D), Chl(+) and cytochrome b₅₅₉ were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.
... At liquid helium temperatures (below 77 K), initial charge separation to form P 680 +@BULLET and Pheo -@BULLET still takes place [10,13,25,26], while electron transfer from Q A -@BULLET to Q B is completely blocked272829. P 680 +@BULLET can drive the oxidation of the side-path electron donors (Car/ Chl Z /Cyt b559 )30313233343536 in most PSII preparations, and it can also drive the oxidation of Tyr Z found recently in intact PSII373839404142434445. It was reported recently that the redox properties of non-heme iron and the existence of Q B significantly affected these secondary electron donors in intact samples [46]. ...
The correlation between the reduction of Q(A) and the oxidation of Tyr(Z) or Car/Chl(Z)/Cyt(b559) in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g=1.95-1.86 Q(A)(-*)EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from Q(A)(-*), Car(+*)/Chl(+*) and oxidized Cyt(b559), while enhancing the amplitude of the S(1)Tyr(Z)* EPR signal in the intact PSII sample. The quantification and stability of the g=1.95-1.86 EPR signal and signals arising from the oxidized Tyr(Z) and the side-path electron donors, respectively, indicate that the EPR-detectable g=1.95-1.86 Q(A)(-*) signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/Chl(Z)/Cyt(b559)), but not of Tyr(Z). These results imply that two types of Q(A)(-*) probably exist in the intact PSII sample. The structural difference and possible function of the two types of Q(A) are discussed.
... of the split signal by P 1/2 studies When examining the split signal (Fig. 4C) at a single applied microwave power, its spectral shape was found to be similar to those examples reported previously [12,21,30,31]. The spectral shape of the split signal was also independent of the presence or absence of exogenous electron acceptors (e.g. ...
Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q(A)((-)) is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (<5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680(+) is observed with the same decay kinetics as the S(1) split signal. Cryogenic donors to P680(+) seen are: (i) transient (t(1/2) approximately 150 s) tyrosine related species, including 'split signals' ( approximately 15% total centers), (ii) reduced cytochrome b(559) ( approximately 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, ( approximately 30% centers).
... They were generated by illuminating PSII in the S 0 , S 1 or S 2 states with visible light or in the S 3 state with near-infrared (NIR) light either at liquid helium temperatures or at 200 K. The species that were formed under these conditions were proposed to correspond to 'non-relaxed' S 0 TyrZ % S 1 TyrZ % and S 2 TyrZ % states (Ioannidis et al. 2002;Koulougliotis et al. 2003Koulougliotis et al. , 2004Zhang & Styring 2003;Zhang et al. 2004;Petrouleas et al. 2005;Havelius et al. 2006;Ioannidis et al. 2006) that could correspond to intermediates in the oxygen-evolving process. ...
The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.
Specialist Periodical Reports provide systematic and critical review coverage in major areas of chemical research. Compiled by teams of leading authorities in the relevant subject, the series creates a unique service for the active research chemist with regular critical in-depth accounts of progress in particular areas of chemistry. Subject coverage of all volumes is very similar and publication is on an annual or biennial basis. As EPR continues to find new applications in virtually all areas of modern science, including physics, chemistry, biology and materials science, this series caters not only for experts in the field, but also those wishing to gain a general overview of EPR applications in a given area.
The Photosystem II (PSII) water oxidation to molecular oxygen process is catalyzed by an oxygen-bridged Mn4CaO5 cluster, known as Oxygen Evolving Complex (OEC) of PSII. The Mn4CaO5 cluster undergoes periodically four one-electron oxidation steps, S0 → S1, S1 → S2, S2 → S3, S3 → S0. The O2 release takes place during the S3-[S4]-S0 transition, S4 being a transient. The intermediates of the S-state transitions are known as metalloradical intermediate states (SiYz) and involve the free radical of Tyrosine Z (Yz, TyrZ). In most metalloradical states it was established that Yz interacts magnetically with the Mn4CaO5 cluster. However, in Ca²⁺- depleted PSII preparations the spin-spin interaction between the Mn4O5 and Yz, during the formation of the S2Yz intermediate state was not strongly supported experimentally. In our effort to investigate the existence of the aforementioned interaction, we took advantance of the NIR sensitivity of the S2 state of Mn4 and examined whether the S2Yz EPR spectrum can be modified. Our EPR experiments, combined with the simulation analysis of the spectra show that reversible modification of the S2 EPR spectrum by NIR irradiation results in a reversible change of the Yz EPR signal shape. These observations strongly support the idea of the magnetic interaction between the Mn4 and tyrosyl radical, upon the formation of the S2Yz metalloradical state in Ca²⁺- depleted PSII membranes.
Virtually all life on our planet is powered by the sun through the process of photosynthesis. Understanding the design and operating principles of natural photosynthesis is a central challenge for fundamental research because biology presents a unique paradigm and possible blueprint for the realization of artificial alternatives. All processes of natural photosynthesis, including light harvesting, charge separation and accumulation, water oxidation, and carbon fixation, embody “solutions” optimized through billions of years of evolution to challenges currently faced by scientists developing components for artificial photosynthetic devices. The present chapter provides an overview of the most important aspects of natural photosynthesis and discusses the ways in which they motivate biomimetic and bioinspired research into artificial photosynthesis.
The oxidation states of the Mn4CaO5 cluster of the photosynthetic oxygen-evolving complex remain controversial. New quantum chemical models for the dark-stable S1 state suggest that a low-valent Mn(III-IV-III-II) form, opposed to the commonly accepted high-valent Mn(III-IV-IV-III) assignment, agrees with available structural data. We examine the magnetic properties of the models and the consequences for electronic structure and catalytic progression of assuming a neutral second-sphere residue D1-His337. The low-valent model reproduces the experimental diamagnetic ground spin-state. However, the protonation assignment introduces a critical flaw because neutral His337 becomes itself the locus of subsequent oxidation, in contradiction to physiological catalytic progression.
The sunlight-powered oxidation of water by photosystem II (PSII) of algae, plants, and cyanobacteria underpins the energy conversion processes that sustain most of life on our planet. Understanding the structure and function of the “engine of life”, the oxygen-evolving complex (OEC) in the active site of PSII, has been one of the great and persistent challenges of modern science. Immense progress has been achieved in recent years through combined contributions of diverse disciplines and research approaches, yet the challenge remains. The improved understanding of the tetramanganese-calcium cluster of the OEC for the experimentally accessible catalytic states often creates a more complex picture of the system than previously imagined, while the various strands of evidence cannot always be unified into a coherent model. This review focuses on selected current problems that relate to structural–electronic features of the OEC, emphasizing conceptual aspects and highlighting topics of structure and function that remain uncertain or controversial. The Mn4CaOx cluster of the OEC cycles through five redox states (S0–S4) to store the oxidizing equivalents required for the final step of dioxygen evolution in the spontaneously decaying S4 state. Remarkably, even the dark-stable state of the OEC, the S1 state, is still incompletely understood because the available structural models do not fully explain the complexity revealed by spectroscopic investigations. In addition to the nature of the dioxygen-evolving S4 state and the precise mechanism of O–O bond formation, major current open questions include the type and role of structural heterogeneity in various intermediate states of the OEC, the sequence of events in the highly complex S2–S3 transition, the heterogeneous nature of the S3 state, the accessibility of substrate or substrate analogues, the identification of substrate oxygen atoms, and the role of the protein matrix in mediating proton removal and substrate delivery. These open questions and their implications for understanding the principles of catalytic control in the OEC must be convincingly addressed before biological water oxidation can be understood in its full complexity on both the atomic and the systemic levels.
Methanol has long being used as a substrate analogue to probe access pathways and investigate water delivery at the oxygen-evolving complex (OEC) of photosystem-II. In this contribution we study the interaction of methanol with the OEC by assembling available spectroscopic data into a quantum mechanical treatment that takes into account the local channel architecture of the active site. The effect on the magnetic energy levels of the Mn4Ca cluster in the S2 state of the catalytic cycle can be explained equally well by two models that involve either methanol binding to the calcium ion of the cluster, or a second-sphere interaction in the vicinity of the "dangler" Mn4 ion. However, consideration of the latest ¹³C hyperfine interaction data shows that only one model is fully consistent with experiment. In contrast to previous hypotheses, methanol is not a direct ligand to the OEC, but is situated at the end-point of a water channel associated with the O4 bridge. Its effect on magnetic properties of plant PS-II results from disruption of hydrogen bonding between O4 and proximal channel water molecules, thus enhancing superexchange (antiferromagnetic coupling) between the Mn3 and Mn4 ions. The same interaction mode applies to the dark-stable S1 state and possibly to all other states of the complex. Comparison of protein sequences from cyanobacteria and plants reveals a channel-altering substitution (D1-Asn87 versus D1-Ala87) in the proximity of the methanol binding pocket, explaining the species-dependence of the methanol effect. The water channel established as the methanol access pathway is the same that delivers ammonia to the Mn4 ion, supporting the notion that this is the only directly solvent-accessible manganese site of the OEC. The results support the pivot mechanism for water binding at a component of the S3 state and would be consistent with partial inhibition of water delivery by methanol. Mechanistic implications for enzymatic regulation and catalytic progression are discussed.
Nature’s water-splitting catalyst moves through a reaction cycle with five catalytic intermediates characterized by different spin ground states, the origin of which is connected to their geometric structures and intermetallic magnetic couplings. The early “inactive” intermediates have low-spin ground states, while the later “active” intermediates transition to high-spin states. The cofactor’s ability to switch from lower to higher-spin states via an open-to-closed cubane conversion is critical for substrate water binding.
This chapter provides an overview of oxygenic photosynthesis, with primary focus on the oxygen-evolving complex (OEC) of photosystem II (PSII). The introduction includes a general discussion of photosynthesis and a brief overview of the metals involved. This is followed by an in-depth description of the metal centers within PSII, in which the structural and electronic characterizations of the OEC are reviewed in detail. The role of chloride in PSII and the proton exit pathway are discussed. Several mechanisms proposed for the O. O bond formation in the OEC are presented, along with a few detailed mechanisms for the complete catalytic cycle obtained from quantum mechanics/molecular mechanics and density functional theory calculations. Model complexes that are functional mimics of the OEC are also briefly discussed. General discussion on plastocyanin, iron-sulfur centers, [FeFe] and [NiFe] hydrogenases, and ribulose-1,5-bisphosphate carboxylase-oxygenase is included. The chapter concludes with the implications of natural photosynthesis on the development of an artificial photosynthetic framework.
We have earlier shown that all electron transfer reactions in Photosystem II are operational up to 800 nm at room temperature [Thapper et al. (2009), Plant Cell 21, 2391-2401]. This led us to suggest an alternative charge separation pathway for far-red excitation. Here we extend these studies to very low temperature (5 K). Illumination of photosystem II (PS II) with visible light at 5 K is known to result in oxidation of almost similar amounts of YZ and the Cyt b559/ChlZ/CarD2 pathway. This is reproduced here using laser flashes at 532 nm and we find the partition ratio between the two pathways to be 1:0.8 at 5 K (the partition ratio is here defined as (yield of YZ/CaMn4 oxidation):(yield of Cyt b559/ChlZ/CarD2 oxidation)). The result using far red laser flashes is very different. We find partition ratios of 1.8 at 730 nm; 2.7 at 740 nm and >2.7 at 750 nm. No photochemistry involving these pathways is observed above 750 nm at this temperature. Thus, far-red illumination preferentially oxidizes YZ while the Cyt b559/ChlZ/CarD2 pathway is hardly touched. We propose that the difference in the partition ratio between visible and far-red light at 5 K reflects the formation of different first stable charge pair. In visible light, the first stable charge pair is considered to be PD1+Qa-. In contrast, we propose that the electron hole is residing on the ChlD1 molecule after illumination by far red at light 5 K resulting in the first stable charge pair being ChlD1+QA-. ChlD1 is much closer to YZ (11.3 Å) than to any component in Cyt b559/ChlZ/CarD2 pathway (closest distance is ChlD1 - CarD2 is 28.8 Å). This would then explain that far-red illumination preferentially drives efficient electron transfer from YZ. We also discuss mechanisms to account for the absorption of the far-red light and the existence of a hitherto unobserved charge transfer states. The involvement of two or more of the porphyrin molecules in the core of the Photosystem II reaction center is proposed.
This article provides an overview of the application of electron paramagnetic resonance (EPR) and related hyperfine spectroscopies for the investigation of the electronic and geometric structure of the tetramanganese cluster that constitutes the oxygen-evolving complex (OEC) of photosystem II. Starting from the spin physics of Mn ions, a quantum-mechanical description of exchange-coupled oligonuclear Mn systems is given. Then, the focus shifts to the characterization of the OEC with emphasis on the two half-integer spin states of its catalytic cycle, namely the S0 and S2. The development of electronic models based on EPR and related spectroscopies, such as electron nuclear double resonance (ENDOR), is described in detail. The role of broken-symmetry density functional theory (BS-DFT) is also outlined. The characterization of Mn ligands and bound substrate waters via electron nuclear magnetic interactions is illustrated. Implications for the mechanism of water oxidation catalysis are discussed.
The role of Ca2+ in the Photosystem IIwateroxidation mechanism has been investigated in the higher redox states of the enzyme. The involvement of Ca2+ in structuring the environment of the tyrosine YZ in the S3 state has been investigated by studying the effect of a Ca/Sr exchange by pulse-EPR spectroscopy. It is shown that Ca and Yz are involved in a common hydrogen bond network in S3. The comparison of the temperature dependence of the rate of the wateroxidation reaction shows that the slowdown of the S3YZ˙ → S0 transition in Sr-PSII mainly arises from a decrease of the entropic part of the ΔG# of the reaction. This suggests that Ca/Sr exchange perturbs the distribution of the conformational microstates and thereby hinders the overall water splitting process.
Illuminating a photosystem II sample at low temperatures (here 5-10 K) yields so called split signals detectable with CW-EPR. These signals reflect the oxidized, deprotonated radical of D1-Tyr161 (YZ(•)) in a magnetic interaction with the CaMn4 cluster in a particular S state. The intensity of the split EPR signals are affected by the addition of the water substrate analogue methanol. This was previously shown by the induction of split EPR signals from the S1, S3 and S0 states [Su, J-H. et al. (2006) Biochemistry 45, 7617-7627.]. Here, we use two split EPR signals induced from photosystem II trapped in the S2 state to further probe the binding of methanol in an S state dependent manner. The signals are induced with either visible or near-infrared light illumination provided at 5-10 K where methanol cannot bind or un-bind from its site. The results imply that the binding of methanol not only changes the magnetic properties of the CaMn4 cluster but also the hydrogen bond network in the OEC, thereby affecting the relative charge of the S2 state. The induction mechanisms for the two split signals are different resulting in two different redox states, S2YZ(•) and S1YZ(•) respectively. The two states show different methanol dependence for their induction. This indicates the existence of two binding sites for methanol in the CaMn4 cluster. It is proposed that methanol binds to MnA with high affinity and to MnD with lower affinity. The molecular nature and S-state dependence of the methanol binding to each respective site is discussed.
Charge recombination in the light-induced radical pair SnTyrZ•QA-• in Photosystem II (PSII) from Thermosynechococcus elongatus has been studied at cryogenic temperatures by time-resolved EPR for different configurations of PSII that are expected to affect the driving force of the reaction (oxidation states S0, S1 or S2 of the Mn4CaO5 cluster; PsbA1, PsbA2 or PsbA3 as D1 protein). The kinetics were independent of temperature in the studied range from 4.2 to 50 K and were not affected by exchange of H2O for D2O, consistent with single-step electron tunneling over the distance of ≈32 Å without any repopulation through Boltzmann equilibration of intermediates lying higher in energy. In PsbA1-PSII, the charge recombinations in the radical pairs SnTyrZ•QA-• (ket = 3.4 10-3 s-1 for S1) were slower than in PsbA3-PSII despite an expected lower driving force owing to a downshifted Em(QA/QA-•) in PsbA1-PSII. Conversely, the reaction was slower in the presence of S2 than in the presence of S1, despite an expected larger driving force due to an upshifted Em(TyrZ•/TyrZ) in S2. These observations indicate that the charge recombination occurs in the Marcus inverted region. Assuming that the driving force of the reaction (-ΔG0 ≈ 1.2 eV at room temperature for S1) does not vary strongly with temperature, the data indicate an optimal electron transfer rate (for a hypothetical -ΔG0 = λ) substantially faster than would be predicted from extrapolation of room temperature intra-protein ET rates over shorter distances. Possible origins of this deviation are discussed, including a possible enhancement of the electronic coupling of TyrZ• and QA-• by aromatic cofactors located in between. Observed similar S1TyrZ•QA-• charge recombinations in PsbA2-PSII and PsbA3-PSII predict that Em(QA/QA-•) in PsbA2-PSII is similar to that in PsbA3-PSII.
The redox-active tyrosine residue (YZ) plays a crucial role in the mechanism of the water oxidation. Metalloradical electron paramagnetic resonance (EPR) signals
reflecting the light-induced YZ· in magnetic interaction with the CaMn4-cluster in the particular S-state, YZ·SX intermediates, have been found in intact photosystem II. These so-called split EPR signals are induced by illumination at
cryogenic temperatures and provide means to both study the otherwise transient YZ· and to probe the S-states with EPR spectroscopy. The illumination used for signal induction grouped the observed split EPR signals in two categories:
(i) YZ in the lower S-states was oxidized by P680+ formed via charge separation, while (ii) YZ in the higher S-states was oxidized by an excited, highly oxidizing Mn species. Applied mechanistic studies of the YZ·SX intermediates in the different S-states are reviewed and compared to investigations in photosystem II at physiological temperature. Addition of methanol induced
S-state characteristic changes in the split signals’ formation which reflect changes in the magnetic coupling within the CaMn4-cluster due to methanol binding. The pH titration of the split EPR signals, on the other hand, could probe the proton-coupled
electron transfer properties of the YZ oxidation. The apparent pK
as found for decreased split signal induction were interpreted in the fate of the phenol proton.
The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr(Z)(•) in the (S(3)Tyr(Z)(•))' is slightly modified; (ii) the split EPR signal arising from Tyr(Z)(•) in the (S(2)Tyr(Z)(•))' state induced by near-infrared illumination at 4.2 K of the S(3)Tyr(Z) state is significantly modified; and (iii) the slow phases of P(680)(+) reduction by Tyr(Z) are slowed down from the hundreds of μs time range to the ms time range, whereas both the S(1)Tyr(Z)(•) → S(2)Tyr(Z) and the S(3)Tyr(Z)(•) → S(0)Tyr(Z) + O(2) transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr(Z) phenol and its environment, likely the Tyr-O···H···Nε-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr(Z) being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr(Z) and between the two α-helices bearing Tyr(Z) and its hydrogen-bonded partner, His-190, are responsible for these changes.
The X-ray crystallographic structure of the native R2F subunit of the ribonucleotide reductase (RNR) of Corynebacterium ammoniagenes ATCC 6872 is reported, with a resolution of 1.36 A. The metal site contains an oxo/hydroxo-bridged manganese dimer, located near a tyrosine residue (Y115). The coordination of the manganese dimer and its distance to a nearby tyrosine residue resemble the di-iron metalloradical cofactor of class I RNR from Escherichia coli . Multifrequency EPR measurements of the highly active C. ammoniagenes R2F subunit show that the metal site contains a ferromagnetically exchange-coupled Mn(III)Mn(III) dimer weakly coupled to a tyrosyl radical. A mechanism for the metalloradical cofactor (Mn(III)Mn(III)Y(*)) generation is proposed. H(2)O(2) (HO(2)(-)) instead of O(2) is hypothesized as physiological oxidant for the Mn dimer which in turn oxidizes the tyrosine Y115. Changes in the ligand sphere of both manganese ions during metalloradical generation direct the complex formation of this cofactor, disfavoring alternate reaction pathways such as H(2)O(2) dismutation, as observed for manganese catalase, a structural analogue of the R2F metal site. The presented results demonstrate the importance of manganese for radical formation in this RNR and confirm the assignment of this enzyme to class Ib.
Genome sequence of Arabidopsis thaliana (Arabidopsis) revealed two psbO genes (At5g66570 and At3g50820) which encode two distinct PsbO isoforms: PsbO1 and PsbO2, respectively. To get insights into the function of the PsbO1 and PsbO2 isoforms in Arabidopsis we have performed systematic and comprehensive investigations of the whole photosynthetic electron transfer chain in the T-DNA insertion mutant lines, psbo1 and psbo2. The absence of the PsbO1 isoform and presence of only the PsbO2 isoform in the psbo1 mutant results in (i) malfunction of both the donor and acceptor sides of Photosystem (PS) II and (ii) high sensitivity of PSII centers to photodamage, thus implying the importance of the PsbO1 isoform for proper structure and function of PSII. The presence of only the PsbO2 isoform in the PSII centers has consequences not only to the function of PSII but also to the PSI/PSII ratio in thylakoids. These results in modification of the whole electron transfer chain with higher rate of cyclic electron transfer around PSI, faster induction of NPQ and a larger size of the PQ-pool compared to WT, being in line with apparently increased chlororespiration in the psbo1 mutant plants. The presence of only the PsbO1 isoform in the psbo2 mutant did not induce any significant differences in the performance of PSII under standard growth conditions as compared to WT. Nevertheless, under high light illumination, it seems that the presence of also the PsbO2 isoform becomes favourable for efficient repair of the PSII complex.
Tyrosine Z (Tyr(Z)) oxidation observed at liquid helium temperatures provides new insights into the structure and function of Tyr(Z) in active Photosystem II (PSII). However, it has not been reported in PSII core complex from higher plants. Here, we report Tyr(Z) oxidation in the S(1) and S(2) states in PSII core complex from spinach for the first time. Moreover, we identified a 500 G-wide symmetric EPR signal (peak position g = 2.18, trough position g = 1.85) together with the g = 2.03 signal induced by visible light at 10 K in the S(1) state in the PSII core complex. These two signals decay with a similar rate in the dark and both disappear in the presence of 6% methanol. We tentatively assign this new feature to the hyperfine structure of the S(1)Tyr(Z)(*) EPR signal. Furthermore, EPR signals of the S(2) state of the Mn-cluster, the oxidation of the non-heme iron, and the S(1)Tyr(Z)(*) in PSII core complexes and PSII-enriched membranes from spinach are compared, which clearly indicate that both the donor and acceptor sides of the reaction center are undisturbed after the removal of LHCII. These results suggest that the new spinach PSII core complex is suitable for the electron transfer study of PSII at cryogenic temperatures.
Methanol binds to the CaMn4 cluster in photosystem II (PSII). Here we report the methanol dependence of the split EPR signals originating from the magnetic interaction between the CaMn4 cluster and the Y(Z)* radical in PSII which are induced by illumination at 5 K. We found that the magnitudes of the "split S1" and "split S3" signals induced in the S1 and S3 states of PSII centers, respectively, are diminished with an increase in the methanol concentration. The methanol concentrations at which half of the respective spectral changes had occurred ([MeOH](1/2)) were 0.12 and 0.57%, respectively. By contrast, the "split S0" signal induced in the S0 state is broadened, and its amplitude is enhanced. [MeOH](1/2) for this change was found to be 0.54%. We discuss these observations with respect to the location and nature of the methanol binding site. Furthermore, by comparing this behavior with methanol effects reported for other EPR signals in the different S states, we propose that the observed methanol-dependent changes in the split S1 and split S0 EPR signals are caused by an increase in the extent of magnetic coupling within the cluster.
S-State-dependent split EPR signals that are induced by illumination at cryogenic temperatures (5 K) have been measured in spinach photosystem II without interference from the Y(D)* radical in the g approximately 2 region. This allows us to present the first decay-associated spectra for the split signals, which originate from the CaMn4 cluster in magnetic interaction with a nearby radical, presumably Y(Z)*. The three split EPR signals that were investigated, "Split S1", "Split S3", and Split S0", all exhibit spectral features at g approximately 2.0 together with surrounding characteristic peaks and troughs. From microwave relaxation studies we can reach conclusions about which parts of the complex spectra belong together. Our analysis strongly indicates that the wings and the middle part of the split spectrum are parts of the same signal, since their decay kinetics in the dark at 5 K and microwave relaxation behavior are indistinguishable. In addition, our decay-associated spectra indicate that the g approximately 2.0 part of the "Split S1" EPR spectrum contains a contribution from magnetically uncoupled Y(Z)* as judged from the g value and 22 G line width of the EPR signal. The g value, 2.0033-2.0040, suggests that the oxidation of Y(Z) at 5 K results in a partially protonated radical. Irrespective of the S state, a small amount of a carotenoid or chlorophyll radical was formed by the illumination. However, this had relaxation and decay characteristics that clearly distinguish this radical from the split signal spectra. In this paper, we present the "clean" spectra from the low-temperature illumination-induced split EPR signals from higher plants, which will provide the basis for further simulation studies.
The S2 state of the oxygen-evolving complex (OEC) of photosystem II is heterogeneous, exhibiting two main EPR spectral forms, the multiline and the g = 4.1 signal. It is not clearly established whether this heterogeneity develops during the S1 to S2 transition or is already present in the precursor states. We have compared the spectra of the S1YZ* intermediate, obtained by visible light excitation (induction of charge separation) of the S1 state at liquid He temperatures, (S1YZ*)vis, or by near-infrared (NIR) light excitation of the S2 state (utilization of the unusual property of the Mn cluster to act as an oxidant of Yz when excited by NIR), (S1YZ*)NIR. The decay kinetics of the (S1YZ*)vis spectrum at 11 K was also studied by the application of rapid-scan EPR. The two spectra share in common a signal with a characteristic feature at g = 2.035, but the (S1YZ*)vis spectrum contains in addition a fast decaying component 26 G wide. The analysis of the surface of the rapid-scan spectra yielded 270 +/- 35 and 90 +/- 15 s for the respective half-times of the two components of the (S1YZ*)vis spectrum at 11 K. (S1YZ*)vis advances efficiently to S2 when annealed at 200 K; notably the g = 2.035 signal advances to the multiline while the 26 G component advances to the g = 4.1 conformation. The "26 G" component is absent or very small, respectively, in thermophilic cyanobacteria or glycerol-containing spinach samples, in correlation to vanishing or very small amounts of the g = 4.1 component in the S2 spectrum. The results validate the assignment of S1YZ* to a true S1 to S2 intermediate and imply that the heterogeneity observed in S2 is already present in S1. Tentative valences are assigned to the individual Mn ions of the OEC in the two heterogeneous conformations of S1.
Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment of the Mn4Ca cluster of photosystem II. Since the discovery of the multiline signal from the S2 state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals from nearly all of the S-states of the Mn4Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various EPR signals obtained from the Mn4Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic radical.
The photosystem II (PSII) reaction center contains two redox active tyrosines, YZ and YD, situated on the D1 and D2 proteins, respectively. By illumination at 5 K, oxidation of YZ in oxygen-evolving PSII can be observed as induction of the Split S1 EPR signal from YZ* in magnetic interaction with the CaMn4 cluster, whereas oxidation of YD can be observed as the formation of the free radical EPR signal from YD*. We have followed the light induced induction at 5 K of the Split S1 signal between pH 4-8.5. The formation of the signal, that is, the oxidation of YZ, is pH independent and efficient between pH 5.5 and 8.5. At low pH, the split signal formation decreases with pKa approximately 4.7-4.9. In samples with chemically pre-reduced YD, the pH dependent competition between YZ and YD was studied. Only YZ was oxidized below pH 7.2, but at pH above 7.2, the oxidation of YD became possible, and the formation of the Split S1 signal diminished. The onset of YD oxidation occurred with pKa approximately 8.0, while the Split S1 signal decreased with pKa approximately 7.9 demonstrating that the two tyrosines compete in this pH interval. The results reflect the formation and breaking of hydrogen bonds between YZ and D1-His190 (HisZ) and YD and D2-His190 (HisD), respectively. The oxidation of respective tyrosine at 5 K demands that the hydrogen bond is well-defined; otherwise, the low-temperature oxidation is not possible. The results are discussed in the framework of recent literature data and with respect to the different oxidation kinetics of YZ and YD.
The interaction EPR split signals from photosystem II (PSII) have been reported from the S0, S1, and S3 states. The signals are induced by illumination at cryogenic temperatures and are proposed to reflect the magnetic interaction between YZ* and the Mn4Ca cluster. We have investigated the formation spectra of these split EPR signals induced in PSII enriched membranes at 5 K using monochromatic laser light from 400 to 900 nm. We found that the formation spectra of the split S0, split S1, and split S3 EPR signals were quite similar, but not identical, between 400 and 690 nm, with maximum formation at 550 nm. The major deviations were found between 440 and 480 nm and between 580 and 680 nm. In the regions around 460 and 680 nm the amplitudes of the formation spectra were 25-50% of that at 550 nm. A similar formation spectrum was found for the S2-state multiline EPR signal induced at 0 degrees C. In general, the formation spectra of these signals in the visible region resemble the reciprocal of the absorption spectra of our PSII membranes. This reflects the high chlorophyll concentration necessary for the EPR measurements which mask the spectral properties of other absorbing species. No split signal formation was found by the application of infrared laser illumination between 730 and 900 nm from PSII in the S0 and S1 states. However, when such illumination was applied to PSII membranes poised in the S3 state, formation of the split S3 EPR signal was observed with maximum formation at 740 nm. The quantum yield was much less than in the visible region, but the application of intensive illumination at 830 nm resulted in accumulation of the signal to an amplitude comparable to that obtained with illumination with visible light. The split S3 EPR signal induced by NIR light was much more stable at 5 K (no observable decay within 60 min) than the split S3 signal induced by visible light (50% of the signal decayed within 30 min). The split S3 signals induced by each of these light regimes showed the same EPR spectral features and microwave power saturation properties, indicating that illumination of PSII in the S3 state by visible light or by NIR light produces a similar configuration of YZ* and the Mn4Ca cluster.
Tyr Z of photosystem II mediates electron transfer from the water splitting site, a Mn4Ca cluster, to the specialized chlorophyll assembly P680. Due to its proton-limited redox properties and the proximity to the Mn cluster, it is thought to play a critical role in the proton-coupled electron transfer reactions that constitute the four-step oxidation mechanism (so-called S-state transitions) of water to molecular oxygen. Spectroscopic evidence for the Tyr Z radical has been scarce in intact preparations (it is difficult to probe it optically, and too short-lived for EPR characterization) until recently. Advances in recent years have allowed the trapping at liquid helium temperatures and EPR characterization of metalloradical intermediates, attributed to tyrosyl Z* magnetically interacting with the Mn cluster. We have extended these studies and examined the evolution of the spectra of five intermediates: S0YZ*, S0YZ* (with 5% MeOH), S1YZ*, S2YZ*, and S2YZ* (with 5% MeOH) in the temperature range of 11-230 K. A rapid-scan EPR method has been applied at elevated temperatures. The tyrosyl radical decouples progressively from Mn, as the Mn relaxation rate increases with an increase in temperature. Above approximately 100 K, the spectra collapse to the unperturbed spectrum of Tyr Z*, which is found to be somewhat broader than that of the stable Tyr D* radical. This study provides a simple means for recording the spectrum of Tyr Z* and extends earlier observations that link the photochemistry at liquid helium temperatures to the photochemistry at temperatures that support S-state transitions.
The O2-evolving complex of photosystem II, Mn 4Ca, cycles through five oxidation states, S0,..., S4, during its catalytic function, which involves the gradual abstraction of four electrons and four protons from two bound water molecules. The direct oxidant of the complex is the tyrosine neutral radical, YZ(*), which is transiently produced by the highly oxidizing power of the photoexcited chlorophyll species P680. EPR characterization of YZ(*) has been limited, until recently, to inhibited (non-oxygen-evolving) preparations. A number of relatively recent papers have demonstrated the trapping of YZ(*) in O2-evolving preparations at liquid helium temperatures as an intermediate of the S0 to S1, S1 to S2, and S2 to S3 transitions. The respective EPR spectra are broadened and split at g approximately 2 by the magnetic interaction with the Mn cluster, but this interaction collapses at temperatures higher than about 100K [Zahariou et al. (2007) Biochemistry 46, 14335 -14341]. We have conducted a study of the Tyr Z(*) transient in the temperature range 77-240 K by employing rapid or slow EPR scans. The results reveal for the first time high-resolution X-band spectra of Tyr Z(*) in the functional system and at temperatures close to the onset of the S-state transitions. We have simulated the S 2Y Z(*) spectrum using the simulation algorithm of Svistunenko and Cooper [(2004) Biophys. J. 87, 582 -595]. The small g(x) = 2.00689 value inferred from the analysis suggests either a H-bonding of Tyr Z (*) (presumably with His190) that is stronger than what has been assumed from studies of Tyr D(*) or Tyr Z(*) in Mn-depleted preparations or a more electropositive environment around Tyr Z(*). The study has also yielded for the first time direct information on the temperature variation of the YZ(*)/QA(-) recombination reaction in the various S states. The reaction follows biphasic kinetics with the slow phase dominating at low temperatures and the fast phase dominating at high temperatures. It is tentatively proposed that the slow phase represents the action of the YZ(*)/YZ(-) redox couple while the fast phase represents that of the YZ(*)/YZH couple; it is inferred that Tyr Z at elevated temperatures is protonated at rest. It is also proposed that YZ(*)/YZH is the couple that oxidizes the Mn cluster during the S1-S2 and S2-S3 transitions. A simple mechanism ensuring a rapid (concerted) protonation of Tyr Z upon oxidation of the Mn cluster is discussed, and also, a structure-based molecular model suggesting the participation of His190 into two hydrogen bonds is proposed.
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.
Recent magnetic-resonance work on YŻ suggests that this species exhibits considerable motional flexibility in its functional site and that its phenol oxygen is not involved in a well-ordered hydrogen-bond interaction (Tang et al., submitted; Tommos et al., in press). Both of these observations are inconsistent with a simple electron-transfer function for this radical in photosynthetic water oxidation. By considering the roles of catalytically active amino acid radicals in other enzymes and recent data on the water-oxidation process in Photosystem II, we rationalize these observations by suggesting that YŻ functions to abstract hydrogen atoms from aquo- and hydroxy-bound managanese ions in the (Mn)4 cluster on each S-state transition. The hydrogen-atom abstraction process may occur either by sequential or concerted kinetic pathways. Within this model, the (Mn)4/YZ center forms a single catalytic center that comprises the Oxygen Evolving Complex in Photosystem II.
Electron spin echo electron-nuclear double resonance (ESE-ENDOR) experiments performed on a broad radical electron paramagnetic resonance (EPR) signal observed in photosystem II particles depleted of Ca2+ indicate that this signal arises from the redox-active tyrosine YZ. The tyrosine EPR signal width is increased relative to that observed in a manganese-depleted preparation due to a magnetic interaction between the photosystem II manganese cluster and the tyrosine radical. The manganese cluster is located asymmetrically with respect to the symmetry-related tyrosines YZ and YD. The distance between the YZ tyrosine and the manganese cluster is estimated to be approximately 4.5 A. Due to this close proximity of the Mn cluster and the redox-active tyrosine YZ, we propose that this tyrosine abstracts protons from substrate water bound to the Mn cluster.
Oxygenic photosynthesis is the principal energy converter on earth. It is driven by photosystems I and II, two large protein-cofactor complexes located in the thylakoid membrane and acting in series. In photosystem II, water is oxidized; this event provides the overall process with the necessary electrons and protons, and the atmosphere with oxygen. To date, structural information on the architecture of the complex has been provided by electron microscopy of intact, active photosystem II at 15-30 A resolution, and by electron crystallography on two-dimensional crystals of D1-D2-CP47 photosystem II fragments without water oxidizing activity at 8 A resolution. Here we describe the X-ray structure of photosystem II on the basis of crystals fully active in water oxidation. The structure shows how protein subunits and cofactors are spatially organized. The larger subunits are assigned and the locations and orientations of the cofactors are defined. We also provide new information on the position, size and shape of the manganese cluster, which catalyzes water oxidation.
Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.
The coupling of proton chemistry with redox reactions is important in many enzymes and is central to energy transduction in biology. However, the mechanistic details are poorly understood. Here, we have studied tyrosine oxidation, a reaction in which the removal of one electron from the amino acid is linked to the release of its phenolic proton. Using the unique photochemical properties of photosystem II, it was possible to oxidize the tyrosine at 1.8 K, a temperature at which proton and protein motions are limited. The state formed was detected by high magnetic field EPR as a high-energy radical intermediate trapped in an unprecedentedly electropositive environment. Warming of the protein allows this state to convert to a relaxed, stable form of the radical. The relaxation event occurs at 77 K and seems to involve proton migration and only a very limited movement of the protein. These reactions represent a stabilization process that prevents the back-reaction and determines the reactivity of the radical.
Photosynthesis uses light energy to drive the oxidation of water at an oxygen-evolving catalytic site within photosystem II (PSII). We report the structure of PSII of the cyanobacterium Thermosynechococcus elongatus at 3.5 angstrom resolution. We have assigned most of the amino acid residues of this 650-kilodalton dimeric multisubunit complex and refined the structure to reveal its molecular architecture. Consequently, we are able to describe details of the binding sites for cofactors and propose a structure of the oxygen-evolving center (OEC). The data strongly suggest that the OEC contains a cubane-like Mn3CaO4 cluster linked to a fourth Mn by a mono-micro-oxo bridge. The details of the surrounding coordination sphere of the metal cluster and the implications for a possible oxygen-evolving mechanism are discussed.
The authors report the detection of a new electron paramagnetic resonance (EPR) signal that demonstrates the presence of a paramagnetic intermediate in the resting (S{sub 1}) state of the photosynthetic oxygen-evolving complex. The signal was detected using the method of parallel polarization EPR, which is sensitive to {Delta}m = 0 transitions in high spin systems. The properties of the parallel polarization EPR signal in the S{sub 1} state are consistent with an S=1 spin state of and exchange-coupled manganese center that corresponds to the reduced form of the species giving rise to the multiline EPR signal in the light-induced S{sub 2} state. The implications for the electronic structure of the oxygen-evolving complex are discussed. 36 refs., 2 fig., 1 tab.
We report the detection of a “split” electron paramagnetic resonance (EPR) signal during illumination of dark-adapted (S1 state) oxygen-evolving photosystem II (PSII) membranes at <20 K. The characteristics of this signal indicate that it arises from an interaction between an organic radical and the Mn cluster of PSII. The broad radical signal decays in the dark following illumination either by back-reaction with Qa•- or by forward electron transfer from the Mn cluster. The forward electron transfer (either from illumination at 11 K followed by incubation in the dark at 77 K or by illumination at 77 K) results in the formation of a multiline signal similar to, but distinct from, other well-characterized multiline forms found in the S0 and S2 states. The relative yield of the “S1 split signal”, which we provisionally assign to S1X•, where X could be YZ• or Car•+, and that of the 77 K multiline signal indicate a relationship between the two states. An approximate quantitation of the yield of these signals indicates that up to 40−50% of PSII centers can form the S1 split signal. Ethanol addition removes the ability to observe the S1 split signal, but the multiline signal is still formed at 77 K. The multiline forms with <700 nm light and is not affected by near-infrared (IR) light, showing that we are detecting electron transfer in centers not responsive to IR illumination. The results provide important new information about the mechanism of electron abstraction from the water oxidizing complex (WOC).
The light-driven oxidation of water to dioxygen is catalyzed by the enzyme photosystem II. A four-manganese ion cluster and a tyrosine, YZ, are present in the catalytic site. In preparations inhibited by addition of acetate or removal of the calcium cofactor, it is possible to trap the tyrosyl radical in interaction with the metal cluster. The coupled species is characterized by a broad split EPR signal at 9 GHz. In this work, high-field EPR has been used for further characterization of the coupling. The 285, 190 and 95 GHz EPR spectra of the interacting system are reported. Analysis of these spectra yielded exchange and dipolar couplings of the same magnitude as those found with 9 GHz EPR. However, the high-field spectra show that the coupling between the radical and the manganese cluster has opposite sign in acetate-treated compared to calcium-depleted samples. The sign difference indicates differences in the electronic structure of the radical−metal center pair. Comparisons are made between photosystem II and other enzymes containing radicals interacting with metal centers. Possible explanations for the difference in sign are proposed. The difficulty in obtaining reliable structural information for the spin coupled system is addressed.
Upon room-temperature illumination, acetate-inhibited photosystem II membranes are known to exhibit a 240 G wide X-band (9.5 GHz) electron paramagnetic resonance (EPR) signal at 10 K. This EPR signal arises from an interaction between the S = 1/2 multiline S2 state of the tetranuclear manganese cluster and an oxidized tyrosine residue, YZ•. In the present study, the exchange and dipolar interactions between the two paramagnetic species are simulated at X- and Q-band (33 GHz) frequencies utilizing second-order perturbation theory. The positions and relative intensities of the hyperfine lines in the S = 1/2 S2 state multiline EPR signal of the noninteracting Mn4 cluster are accurately simulated by including g anisotropy and four sets of axially symmetric 55Mn hyperfine tensors. These parameters are then used to simulate the dipolar and exchange interactions giving rise to the interacting S2YZ• (formerly referred to as S3) EPR signal. Relative intensities of components of the S2YZ• EPR spectrum, at both X- and Q-band frequencies, are best reproduced with a dipolar coupling corresponding to an interspin distance of 7.7 Å and an exchange coupling (J) of −280 × 10-4 cm-1.
The photosynthetic oxidation of water to oxygen occurs in photosystem II (PSII) at an active site composed of a tetranuclear cluster of manganese ions, a redox active tyrosine, YZ, and two essential cofactors, calcium and chloride. Recently, several experimental observations have led to the proposal of a metalloradical catalytic cycle in which water oxidation occurs via hydrogen-atom abstraction by the tyrosyl radical from water bound to the manganese cluster. This model predicts a close proximity between the radical tyrosine, Yz•, and the Mn cluster and the involvement of the radical in a bifurcated hydrogen bond. Magnetic resonance techniques have been used in this work to probe the interaction of the tyrosyl radical with its environment in PSII samples in which the catalytic cycle is blocked by acetate treatment and the enzyme is trapped in a paramagnetic S2Yz• state. Radical interaction with the metal cluster has been studied via simulations of the EPR spectra obtained for this state. The simulations were based on a radical-pair model and included terms for both electron−electron dipolar and exchange interactions. The results show a dominant exchange interaction between the radical and the manganese cluster in these preparations and led to an estimate of 8−9 Å for the spin−spin distance. ESEEM spectroscopy and 1H2O/2H2O exchange were used to study interactions of the S2Yz• state with exchangeable hydrogen nuclei in the site. Two-pulse ESEEM data show features expected for a radical-pair species, in support of our analysis of the continuous-wave EPR spectrum. An ESEEM analysis based on an electron spin 1/2, nuclear spin 1 model shows that both two- and three-pulse ESEEM data are consistent with four deuterons that exhibit an electron−nuclear dipole−dipole coupling of 0.42 MHz. The validity of this analysis and its implications for the oxygen-evolving apparatus are discussed.
The carotenoid (Car•+) and chlorophyll z cation (ChlZ•+) radicals in hydroxylamine treated PSII membranes from spinach were studied by 285 GHz high-field EPR spectroscopy. Car•+ was generated by 5 K illumination in the magnet. The spectrum was characterized by a g-tensor, the principal values of which were 2.00322, 2.00252, and 2.00211. The spectrum of the ChlZ•+ generated by illumination at 198 K was clearly different from the Car•+, showing less resolution. The g-values were 2.00308, 2.00253, and 2.00216. An identical ChlZ•+ spectrum was obtained when the sample exhibiting the Car•+ spectrum was dark-adapted at 198 K. This observation is consistent with the electron transfer from ChlZ to Car•+ that occurs upon raising the temperature as proposed earlier [Hanley et al., Biochemistry, 1999, 38, 8189]. The spectra of the one-dimensional oriented samples were obtained to determine the angular orientation of ChlZ•+. If one assumes that the g-tensor of the ChlZ cation radical is oriented similar to the well-characterized bacteriochlorophyll a, the data show that the ring plane of ChlZ•+ is oriented perpendicular to the membrane plane.
In photosystem II (PS II), chlorophyll, β-carotene, and cytochrome b559 are alternate electron donors that may be involved in a photoprotection mechanism. The present study describes the use of high-field EPR spectroscopy to characterize the low-temperature photooxidation of ChlZ and Car cofactors in PS II. The EPR signals of the individual species, previously not resolved at X-band frequency (9 GHz), are resolved at higher D-band frequency (130 GHz) in deuterated Synechococcus lividus PS II. Deuteration of PS II results in significant narrowing of the EPR lines, yielding well-resolved EPR spectra of the Car+ and ChlZ+ radicals at 130 GHz. The g tensors of the individual species were determined by EPR spectral simulations. The g tensor determined for the Car+ radical (gxx = 2.00335, gyy = 2.00251, gzz = 2.00227) is similar to that previously observed for a canthaxanthin cation radical but with a slightly rhombic tensor. The ChlZ+ g tensor (gxx = 2.00312, gyy = 2.00263, gzz = 2.00202) is similar to that of a chlorophyll a cation radical. This study shows that both the carotenoid and chlorophyll radicals are generated in PS II by illumination at temperatures from 6 to 190 K and that there is no interconversion of Car+ and ChlZ+ radicals upon dark annealing at temperatures up to 160 K. This study also establishes the feasibility of using deuteration and high-field EPR to resolve previously unresolvable cofactor signals in PS II.
A quantitative method for the analysis of EPR spectra from dinuclear Mn(II) complexes is presented. The complex [(Me(3)TACN)(2)Mn(II)(2)(mu-OAc)(3)]BPh(4) (1) (Me(3)TACN=N, N('),N(")-trimethyl-1,4,7-triazacyclononane; OAc=acetate(1-); BPh(4)=tetraphenylborate(1-)) was studied with EPR spectroscopy at X- and Q-band frequencies, for both perpendicular and parallel polarizations of the microwave field, and with variable temperature (2-50K). Complex 1 is an antiferromagnetically coupled dimer which shows signals from all excited spin manifolds, S=1 to 5. The spectra were simulated with diagonalization of the full spin Hamiltonian which includes the Zeeman and zero-field splittings of the individual manganese sites within the dimer, the exchange and dipolar coupling between the two manganese sites of the dimer, and the nuclear hyperfine coupling for each manganese ion. All possible transitions for all spin manifolds were simulated, with the intensities determined from the calculated probability of each transition. In addition, the non-uniform broadening of all resonances was quantitatively predicted using a lineshape model based on D- and r-strain. As the temperature is increased from 2K, an 11-line hyperfine pattern characteristic of dinuclear Mn(II) is first observed from the S=3 manifold. D- and r-strain are the dominate broadening effects that determine where the hyperfine pattern will be resolved. A single unique parameter set was found to simulate all spectra arising for all temperatures, microwave frequencies, and microwave modes. The simulations are quantitative, allowing for the first time the determination of species concentrations directly from EPR spectra. Thus, this work describes the first method for the quantitative characterization of EPR spectra of dinuclear manganese centers in model complexes and proteins. The exchange coupling parameter J for complex 1 was determined (J=-1.5+/-0.3 cm(-1); H(ex)=-2JS(1).S(2)) and found to be in agreement with a previous determination from magnetization. The phenomenon of exchange striction was found to be insignificant for 1.
In high‐accuracy work, electron‐spin‐resonance (ESR) g values are generally determined by calibrating against the accurately known proton nuclear magnetic resonance (NMR). For that method—based on leakage of microwave energy out of the ESR cavity—a convenient technique is presented to obtain accurate g values without needing conscientious precalibration procedures or cumbersome constructions. As main advantages, the method allows the easy monitoring of the positioning of the ESR and NMR samples while they are mounted as close as physically realizable at all time during their simultaneous resonances. Relative accuracies on g of ≊2×10<sup>-</sup><sup>6</sup> are easily achieved for ESR signals of peak‐to‐peak width ΔB pp ≲0.3 G. The method has been applied to calibrate the g value of conduction electrons of small Li particles embedded in LiF—a frequently used g marker—resulting in g LiF Li =2.002 293±0.000 002.
Detergent-treatment of higher plant thylakoids with Triton X-100 at pH 6.3 has been used to purify a PS2 fraction with very high rates of oxygen evolution (1000 μmol.mg chl−1.h−1). A photosynthetic unit size of about 300 chlorophyll (chl) molecules has been determined by optical methods, suggesting an average turnover time for PS2 of about 2 ms. The donor system for P680+ is particularly well preserved in the preparation, as judged by P680+ reduction kinetics, the detection by EPR of Signal IILT and the presence of the high potential form of cytochrome b-559 (at a ratio of 1:1 with the reaction centre).
Using EPR and EXAFS spectroscopies we show that high concentrations of ammonium cations at alkaline pH are required for (1) inhibition of oxygen evolution: (2) an alteration of the EPR properties of the oxygen evolving complex: (3) the ability to detect Y.Z; and (4) the slow reduction of the Mn complex leading to the appearance of EPR detectable Mn2+. The inhibition of S state cycling, slowing of Y.Z reduction, appearance of Mn2+ and the yield of a Hpp < 10 mT S3 type EPR signal are decreased by calcium addition. This indicates that these effects were probably associated with calcium depletion arising from the high concentration of ammonium cation. The ammonia-induced changes to the S2 multiline EPR signal are not affected by calcium addition. The appearance of Mn2+ is shown to be reversible on illumination, suggesting that the Mn reduced from the native state is located at or near the native site. Simulations of the interaction which gives rise to the S3 EPR signal are also presented and discussed. These indicate that lineshape differences occur through small changes in the exchange component of the interaction between the manganese complex and organic radical, probably through minor structural changes between the variously treated samples.
An S1-state parallel polarization "multiline" EPR signal arising from the oxygen-evolving complex has been detected in spinach (PSII) membrane and core preparations depleted of the 23 and 17 kDa extrinsic polypeptides, but retaining the 33 kDa extrinsic protein. This S1-state multiline signal, with an effective g value of 12 and at least 18 hyperfine lines, has previously been detected only in PSII preparations from the cyanobacterium sp. Synechocystis sp. PCC6803 [Campbell, K. A., Peloquin, J. M., Pham, D. P., Debus, R. J., and Britt, R. D. (1998) J. Am. Chem. Soc. 120, 447-448]. It is absent in PSII spinach membrane and core preparations that either fully retain or completely lack the 33, 23, and 17 kDa extrinsic proteins. The S1-state multiline signal detected in spinach PSII cores and membranes has the same effective g value and hyperfine spacing as the signal detected in Synechocystis PSII particles. This signal provides direct evidence for the influence of the extrinsic PSII proteins on the magnetic properties of the Mn cluster.
Electron paramagnetic resonance (EPR) spectroscopy has been used to analyze the ascorbate peroxidase Fe3+ resting state and to compare the reaction product between the enzyme and H2O2, compound I, with that of cytochrome c peroxidase. Because ascorbate peroxidase has a Trp residue in the proximal heme pocket at the same location as the Trp191 compound I free radical in cytochrome c peroxidase [Patterson, W. R., & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], it was anticipated that ascorbate peroxidase compound I might also contain a Trp-centered radical. However, the ascorbate peroxidase compound I EPR spectrum is totally different from that of cytochrome c peroxidase. Immediately after the addition of H2O2, the 7.5 K EPR spectrum of ascorbate peroxidase compound I exhibits an axial resonance extending from g perpendicular = 3.27 to g parallel approximately 2 that disappears within 30 s, presumably due to endogenous reduction of compound I. In contrast, cytochrome c peroxidase compound I exhibits a long-lived g approximately 2 signal associated with the Trp191 cation free-radical [Houseman, A. L. P., et al. (1993) Biochemistry 32, 4430-4443]. Recently, the 2 K EPR spectrum of a catalase compound I was found to exhibit a broad signal extending from g perpendicular = 3.45 to g parallel approximately 2 and was interpreted as a porphyrin pi cation radical [Benecky, M. J., et al. (1993) Biochemistry 32, 11929-11933]. On the basis of these comparisons, we conclude that ascorbate peroxidase forms an unstable compound I porphyrin pi cation radical, even though it has a Trp residue positioned precisely where the Trp191 radical is located in cytochrome c peroxidase.
We present the first EPR and ENDOR examination of a catalase compound I (Cat I), the one formed by peracetic acid treatment of Micrococcus lysodeikticus catalase. The Cat I rapid-passage EPR signal (g perpendicular eff = 3.32; g parallel eff approximately 2) appears quite different from those reported previously for the compounds I from horseradish peroxidase (HRP I) and chloroperoxidase. Nonetheless, all three signals can be explained by the same model for exchange coupling between an S = 1 oxoferryl [Fe = O]2+ moiety and a porphyrin pi-cation radical (S' = 1/2) (Schulz, C. E., et al. (1979) FEBS Lett. 103, 102-105). The signal for Cat I is unlike those for the two peroxidases in that it reflects a ferromagnetic rather than antiferromagnetic exchange. Preliminary 1H ENDOR spectra for Cat I appear to differ from the proton (1H) ENDOR spectra of HRP I; the latter, along with the 14N ENDOR spectra, indicate that the porphyrin radical in HRP I exhibits a predominantly A2u-like state having large spin densities on porphyrin N and C(beta). The proton ENDOR spectrum of Cat I is insensitive to H/D exchange, which indicates that the [Fe = O]2+ moiety is not protonated. Consideration of the EPR results for a series of compounds I suggests that the sign and magnitude of the exchange parameter (J) is correlated with the nature of the proximal axial ligand.
Although the cytochrome c peroxidase/H2O2 reaction product, compound ES, has been a long-standing subject of research, only recently has its broad EPR signal been proven to arise from a radical at Trp-191. Despite this advance, no model has satisfactorily explained the anomalous breadth and shape of this signal, which is conventionally interpreted as having axial symmetry with g∥ ≈ 2.04 > g⊥ ≈ 2.01, contrary to expectations for a planar π radical. Furthermore, these g values exhibit marked temperature and preparation dependencies as well as an unexplained high-field "tail" extending from the g = 2.01 peak. We have reexamined the EPR and ENDOR spectra of compound ES at 35 GHz, as well as those of compound ES in the mutant D235E. This mutation significantly alters the line shape of the Trp-191 free radical. We present a comprehensive model that successfully accounts for the properties of this unusual protein free radical. We show that the EPR spectra of both proteins can be described in terms of a weak exchange interaction between the S = 1 oxyferryl (Fe=O)2+ moiety and a radical on Trp-191; a distribution in protein conformation leads to a distribution in the coupling, which ranges from ferromagnetic to antiferromagnetic. We also derive, for the first time, explicit expressions for frozen-solution and single-crystal spectra of such spin-coupled systems and show that the model accounts for all the data that previously led to apparent anomalies in the interpretation of the frozen-solution and single-crystal [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 349-355] EPR properties. Finally, we have used the CW EPR and pulsed-EPR saturation-recovery methodology to address reports that the broad signal from the spin-coupled Trp-191 radical is accompanied by a minority (∼ 10%), narrow signal that is associated with a radical site other than Trp-191. We find no evidence for such a species and discuss the earlier reports in light of our model.
A 245 GHz 8.7 T high-field EPR study of tyrosine-D (TyrD zero) and tyrosine-Z (TyrZ zero) radicals of photosystem II (PSII) from Synechocystis PCC 6803 was carried out. Identical principal g values for the wild-type Synechocystis and spinach TyrD zero showed that the two radicals were in similar electrostatic environments. By contrast, the principal g values of the TyrD zero in the D2-His189Gln mutant of Synechocystis were different from those of the wild-type and spinach radicals and were similar to those of the tyrosyl radical in ribonucleotide reductase. These comparisons indicate that the D2-His189Gln mutant TyrD zero is not hydrogen-bonded or is only weakly so. The HF-EPR spectrum of TyrZ zero was obtained from the D2-Tyr160Phe mutant that lacks TyrD zero. The principal g values were nearly identical to those of the wild-type TyrD zero. The low-field edge of the TyrZ zero spectrum was much broader than at the other two principal g values and was also much broader than the TyrD zero spectrum. From the identical g values and previous work on tyrosyl radical g values [Un S., Atta M., Fontecave, M., & Rutherford, A. W. (1995) J. Am. Chem. Soc. 117, 10713-10719], it was concluded that TyrZ zero, like TyrD zero, is hydrogen-bonded The broadness of the gx component was interpreted as a distribution in strength of the hydrogen-bonding due to disorder in the protein environment about TyrZ zero.
Magnetic properties of the S1-state manganese cluster in the oxygen-evolving photosystem II were studied by parallel polarization electron paramagnetic resonance spectroscopy. Dark minus light spectra gave rise to a broad S1-state signal with a g value of about 4.9 [Dexheimer, S. L., Klein, M. P. (1992) J. Am. Chem. Soc. 114, 2821-2826]. Temperature variation of the signal intensity between 1.9 and 10 K observed in PS II with a sucrose buffer indicates that the signal originates from an excited state with a spin S of 1 with separation from the ground state (S = 0) of about 2.5 K. The S1-state signal was also observed in the sucrose buffer supplemented by 50% glycerol. However, no S1-state signal was detected by addition of 3% methanol or 30% ethylene glycol in the sucrose buffer, although illumination at 200 K in the presence of these alcohols induced the normal multiline S2 signal. Furthermore, modification of the Mn cluster by Cl- or Ca2+ depletion from PS II membranes failed to produce a detectable S1-state signal. A possible magnetic structure of the Mn cluster responsible for the generation of the S1-state signal is discussed on the basis of these observations.
In plants and algae, photosystem II uses light energy to oxidize water to oxygen at a metalloradical site that comprises a
tetranuclear manganese cluster and a tyrosyl radical. A model is proposed whereby the tyrosyl radical functions by abstracting
hydrogen atoms from substrate water bound as terminal ligands to two of the four manganese ions. Molecular oxygen is produced
in the final step in which hydrogen atom transfer and oxygen-oxygen bond formation occur together in a concerted reaction.
This mechanism establishes clear analogies between photosynthetic water oxidation and amino acid radical function in other
enzymatic reactions.
In oxygen-evolving photosystem II (PSII), a tyrosine residue, D1Tyr161 (YZ), serves as the intermediate electron carrier between the catalytic Mn cluster and the photochemically active chlorophyll moiety P680. A more direct catalytic role of YZ, as a hydrogen abstractor from bound water, has been postulated. That YZox appears as a neutral (i.e. deprotonated) radical, YZ*, in EPR studies is compatible with this notion. Data based on electrochromic absorption transients, however, are conflicting because they indicate that the phenolic proton remains on or near to YZox. In Mn-depleted PSII the electron transfer between YZ and P680+ can be almost as fast as in oxygen-evolving material, however, only at alkaline pH. With an apparent pK of about 7 the fast reaction is suppressed and converted into an about 100-fold slower one which dominates at acid pH. In the present work we investigated the optical difference spectra attributable to the transition YZ --> YZox as function of the pH. We scanned the UV and VIS range and used Mn-depleted PSII core particles and also oxygen-evolving ones. Comparing these spectra with published in vitro and in vivo spectra of phenolic compounds, we arrived at the following conclusions: In oxygen-evolving PSII YZ resembles a hydrogen-bonded tyrosinate, YZ(-).H(+).B. The phenolic proton is shifted toward a base B already in the reduced state and even more so in the oxidized state. The retention of the phenolic proton in a hydrogen-bonded network gives rise to a positive net charge in the immediate vicinity of the neutral radical YZ*. It may be favorable both for the very rapid reduction by YZ of P680+ and for electron (not hydrogen) abstraction by YZ* from the Mn-water cluster.
A mechanism for photosynthetic water oxidation is proposed based on a structural model of the oxygen-evolving complex (OEC) and its placement into the modeled structure of the D1/D2 core of photosystem II. The structural model of the OEC satisfies many of the geometrical constraints imposed by spectroscopic and biophysical results. The model includes the tetranuclear manganese cluster, calcium, chloride, tyrosine Z, H190, D170, H332 and H337 of the D1 polypeptide and is patterned after the reversible O2-binding diferric site in oxyhemerythrin. The mechanism for water oxidation readily follows from the structural model. Concerted proton-coupled electron transfer in the S2-->S3 and S3-->S4 transitions forms a terminal Mn(V)=O moiety. Nucleophilic attack on this electron-deficient Mn(V)=O by a calcium-bound water molecule results in a Mn(III)-OOH species, similar to the ferric hydroperoxide in oxyhemerythrin. Dioxygen is released in a manner analogous to that in oxyhemerythrin, concomitant with reduction of manganese and protonation of a mu-oxo bridge.
Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic resonance (EPR) spectroscopy has been used to characterize radical intermediates in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high-field EPR spectra of the exchange-coupled oxoferryl--trytophanyl radical pair that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp*(+)] were analyzed using a spin Hamiltonian that incorporated a general anisotropic spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discussed. Using numerical solutions of the completely anisotropic Hamiltonian, its was possible to simulate accurately the experimental data from 9 to 285 GHz using a single set of spin parameters. The results are also consistent with previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a transient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degrees C and disappeared after 1 h. High-field EPR was also used to characterize the radical intermediate that was generated by hydrogen peroxide addition to the W191G CcP mutant. The g- values of this radical (g(x)= 2.00660, g(y) = 2.00425, and g(z)= 2.00208), as well as the wild-type transient tyrosyl radical, are essentially identical to those obtained from the high-field EPR spectra of the tyrosyl radical generated by gamma-irradiation of crystals of tyrosine hydrochloride (g(x)= 2.00658, g(y) = 2.00404, and g(z) = 2.00208). The low g(x)-value indicated that all three of the tyrosyl radicals were in electropositive environments. The broadening of the g(x) portion of the HF-EPR spectrum further indicated that the electrostatic environment was distributed. On the basis of these observations, possible sites for the tyrosyl radical(s) are discussed.
Four of the five intermediate oxidation states (S-states) in the catalytic cycle of water oxidation used by O2-evolving photoautotrophs have been previously characterized by EPR and/or ENDOR spectroscopy, with the first reports for the S0, S1, and S3 states available in just the last three years. The first electron density map of the Mn cluster derived from X-ray diffraction measurements of single crystals of photosystem II at 3.8–4.2 Å resolution has also appeared this year. This wealth of new information has provided significant insight into the structure of the inorganic core (Mn4Ox
Ca1Cl1–2), the Mn oxidation states, and the location and function of the essential Ca2+ cofactor within the water-oxidizing complex (WOC). We summarize these advances and provide a unified interpretation of debated structural proposals and Mn oxidation states, based on an integrated analysis of the published data, particularly from Mn X-ray absorption spectroscopy (XAS) and EPR/ENDOR data. Only three magnetic spin-exchange models for the inter-manganese interactions are possible from consideration of the EPR data for the S0, S1, S2 and S–N
(NO-reduced) states. These models fall into one of three types denoted butterfly, funnel, or tetrahedron. A revised set of eight allowed chemical structures for the Mn4Ox
core can be deduced that are shown to be consistent with both EPR and XAS. The popular "dimer-of-dimers" structural model is not compatible with the possible structural candidates. EPR data have identified two inter-manganese couplings that are sensitive to the S-state, suggesting two possible bridging sites for substrate water molecules. Spin densities derived from 55Mn hyperfine data together with Mn K-edge energies from Ca-depleted samples provide an internally consistent assignment for the Mn oxidation states of Mn4(3III,IV) for the S2 state. EPR and XAS data also provide a consistent picture, locating Ca2+ as an integral part of the inorganic core, probably via shared bridging ligands with Mn (aqua/hydroxo/carboxylato/chloro). XAS data reveal that the Ca2+ cofactor increases the Mn(1s→4p) transition energy by 0.6–1 eV with minimal structural perturbation versus the Ca-depleted WOC. Thus, calcium binding appears to increase the Mn-ligand covalency by increasing electron transfer from shared ligands to Mn, suggesting a direct role for Ca2+ in substrate water oxidation. Consideration of both the XAS and the EPR data, together with reactivity studies on two model complexes that evolve O2, suggest two favored structure types as feasible models for the reactive S4 state that is precursor to the O2 evolution step. These are a calcium-capped "cuboidal" core and a calcium-capped "funnel" core.
The S(3) state of the water-oxidizing complex (WOC) of photosystem II (PSII) is the last state that can be trapped before oxygen evolution occurs at the transient S(4) state. A number of EPR-detectable intermediates are associated with this critical state. The preceding paper examined mainly the decay of S(3) at cryogenic temperatures leading to the formation of a proton-deficient configuration of S(2) termed S(2)'. This second paper examines all intermediates formed by the near-IR light (NIR) excitation of the S(3) state and compares these with the light-excitation products of the S(2)' state. The rather complex set of observations is organized in a comprehensive flowchart, the central part of which is the S(3)...Q(A)(-) state. This state can be converted to various intermediates via two main pathways: (A) Excitation of S(3) by NIR light at temperatures below 77 K results presumably in the formation of an excited S(3) state, S(3), which decays via either of two pathways. Slowly at liquid helium temperatures but much faster at 77 K, S(3) decays to an EPR-silent state, denoted S(3)' ', which by raising the temperature to ca. 190 K converts to a spin configuration of the Mn cluster, characterized by g = 21, 3.7 in perpendicular and g = 23 in parallel mode EPR, denoted S(3)'. Upon further warming to 220 K, S(3)' relaxes to the untreated S(3) state. Below about 77 K and more favorably at liquid helium temperatures, an alternative pathway of S(3) decay via the metallo-radical intermediate S(2)'Z*...Q(A)(-) can be traced. This leads to the metastable state S(2)'Z...Q(A) via charge recombination. S(2)'Z* is characterized by a split-radical signal at g = 2, while all S(2)' transients are characterized by the same g = 5/2.9 (S = (7)/(2)) configuration of the Mn cluster with small modifications, reflecting an influence of the tyr Z oxidation state on the crystal-field symmetry at the Mn cluster. (B) S(2)'...Q(A) can be reached alternatively by the slow charge recombination of S(3) and Q(A)(-) at 77 K. White-light illumination of S(2)'.Q(A) below about 20 K results in charge separation, reforming the intermediate S(2)'Z*...Q(A)(-). Thermally activated branches to the main pathways are also described, e.g., at elevated temperatures tyr Z* reoxidizes S(2)' to the S(3) state. The above observations are discussed in terms of a molecular model of the S(3) state of the OEC. Main aspects of the model are the following. Intermediates, isoelectronic to S(3), are attributed to the NIR-induced translocation of the positive hole to different Mn ligands, or to tyr Z. On the basis of a comparison of the electron-donating efficiency of tyr Z and tyr D at cryogenic temperatures, it is inferred that the Mn cluster acts as the main proton acceptor from tyr Z. Water associated with the Mn cluster is assumed to be in hydrogen-bonding equilibrium with tyr Z, and an array comprising this water and adjacent water (or OH or O) ligands to Mn followed by a sequence of proton acceptors is proposed to act as an efficient proton translocation pathway. Oxidation of the tyrosine by P(680)(+) repels protons to and out from the Mn cluster. This proposed role of tyr Z in the water-splitting process is described as a proton repeller/electron abstractor.
Conceptually, photosystem II, the oxygen-evolving enzyme, can be divided into two parts: the photochemical part and the catalytic part. The photochemical part contains the ultra-fast and ultra-efficient light-induced charge separation and stabilization steps that occur when light is absorbed by chlorophyll. The catalytic part, where water is oxidized, involves a cluster of Mn ions close to a redox-active tyrosine residue. Our current understanding of the catalytic mechanism is mainly based on spectroscopic studies. Here, we present an overview of the current state of knowledge of photosystem II, attempting to delineate the open questions and the directions of current research.
Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.
The effect of illumination at 5 K of photosystem II in different S-states was investigated with EPR spectroscopy. Two split radical EPR signals around g approximately 2.0 were observed from samples given 0 and 3 flashes, respectively. The signal from the 0-flash sample was narrow, with a width of approximately 80 G, in which the low-field peak can be distinguished. This signal oscillated with the S(1) state in the sample. The signal from the 3-flash sample was broad, with a symmetric shape of approximately 160 G width from peak to trough. This signal varied with the concentration of the S(0) state in the sample. Both signals are assigned to arise from the donor side of PSII. Both signals relaxed fast, were formed within 10 ms after a flash, and decayed with half-times at 5 K of 3-4 min. The signal in the S(0) state closely resembles split radical signals, originating from magnetic interaction between Y(Z)(*) and the S(2) state, that were first observed in Ca(2+)-depleted photosystem II samples. Therefore, we assign this signal to Y(Z)(*) in magnetic interaction with the S(0) state, Y(Z)(*)S(0). The other signal is assigned to the magnetic interaction between Y(Z)(*) and the S(1) state, Y(Z)(*)S(1). An important implication is that Y(Z) can be oxidized at 5 K in the S(0) and S(1) states. Oxidation of Y(Z) involves deprotonation of the tyrosine. This is restricted at 5 K, and we therefore suggest that the phenolic proton of Y(Z) is involved in a low-barrier hydrogen bond. This is an unusually short hydrogen bond in which proton movement at very low temperatures can occur.
Photosystem II (PSII) is a multisubunit membrane protein complex performing light-induced electron transfer and water-splitting reactions, leading to the formation of molecular oxygen. The first crystal structure of PSII from a thermophilic cyanobacterium Thermosynechococcus elongatus was reported recently [Zouni, A., Witt, H. T., Kern, J., Fromme, P., Krauss, N., Saenger, W. & Orth, P. (2001) Nature 409, 739-743)] at 3.8-A resolution. To analyze the PSII structure in more detail, we have obtained the crystal structure of PSII from another thermophilic cyanobacterium, Thermosynechococcus vulcanus, at 3.7-A resolution. The present structure was built on the basis of the sequences of PSII large subunits D1, D2, CP47, and CP43; extrinsic 33- and 12-kDa proteins and cytochrome c550; and several low molecular mass subunits, among which the structure of the 12-kDa protein was not reported previously. This yielded much information concerning the molecular interactions within this large protein complex. We also show the arrangement of chlorophylls and cofactors, including two beta-carotenes recently identified in a region close to the reaction center, which provided important clues to the secondary electron transfer pathways around the reaction center. Furthermore, possible ligands for the Mn-cluster were determined. In particular, the C terminus of D1 polypeptide was shown to be connected to the Mn cluster directly. The structural information obtained here provides important insights into the mechanism of PSII reactions.