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Spectroscopic and kinetic properties of the transient intermediate acceptor in reaction centers of Rhodopseudomonas sphaeroides
Abstract
The photoreductive trapping of the transient, intermediate acceptor, I⁻, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I⁻ was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 440, 587–599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301–304). The optical changes of I⁻ showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I⁻ were observed: one was a narrow singlet at g = 2.0035, ΔH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I⁻ on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI⁻Q⁻Fe²⁺ and the singlet EPR signal is due to DI⁻Q²⁻Fe²⁺. where D is the primary donor (BChl)⁺2.
... Absorption spectra were recorded at room temperature on Agilent 8453 or Shimadzu UV1800 spectrophotometers. Photoaccumulation of H A − was achieved by continuous illumination of RC samples in TT-buffer at room temperature in the presence of a reductant, sodium dithionite (2 mM), and redox mediators, potassium indigotetrasulfonate (0.1 mM), and neutral red (0.1 mM) to pre-reduce Q A in the dark and rapidly re-reduce P + in the photogenerated radical pair P + H A − (Shuvalov and Klimov 1976;Tiede et al. 1976;van Grondelle et al. 1976;Okamura et al. 1979;Zabelin et al. 2011). The reaction mixture was placed in a vacuum-tight quartz cuvette (optical path length of 1 cm) and degassed by a mild application of vacuum. ...
... aurantiacus RCs against pheophytins, RC/pheophytin mixtures were incubated at room or elevated (45 °C and 50 °C) temperatures. The H A − photoaccumulation reaction (Shuvalov and Klimov 1976;Tiede et al. 1976;van Grondelle et al. 1976;Okamura et al. 1979;Zabelin et al. 2011) was used to test the ability of newly introduced pigments to occupy the H A -binding site and participate in electron transfer in modified Cfl. aurantiacus RCs. ...
... The shape of the light-minus-dark H A − /H A difference spectrum obtained after incubation of RCs with Pheo at room temperature (Fig. 5, red) is virtually identical to the H A − /H A spectrum for native RCs, but the relative amplitude of the absorbance changes is strongly reduced. Since the difference spectra Pheo − /Pheo (Fujita et al. 1978;Klimov et al. 1977;Nanba and Satoh 1987;Barber and Melis 1990;Shkuropatov et al. 1999;Zabelin et al. 2014) and BPheo − /BPheo (Fajer et al. 1975;Shuvalov and Klimov 1976;van Grondelle et al. 1976;Okamura et al. 1979;Zabelin et al. 2011) are characterized by distinctly different spectral features, it follows from Fig. 5 that no signals that would be attributed to a reduced state of the introduced Pheo are discernible for room-temperatureincubated RCs. The small absorbance changes observed for these RCs are apparently associated with the photoaccumulation of the BPheo radical anion in the residual fraction of non-exchanged RCs (and/or in a minor fraction of modified RCs in which only the photochemically inactive BPheo was replaced by Pheo). ...
The pigment composition of isolated reaction centers (RCs) of the green filamentous bacterium Chloroflexus (Cfl.) aurantiacus was changed by chemical exchange of native bacteriopheophytin a (BPheo) molecules with externally added pheophytin a (Pheo) or [3-acetyl]-Pheo upon incubation of RC/pheophytin mixtures at room temperature and 45 °C. The modified RCs were characterized by Vis/NIR absorption spectroscopy, and the effect of pigment exchange on RC photochemical activity was assessed by measuring the photoaccumulation of the reduced pigment at the binding site HA. It is shown that both pheophytins can be exchanged into the HA site instead of BPheo by incubation at room temperature. While the newly introduced Pheo molecule is not active in electron transfer, the [3-acetyl]-Pheo molecule is able to replace functionally the photoreducible HA BPheo molecule with the formation of the [3-acetyl]-Pheo− radical anion instead of the BPheo−. After incubation at 45 °C, the majority (~ 90%) of HA BPheo molecules is replaced by both Pheo and [3-acetyl]-Pheo. Only a partial replacement of inactive BPheo molecules with pheophytins is observed even when the incubation temperature is raised to 50 °C. The results are discussed in terms of (i) differences in the accessibility of BPheo binding sites for extraneous pigments depending on structural constraints and incubation temperature and (ii) the effect of the reduction potential of pigments introduced into the HA site on the energetics of the charge separation process. The possible implication of Pheo-exchanged preparations for studying early electron-transfer events in Cfl. aurantiacus RCs is considered.
... To go further in this analysis, we have considered a decomposition of J overlap (eq. [5]) into atomic contributions. 26 [5] ͗ J overlap ͘ ϭ ͗͟ n exp ( X n ϩ P n ϩ iX n P n ) ͘ [6] X n ϭ Ϫ a n 4 ( x 2n (t) Ϫ x 1n (t) ) 2 [7] P n ϭ Ϫ 1 4a n ប 2 ( p 2n (t) Ϫ p 1n (t) ) 2 ...
... [5]) into atomic contributions. 26 [5] ͗ J overlap ͘ ϭ ͗͟ n exp ( X n ϩ P n ϩ iX n P n ) ͘ [6] X n ϭ Ϫ a n 4 ( x 2n (t) Ϫ x 1n (t) ) 2 [7] P n ϭ Ϫ 1 4a n ប 2 ( p 2n (t) Ϫ p 1n (t) ) 2 ...
... If these terms remain small on the time window necessary for decoherence to occur, the average of the exponential term (eq. [5]) can be approximated by eq. [9]: [9] ͗ J overlap ͘ ≈ exp ͗ ͚ n ( X n ϩ P n ϩ iXP n ) ͘ Furthermore, if we assume that the (complex) positionmomentum coupling term (XP n ) is negligible, we obtain an expression that enables gauging the role of each atom in the decay of the overlap function: ...
The notion of decoherence is particularly adapted to discuss the quantum-to-classical transition in the context of chemical reactions. Decoherence can be modeled by computing the time evolution of nuclear wave packets evolving on distinct potential energy surfaces, here using density functional theory (DFT) and Born–Oppenheimer molecular dynamics simulations. We investigate a redox cofactor of biological interest (tryptophan tryptophylquinone, TTQ) found in the enzyme methylamine dehydrogenase. We also report the first systematic comparison of semi-empirical DFT (tight-binding DFT) and classical force field approaches for estimating decoherence in molecular systems. In the TTQ cofactor, we find that decoherence combines structural and dynamical aspects: it is initiated by the divergent motions of few atoms and then propagates dynamically to the remaining atoms. It is the mass effect of all the atoms that leads to decoherence within a few femtosecond.
... The difference H A -/H A spectrum of native RCs presented in Fig. 4 (curve 1) corresponds well to the H A photoaccumulation spectra previously published for the RCs of Rba. sphaeroides R-26 [24,35,36], demonstrating selective bleaching of the Q y and Q x bands of BPheo H A at 762 and 543 nm, respectively, appearance of characteristic absorption bands of the radical anion H A at 660, 912, and 965 nm, and electrochromic short-wavelength shift and decrease in the dipole strength of the absorption band of monomeric BChl molecules in the region of 800 nm. Figure 4 shows that the difference spectrum for the Pheo-modified RCs obtained by incubation in the TL buffer at 25°C (curve 2) was almost identical in shape to the spectrum for the native RCs and had similar amplitude (the minor difference in the amplitude between curves 1 and 2 was within the measurement error of ±5%). Analogous results were obtained for the Pheo-modified RCs after incubation in the TLT buffer at 25°C (Fig. S2 in the Online Resource 1). ...
... ref lecting selective photoaccumulation of reduced BPheo H A at a low redox potential of the medium[24,35,36] were carried out.The effect of incubation medium temperature and the method of introducing Pheo molecules into the medium on the degree of BPheo to Pheo exchange in the RCs of Rba. sphaeroides R-26 ...
To elucidate the mechanism of site-selective chemical replacement of chromophores in the reaction centers (RCs) of photosynthetic bacteria by external pigments, we investigated how the efficiency of incorporation of plant pheophytin a (Pheo) into the binding sites for bacteriopheophytin a molecules (BPheo) in the isolated Rhodobacter sphaeroides R-26 RCs depended on the incubation medium temperature, Pheo aggregation state, and the presence of organic solvent (acetone). When Pheo was in a form of monomers in free detergent micelles in a water-detergent incubation medium, the degree of selective replacement of photochemically inactive BPheo HB molecules upon incubation of the RC/Pheo mixture at 5°C was ~15%. The exchange efficiency increased to 40% upon incubation at 25°C and reached 100% at the same temperature when 10% acetone was added to the incubation medium. At both 5 and 25°C, the degree of pigment exchange increased approximately twice, when a mixture of Pheo monomers and dimers in the presence of 10% acetone was used as the incubation medium. The removal of acetone from this medium with the preservation of pigment forms led to a significant decrease in the efficiency of Pheo incorporation. The effect of acetone on the pigment exchange was also observed at an elevated incubation temperature (43.5°C), when functionally active BPheo HA molecules were partially replaced. The results are discussed in terms of the mechanism according to which (i) the temperature-dependent internal movements of the RC protein facilitate the release of the BPheo molecule from the binding site with simultaneous insertion of the Pheo molecule into the same site in a coupled process, (ii) the role of temperature largely depends on the steric accessibility of binding pockets in the RC protein, (iii) the incorporation of Pheo occurs from a pool of monomeric molecules included in the RC-detergent micelles, and (iv) the presence of acetone in the incubation medium facilitates the exchange of Pheo monomers between micelles in the solution and the detergent belt of the RC complex.
... Two mechanisms of protonation in the A 1 binding site have been proposed (McConnell et al. 2011). One mechanism resembles the Q A protonation process (Okamura et al. 1979), and one involves the iron-sulfur clusters F A and F B (Setif and Bottin 1989). The first mechanism was not favored as it requires the generation of a P700 + A 0 − A − state, which is unlikely given the short lifetime of P700 + A 1 − Semenov et al. 2000) and current models of charge separation (Holzwarth et al. 2006;Müller et al. 2003Müller et al. , 2010. ...
Time-resolved FTIR difference spectroscopy has been used to study photosystem I (PSI) particles with three different benzoquinones [plastoquinone-9 (PQ), 2,6-dimethyl-1,4-benzoquinone (DMBQ), 2,3,5,6-tetrachloro-1,4-benzoquinone (Cl4BQ)] incorporated into the A1 binding site. If PSI samples are cooled in the dark to 77 K, the incorporated benzoquinones are shown to be functional, allowing the production of time-resolved (P700⁺A1⁻−P700A1) FTIR difference spectra. If samples are subjected to repetitive flash illumination at room temperature prior to cooling, however, the time-resolved FTIR difference spectra at 77 K display contributions typical of the P700 triplet state (³P700), indicating a loss of functionality of the incorporated benzoquinones, that occurs because of double protonation of the incorporated benzoquinones. The benzoquinone protonation mechanism likely involves nearby water molecules but does not involve the terminal iron–sulfur clusters FA and FB. These results and conclusions resolve discrepancies between results from previous low-temperature FTIR and EPR studies on similar PSI samples with PQ incorporated.
... Cytochrome C electron transfer 0.14 ± 0.03 [22,23] Quinones reduction 0.477 [24] Reduction of CoQ by NADH 0.86-1.23 [25] Ubiquinol oxidation by the bc1 complex 0.466-0.518 [26] Quinol oxidation 0.466 [27] Iron-sulfur protein 0.02 [28] Conclusion A miniaturized MFC is built to characterize its current output capability as a function of the temperature. ...
A microbial fuel cell (MFC) is a bioinspired energy converter which directly converts biomass into electricity through the catalytic activity of a specific species of bacteria. The effect of temperature on a miniaturized microbial fuel cell with Geobacter sulfurreducens dominated mixed inoculum is investigated in this paper for the first time. The miniaturized MFC warrants investigation due to its small thermal mass, and a customized setup is built for the temperature effect characterization. The experiment demonstrates that the optimal temperature for the miniaturized MFC is 322–326 K (49–53 °C). When the temperature is increased from 294 to 322 K, a remarkable current density improvement of 282% is observed, from 2.2 to 6.2 Am−2. Furthermore, we perform in depth analysis on the effect of temperature on the miniaturized MFC, and found that the activation energy for the current limiting mechanism of the MFC is approximately between 0.132 and 0.146 eV, and the result suggest that the electron transfer between cytochrome c is the limiting process for the miniaturized MFC.
Ultrafast transient absorption (TA) spectroscopy was used to study electron transfer (ET) at 100 K in native (as isolated) reaction centers (RCs) of the green filamentous photosynthetic bacterium Chloroflexus (Cfl.) aurantiacus. The rise and decay of the 1028 nm anion absorption band of the monomeric bacteriochlorophyll a molecule at the BA binding site were monitored as indicators of the formation and decay of the P+BA- state, respectively (P is the primary electron donor, a dimer of bacteriochlorophyll a molecules). Global analysis of the TA data indicated the presence of at least two populations of the P⁎ excited state, which decay by distinct means, forming the state P+HA- (HA is a photochemically active bacteriopheophytin a molecule). In one population (~65 %), P⁎ decays in ~2 ps with the formation of P+HA- via a short-lived P+BA- intermediate in a two-step ET process P⁎ → P+BA- → P+HA-. In another population (~35 %), P⁎ decays in ~20 ps to form P+HA- via a superexchange mechanism without producing measurable amounts of P+BA-. Similar TA measurements performed on chemically modified RCs of Cfl. aurantiacus containing plant pheophytin a at the HA binding site also showed the presence of two P⁎ populations (~2 and ~20 ps), with P⁎ decaying through P+BA- only in the ~2 ps population. At 100 K, the quantum yield of primary charge separation in native RCs is determined to be close to unity. The results are discussed in terms of involving a one-step P⁎ → P+HA- superexchange process as an alternative highly efficient ET pathway in Cfl. aurantiacus RCs.
The differences in temperature dependent behaviour and microwave power saturation characteristics between the g=1.9 and g=1.8 QA -Fe2+ signals are described. The dependence of these behaviourial differences on the presence or absence of bicarbonate is emphasised. By studying the EPR signals of QA-Fe2+, Q-Fe2+, Q-Fe2+TBTQ- and the oxidised non-haem iron I have found that detergent solubilisation of BBY PS2 preparations with the detergent OGP, at pH 6.0, results in loss of bicarbonate binding. New preparations, including a dodecylmaltoside prepared CP47, CP4 3, D1, D2, cytochrome bgsg complex, are described which at pH 7.5 retain native bicarbonate binding. These preparations provide a new system for studies into the "bicarbonate effect" because bicarbonate depletion can now be achieved without displacement by another anion. The new OGP particles have been used to investigate both the split pheophytin signal and the two step redox titration phenomenon associated with this signal. The low potential step of the titration was concluded to be independent of the QA/QA- mid-point potential but was found to be linked to the ability to photoreduce pheophytin; once the low potential component, suggested here to be the fluorescence quencher QL, was reduced, pheophytin photoreduction increased. A model is described to explain the two step titration and, from analysis of the signal splitting in +/- HCO3- samples, a possible structural role for bicarbonate is proposed. I have probed the structure of the PS2 electron acceptor region with the protease trypsin. The QA, iron-semiquinone; oxidised non-haem iron and cytochrome bss, EPR signals were all found to be susceptible to trypsin damage, while oxygen evolution with ferricyanide was enhanced by protease treatment. The protective effect of calcium ions against trypsin damage was demonstrated and a possible Ca2+ binding site in the binding region identified.
The photoinduced charge separation and charge recombination in a set of four molecular dyads consisting of a triarylamine donor and a naphthalene diimide acceptor were investigated by time resolved transient absorption spectroscopy with fs and ns time resolution. In these dyads donor and acceptor are bridged by a meta-conjugated diethynylbenzene bridge whose electronic nature was tuned by small electron donating (OMe, Me) or electron withdrawing (Cl, CN) substituents. While the formation of the transient charge separated states is complete within tens of ps, charge recombination is biphasic with a shorter component of several hundred ns and a longer component with several microseconds. This behavior could be rationalized by assuming an equilibrium of singlet and triplet charge separated states. Magnetic field dependent measurements showed a strong influence on the biphasic decay kinetics and also a pronounced level crossing effect in the magnetic field affected reaction yield (MARY) spectra caused by a significant exchange coupling. An analysis of the observed kinetics using classical kinetic rate equations yields rate constants for charge separation and charge recombination as well as the exchange interaction splitting in the radical ion pair, all of them showing a delicate dependence on the bridge substituents.
Volume 204 of the Journal of Electroanalytical Chemistry is dedicated to the memory of the late Professor R. R. Dogonadze. It contains an appreciation and bibliography,(1) and articles in areas related to many aspects of his work, including proton and electron transfer(2) and adiabatic electron transfer at electrodes.(3)
Photosystem II is the photosynthetic apparatus which oxidizes water and supplies photosystem I with an electron source which is less difficult to oxidize. In addition system II helps to create the proton concentration difference between the in-and outside of the photosynthetic membrane system, which provides the driving force for ATP synthesis. The proton translocation is due to the fact that water is deprotonated at the inside of the membrane system and the intermediate electron carriers between the two photosystems are protonated on the outside. The mechanism of water oxidation is poorly understood, but considerable progress may soon be expected, since it appears that the oxygen evolving enzyme has now been isolated [1]. The kinetics of the process have been studied extensively [2]. The liberation of one oxygen molecule requires four successive photoacts in the same system II reaction center. Of the corresponding oxidation states of the enzyme, designated S0 to S4 only S0 and S1 are stable in darkness, S2 and S0 have lifetimes in the order of a minute, and S4 releases oxygen in a millisecond.
The optical detection of recombination dynamics and its dependence on external magnetic fields gives access to kinetic and structural features of short-lived radical pairs (RPs) In reaction centers (RCs) of purple bacteria this RP consists of the cation of the bacteriochlorophyll dimer, (BC)+2 and the anion of the bacteriopheophytin, BP−and is formed within a few picoseconds by electron-transfer from the singlet excited dimer state. In quinone-depleted RCs at room temperature the RP is stabilized for approximately 10 ns. The initially formed RP is singlet-phased as its precursor state and recombines via the rate ks to form the ground state species. This is illustrated in the following kinetic scheme: Hyperfine interaction HFI of the two electron spins with their different nuclear spin environments can change the multiplicity of the RP spin state. This leads to a RP triplet state,thus providing a new channel of recombination forming the dimer triplet state with the rate kT . Such singlet-triplet transitions,however, can be hindered by exchange and spin dipolar interaction (J and D) between the radical electrons as well as by the recombination rates, which are responsible for the lifetime broadening of the RP states.
A model for electron transfer is presented. It is based on a selfconsistent treatment of the initial (final)-state charge distribution and the equilibrium arrangements tot the molecular coordinates. The theory comprises the nonadiabatic electron transfer and internal conversion processes as special limits within the same frame. It is shown that the initial state contains a small degree of charge delocalization, which is a resonance like function of the free energy change. It peaks when the free energy change matches the nuclear reorganisation energy. The physical origin for this resonance like enhancement is a non-Condon- effect. The analysis of exact eigenstates for an asymmetric dimer is used to interrelate this soliton approach with others based on the small polaron transformation.
The pathway of electron transfer from the primary electron donor, P, to the primary electron acceptor, QA, in the photoreaction centers of various organisms has been unraveled by two main approaches: very fast kinetic measurements and phototrapping. While the first technique has been the main source of information about the identity of the intermediates and about the sequence of events they undergo, the latter has advantages that have made it an indispensable complement. Its main value resides in its greater sensitivity that permits the identification of products that are formed with quantum yields too low to be detectable by flash photolysis. Perhaps the principal asset of phototrapping is that it renders amenable the intermediary states it captures to detailed spectroscopic scrutiny. The identification of one of the pheophytin molecules (Φ A) as an intermediary of the primary reaction owes a great deal to phototrapping1-3. The following is an illustration of the power of phototrapping as applied to the photoreaction center of Ectothiorhodospira sp.
We report a magneto-structural study using X-ray diffraction and electron paramagnetic resonance (EPR) of three new pyrophosphate-bridged CuII complexes, [Cu2(terpy)2(HP2O7)(H2PO4)(H3PO4)(H2O)], 1, terpy = 2,2':6',2"-terpyridine, [Cu2(bpa)2(P2O7)(H2O)2].2.5H2O, 2, and [Cu(bpa)(H2P2O7)]2, 3, bpa = 2,2'-bipyridylamine, were synthesized and their crystal structures determined by single- crystal X-ray diffraction. Two other copper pyrophosphate compounds already reported, [Cu(bipy)(cis-H2P2O7)]2.3(H2O),4, whose crystal structure was refined along this work, and [Cu(bipy)(trans-H2P2O7)]2, 5, bipy = 2,2'-bipyridine, were added to the previous ones. The five compounds are triclinic, space group , and contain dinuclear copperII units bridged by pyrophosphate anions.
EPR spectra were collected in three planes of single crystal samples as a function of the orientation of the magnetic field B0 at 293 K for compounds 1, 3, 4 and 5, and at T = 4.7, 50 and 293 K for 2. Also, the spectra of the five compounds were studied as a function of temperature (T) between ~ 4 and 293 K for fixed orientations of B0. In the compounds 1, 3, 4 y 5 the EPR spectra display a single resonance for any field orientation and temperature T between 4 and 293 K, as in mononuclear spin systems, without hyperfine structure. The calculated g-matrices are discussed in terms of the molecular structures and of the results for CuII ions in related compounds. In compounds 1, 3, 4 y 5 the temperature dependences of the intensity of the EPR signals observed above 4 K display a paramagnetic Curie behavior, indicating that |J0| < 2 K (defined as Hex = -J0 S1.S2), with no structure arising from hyperfine coupling or intradinuclear exchange interactions. The absence of hyperfine structure and dinuclear splitting of the units is explained considering exchange narrowing processes where interdinuclear exchange interaction average out the intradinuclear interactions, a model that allows setting a lower limit of the interactions between neighbor dinuclear units. Mean while, the EPR signals observed in compound 2 display a rich T- dependent structure. Below ~8 K the spectra are assigned to two types of mononuclear crystal defects hyperfine-coupled to one copper and two nitrogen nuclei. The g-matrices and hyperfine couplings at these T provide information about the structures of the defects. Above 10 K the spectra are dominated by the response of the bulk binuclear CuII material, showing hyperfine interactions with two copper nuclei, collapsing to a single peak above 18 K when the units are magnetically connected, and the magnetic behavior becomes 3D. We attribute the results above 10 K to the interplay of an AFM intradinuclear exchange interaction J0 = -28(3) cm-1, and a three orders of magnitude weaker exchange coupling with average magnitude |J1| ~ 0.022 cm-1 between CuII ions in neighbor dinuclear units. The interplays between structure, exchange couplings, magnetic dimension and spin dynamics in the dinuclear compound are discussed. A previously unreported situation where the structure of the spectra arising from the anisotropic spin-spin interaction term (D) within the dinuclear unit is averaged out, but the forbidden half field transition is not, is observed and explained.
When bacteriochlorophyll absorbs a photon, it becomes an extremely active reductant (see Sauer, Chap. 2, this Vol. for an overview of the associated events). The effective midpoint potential (Em) of the redox couple BChl*/BChl+ is more negative than that of BChl/BChl+ by approximately hv0/e, where BChl* and BChl are bacteriochlorophyll in the excited electronic state and ground state, BChl+ is a 7r-cation radical, hv0 is the o ?o transition energy for excitation of BChl to BChl*, and e is the electronic charge (Parson 1978). For the photo-chemically reactive BChl complex (P) in bacterial reaction centers (RC?s), the Em in the ground state is about +0.45 V and hv0 of the lowest excited singlet state (P*) is about 1.38 eV. The Em of P* is therefore approximately ?0.9 V. This means that P* should be able to transfer an electron to another molecule of BChl or bacteriopheophytin (BPh), because the Em values for BChl-/BChl and BPh-/BPh are on the order of 0.85 V and 0.55 V (Fajer et al. 1975). However, the Em values for the reductions of BChl and BPh have been measured only for the molecules in solution. The pigments in the RC could interact with charged amino acids, P+ , or the quinone (QA) that serves as a later electron acceptor.
The electron transport chain of photosystem I (PS I) comprises the primary donor P700 and five electron acceptors: the primary electron acceptor A0, which is a chlorophyll molecule, a secondary acceptor A1 and 3 iron-sulfur centers Fe-SA, Fe-SB and Fe-SX which act as tertiary electron acceptors. The knowledge of electron transfer pathways is far below in PS I compared to purple bacteria and PS II. Major controversial points are constituted by the electron transfer reactions concerning the secondary acceptor A1 and moreover by the chemical nature of this acceptor, although a lot of new data have accumulated in this field during the last few years.
Continuous illumination of PS-I reaction centers in the presence of dithionite and at high pH is known to induce the reduction of the iron-sulfur electron acceptors Fe-SA. Fe-SB and Fe-SX (1). Under such conditions, a flash induces the charge separation state P-700+…A1- (A1 has been identified with phylloquinone (2)), characterized by its recombination half-time (t1/2 < 1µs in spinach)(3). It has been recently shown that a strong illumination induces the stable double reduction of the acceptor A1 (2). In the present work, it is shown that the incubation in total darkness of PS-I from the cyanobacterium Synechocystis PCC 6803 at high pH, in the presence of dithionite and electron mediators such as methylviologen, induces the inhibition of the electron transfer after the primary electron acceptor A0. Results are interpreted as due to A1 double reduction. The reduced reaction acceptor can be totally re-oxidized by ferricyanide or oxygen without any damage.
The photoreductive trapping method (1–3) was used to study electron transfer reactions of the primary electron donor, P, in the photoreaction center of Ectothiorhodospira sp. This photoreaction center contains a tightly bound c-type (four heme) cytochrome (4) which can reduce P+ before it undergoes charge recombination with the reduced primary acceptor. By holding the preparation at low redox potential to insure reduction of the quinones, intermediary reduced acceptors can be photo-trapped. If the lifetime of the trapped state is very long compared with its trapping time, continuous illumination can lead to its trapping in large amounts. The photochemical trapping technique can detect acceptors which are reduced by P with very low quantum yields. Using this method, we found that P can transfer electrons to both bacteriopheophytin molecules (BPh) which can accept either one or two electrons.
This chapter reveals that the photosynthetic bacterium Rhodopseudomonas sphaeroides has a light-driven cyclic electron transport system organized to generate an electric potential and a pH gradient across the cytoplasmic membrane. Although the details are lacking, it is widely accepted that the electrochemical gradients so generated are harnessed for ATP production, NAD+ reduction, and solute transport. The results from several approaches together contribute to the view of the RC as a light-activatable redox protein that generates both a redox potential (ΔEh) and a membrane potential (Δ↓). There is evidence that describes the Q-b/c2 oxidoreductase as a protein capable of using the ΔEh generated by the RC to drive the formation of further Δ↓ and the translocation of protons across the membrane.
For magnetic resonance studies of paramagnetic species electron paramagnetic resonance (EPR) is a well-established method. However, when trying to elucidate the electronic structure of large and lowsymmetry radicals, as they typically occur in biological systems, one is often hampered by problems of spectral resolution. It was as early as 1956 when Feher (1956) demonstrated that by electron nuclear double resonance (ENDOR) the spectral resolution can be greatly improved. ENDOR signals are obtained by monitoring the changes of the amplitude of a saturated EPR line that occur when sweeping the frequency of an additionally applied rf field through the nuclear (NMR) region. This first ENDOR experiment was technically feasible only because the sample—phosphorus doped silicon—was studied at low temperature, where all the relaxation times are sufficiently long to easily obtain saturation. For radicals in liquid solution, however, these relaxation times are much shorter—on the order of 10-5-10-7 sec—and, consequently, ENDOR-msolution experiments are technically much more sophisticated since much larger saturating microwave and rf fields have to be applied. This probably explains why the first ENDOR-in-solution experiments required many more years before they could be successfully performed by Cederquist (1963) and by Hyde and Maki (1964).
An introduction to electron magnetic resonance (EMR) with applications in biophysical studies is presented at the level of nonspecialist or beginning graduate student. The first half of the chapter briefly introduces the resonance phenomenon, a typical EMR spectrum and its interpretation, and describes fundamental applications of electron resonance spectroscopy in free radical research, identification and characterization of metalloproteins and reaction intermediates, spin probes, and imaging. The second half of the chapter describes the magnetochemical origins of resonance spectroscopy and the steps that have led to modern EMR techniques.
This chapter discusses electron transfer, proton translocation, and ATP synthesis in bacterial chromatophores. The study of bacterial photosynthesis, although dealing with a unique process confined to a relatively small group of organisms, offers the possibility of tackling experimentally many of the most critical problems in the field of membrane bioenergetics. This is because of the essentiality of the system, which combines features common to photosynthetic and respiratory electron-transfer apparatuses, that is a photosynthetic reaction center and aubiquinone–cytochromec oxidoreductase complex, and to the experimental advantages that this system offers for the studies of fast steps of oxidoreduction reactions, activated by single-turnover flashes of actinic light. The energy of light, captured by the reaction center and converted into redox energy of a cyclic electron-transfer system, is utilized for the synthesis of ATP by a proton-translocating ATP synthetase, which has many chemical and physical properties common to the ATP synthetase present in other photosynthetic and respiratory membranes
The idea that the initial photochemical reaction in bacterial photosynthesis is the oxidation of a bacteriochlorophyll (BChl) complex in a special site, or “reaction center,” developed from pioneering studies by L.N.M. Duysens and R.K. Clayton. Reaction centers of the purple, nonsulfur photosynthetic bacteria (Rhodospirillaceae) have proved easier to isolate than those of other photosynthetic organisms. In a typical procedure, chromatophore membranes are first collected from broken cells by differential centrifugation. Mild disruption of the membranes with lauryldimethylamine-N-oxide or other non-ionic detergent solublizes the reaction centers, leaving most of the antenna BChl in a particulate fraction that is removed by centrifugation. Sucrose-gradient centrifugation or fractionation with ammonium sulfate is sometimes used at this point. The reaction centres are then purified by column chromatography on a cationic resin, such as DEAE-Sephacryl in the continuing presence of a low concentration of detergent, and are concentrated by pressure dialysis. Reaction centres isolated from Rb. sphaeroides, Rb. capsulatus and Rs. Rubrum contain three polypeptides in 1:1:1 stoichiometry, with a total molecular weight of about 105.
To understand quantitatively the electron transfer kinetics in reaction centers (RCs) one needs to know both the spatial, three-dimensional, structure as well as the electronic structure of the reactants. The advances made in the determination of the three-dimensional structure of RCs in Rp. viridis and Rb. sphaeroides were presented earlier at this Conference by H. Deisenhofer, D. Tiede and our group. In this communication we would like to report on the results of investigations of the electronic structure of the intermediate acceptor I⨪. The acceptor, I, is believed to be a bacteriopheophytin a (Bphe a), that receives an electron from the singlet excited primary donor in ~ 4 picoseconds and passes it on to a quinone acceptor with a characteristic time of ~ 200 ps (for a review see ref. 1). In general, the charge transfer in photosynthesis is a one electron process that results in the formation of donor cation and acceptor anion radicals.
Publisher Summary This chapter discusses electron paramagnetic resonance (EPR) in photosynthesis. Practically, all aspects of EPR spectroscopy come to the fore, individually or in combination, in the various photosynthetic systems of plants and bacteria, in intact cells or in isolated subcellular particles or purified reaction center proteins. EPR has been instrumental in the demonstration that the primary electron donor, P, in bacterial reaction centers (RC) is a bacteriochlorophyll dimer. In normal photosynthesis, in all photosystems the charge on the photoreduced intermediary acceptor is quickly transported to the next primary acceptor. When this acceptor is (photo) chemically pre-reduced or removed by extraction, however, this negative charge cannot be further transported, and recombines with the positive charge on the primary donor. The recombination product is either the singlet ground or excited state, or the triplet excited state of P. The primary donor of photosystem II (P-680) is much more difficult to observe with EPR than that of photosystem PS I, because in normally functioning PS II the photo-oxidized donor is very rapidly (within at most a few hundred ns) reduced by an electron donor called Z. The reduced intermediary acceptor I (BPh) is normally too short-lived to be observable by EPR. However, it can be photoaccumulated at cryogenic temperatures in isolated RCs of, for example, Rb. Sphaeroides, when reduced Cyt c is added, because of slow, irreversible electron donation to P + .
After enzymes were first discovered in the late XIX century, and for the first seventy years of enzymology, kinetic experiments were the only source of information about enzyme mechanisms. Over the following fifty years, these studies were taken over by approaches that give information at the molecular level, such as crystallography, spectroscopy and theoretical chemistry (as emphasized by the Nobel Prize in Chemistry awarded last year to M. Karplus, M. Levitt and A. Warshel). In this review, we thoroughly discuss the interplay between the information obtained from theoretical and experimental methods, by focussing on enzymes that process small molecules such as H2 or CO2 (hydrogenases, CO-dehydrogenase and carbonic anhydrase), and that are therefore relevant in the context of energy and environment. We argue that combining theoretical chemistry (DFT, MD, QM/MM) and detailed investigations that make use of modern kinetic methods, such as protein film voltammetry, is an innovative way of learning about individual steps and/or complex reactions that are part of the catalytic cycles. We illustrate this with recent results from our labs and others, including studies of gas transport along substrate channels, long range proton transfer, and mechanisms of catalysis, inhibition or inactivation.
Time-resolved electron paramagnetic resonance (TREPR) spectroscopy was used for studying photoinduced intramolecular electron transfer (ET) and energy transfer (EnT) processes in three mechanically interlocked molecules in which zinc(II)porphyrin (ZnP) and C60 fullerene moieties are arrayed around a central Cu(I) bisphenanthroline core used to assemble these donor–acceptor (D–A) systems. The specific molecules studied include a “long” (ZnP)2–Cu(I)(phen)2–C60 rotaxane as well as ZnP–Cu(I)(phen)2 and ZnP–Cu(I)(phen)2–C60 catenanes, embedded in different phases of nematic liquid crystal and frozen isotropic solvents. It was demonstrated that the routes and rates of the transfer processes in these supramolecules strongly depend on the characteristics of their microenvironment and the molecular entity which was selectively photoexcited. This is reflected by formation of distinct long-lived charge-separated species such as the radical ion pair (ZnP)2•+–Cu(I)(phen)2•– and the metal-to-ligand charge-separated state Cu(II)–(phen)2•– under the various experimental conditions. The results are discussed in terms of the correlation between the chemical structure, conformational mobility, and relaxation pathways of the photoexcited states in these mechanically interlocked systems. Results are compared with previously reported TREPR data on related interlocked D–A porphyrin/fullerene systems.
Two different dyads containing a triarylamine (TAA) donor and a naphthalene-1,8:4,5-bis(dicarboximide) (NDI) acceptor bridged by either a [2.2]- or a [3.3]paracyclophane (CP) were synthesized. These dyads show a high population of long-lived charge separated (CS) singlet and triplet states. The lifetimes of these different spin states vary only by 1 order of magnitude. This unique situation is a consequence of both a large electronic coupling V and a large exchange coupling 2J. The population of the different CS spin states and therefore the charge recombination (CR) and intersystem crossing (ISC) kinetics were monitored by standard ns-transient absorption spectroscopy. Together with fs-transient absorption spectroscopy supported by electrochemistry, steady state fluorescence and steady state absorption spectroscopy a detailed model of the photoinduced processes was derived.
Comparison of the optical, paramagnetic and redox properties of the reduced primary acceptors of Photosystems (PS) I and II with those of chlorophyll and pheophytin anions suggests that chlorophyll is the primary reduced product in PS I and pheophytin in PS II. Molecular orbital calculations provide a description of the electronic profiles of the radicals, and indicate that the protein environment of the chromophores in the reaction center may impose specific orientations and induce hydrogen bonding to some of the chlorophyll substituent groups. Examination of all the primary acceptors postulated for green plants and purple bacteria suggests that (bacterio)chlorophyll‐like acceptors may be obligatory to effect the rapid primary charge separation, because they allow favorable orbital overlap between donor and acceptor. Extrapolation of these results to green bacteria suggests that bacteriochlorophyll a and bacteriopheophytin c may act as transient electron acceptors in these organisms.
The spin alignment in a charged molecular field is important research issue in the molecular magnetism. In order to clarify the interrelation between spin alignment and the charged molecular field, we have investigated intra- and inter-molecular exchanges on some Hydrazine diradical dictations 1–3 shown in Scheme 1 (see texts) by ESR and magnetic susceptibility measurement. The magnetic behavior of the dication salt 1 has been well analyzed using the alternating linear chain models with Jintra/kB= −106 K, Jinter/kB= −49 K and an alternating parameter α=0.46. The magnetic property of 3 has been also fitted to the alternating chain model with J/kB= −106 K Jinter/kB= −42 K (α=0.40). On the other hand, 2 gives a robust triplet ground state with larger energy separation from other spin states. The energy separation has been estimated to be larger than 300 cm−1 (Jintra/kB > ±190 K) from the temperature dependence of the ESR signal intensity. These findings indicate that the sign of the intramolecular exchange depends on the linking position (m- or p-) of the hydrazine cation group, i.e. the topology of the π orbital network even in the cationic molecular field. The magneto-optical correlation is also discussed based on the intramolecular electron transfer (ET) parameters.
Gemischtvalente (MV) Verbindungen sind ausgezeichnete Modellsysteme, um grundlegende Elektronentransport(ET)- und Ladungstransfer(CT)-Phänomene zu untersuchen. Diese sind in komplexen biophysikalischen Prozessen wie der Photosynthese, aber auch in artifiziellen elektronischen Bauteilen von großer Bedeutung. So sind organische MV-Verbindungen effiziente Lochtransportmaterialien in organischen lichtemittierenden Dioden (OLEDs), Solarzellen und photochromen Fenstern. Die Bedeutung der organischen gemischtvalenten Chemie sollte jedoch weniger in ihrer direkten Anwendbarkeit gesehen werden, als vielmehr in dem riesigen Kenntnisschatz über Elektronentransferphänomene, der durch ihr Studium gewonnen wurde. Die große Vielfalt an organischen Redoxzentren und Brückeneinheiten, die zu MV-Verbindungen kombiniert werden können, sowie die stete Weiterentwicklung von ET-Theorien und ET-Untersuchungsmethoden förderten das enorme Interesse an organischen MV-Verbindungen in den letzten Jahrzehnten und zeigen das große Potential dieser Verbindungsklasse auf. Das Ziel dieses Aufsatzes ist es, das letzte Jahrzehnt der organischen gemischtvalenten Chemie zusammenzufassen und ihren Einfluss auf moderne funktionelle Materialien hervorzuheben.
Our understanding of photosynthesis has been greatly advanced by the elucidation of the structure and function of the reaction center (RC), the membrane protein responsible for the initial light-induced charge separation in photosynthetic bacteria and green plants. Although today we know a great deal about the details of the primary processes in photosynthesis, little was known in the early days. George Feher made pioneering contributions to photosynthesis research in characterizing RCs from photosynthetic bacteria following the ground-breaking work of Lou Duysens and Rod Clayton (see articles in this issue by van Gorkom and Wraight). The work in his laboratory at the University of California, San Diego, started in the late 1960s and continued for over 30 years. He isolated a pure RC protein and used magnetic resonance spectroscopy to study the primary reactants. Following this pioneering work, Feher studied the detailed structure of the RC and the basic electron and proton transfer functions that it performs using a wide variety of biophysical and biochemical techniques. These studies, together with work from many other researchers, have led to our present detailed understanding of these proteins and their function in photosynthesis. The present article is a brief historical account of his pioneering contributions to photosynthesis research. A more detailed description of his work can be found in an earlier biographical paper (Feher in Photosynth Res 55:1-40, 1998a).
Calculations on a system consisting of three electron spins and one nuclear spin are presented and their implications for bacterial photosynthesis discussed. Comparison with experimental measurements of electron spin polarization in pre-reduced photosynthetic reaction centres leads to conclusion that the exchange interaction within the primary radical pair is positive and less than 0.8 mT when the g values of the photoinduced radicals are taken to be those measured for the isolated radical species.
The electron paramagnetic resonance (EPR) signal associated with the photo‐accumulated radical anion of the primary electron acceptor (I⋅−, a bacteriopheophytin radical) in reaction centers (RCs) of the photosynthetic purple bacterium rhodopseudomonas viridis shows a characteristic splitting of about 14.0 mT. This splitting has been attributed to an exchange interaction between I⋅− and the reduced complex of the second electron acceptor, a quinone molecule, and a divalent, high‐spin (S=2) Fe‐ion, [Q⋅−AFe++]. The magnetic structure of the three‐spin complex, Fe++ (S=2), Q⋅−A (S=1/2), and I⋅− (S=1/2), is assessed by Q‐band (34.8 GHz) and X‐band (9.2 GHz) EPR spectroscopy. The EPR spectrum of [I⋅−Q⋅−AFe++] is simulated accurately for the first time, using the magnetic parameters for the quinone‐iron complex [W. F. Butler, R. Calvo, D. R. Fredkin, M. Y. Okamura, and G. Feher [Biophys. J. 45, 947 (1984)]. A largely isotropic interaction between I⋅− and Q⋅−A is required (JIQ=−7.5 mT), together with an anisotropic interaction between I⋅− and the Fe++‐ion, whose y‐component, CIFe,y, is −3.5 mT. The simulations were insensitive to the magnitude of the x,z‐components, CIFe,x/z. The experimental magnetic interactions correspond very well with values calculated from the distances in the RC crystal structure. Thus, the interaction between I⋅− and Q⋅−A is largely isotropic (exchange), whereas the interaction between I⋅− and Fe++ has a purely dipolar character. This result is used to determine the principal directions of the magnetic interaction tensors of the Fe++‐ion.
The photoexcited triplet states of bacteriochlorophyll a, 3BChl a, and of the primary donor in reaction centers of Rhodobacter sphaeroides R-26, 3P865, are investigated by pulsed EPR and ENDOR spectroscopy. In 3P865 a splitting of ENDOR lines and reduction of corresponding positive and negative hyperfine couplings as compared with the monomeric 3BChl a is observed. This indicates an asymmetric distribution of the triplet excitation over the two BChl a moieties, PL and PM, forming 3P865. Based on the signs of the hyperfine couplings and on a comparison with the cation and anion radical of BChl a an assignment to nuclei in the different dimer halves is proposed. This yields an estimate for the extent of delocalization of the triplet excitation over PL and PM and for the charge transfer contribution of 3P865.
This paper considers electron transfer between biological molecules in terms of a nonadiabatic multiphonon nonradiative decay process in a dense medium. This theoretical approach is analogous to an extended quantum mechanical theory of outer sphere electron transfer processes, incorporating the effects of both low-frequency medium phonon modes and the high-frequency molecular modes. An explicit, compact and useful expression for the electron transfer probability is derived, which is valid throughout the entire temperature range, exhibiting a continuous transition from temperature independent tunneling between nuclear potential surfaces at low temperatures to an activated rate expression at high temperatures. This result drastically differs at low temperatures from the common, semiclassical, Gaussian approximation for the transition probability. The experimental data of De Vault and Chance [Biophys. J. 6, 825 (1966)] on the temperature dependence of the rate of electron transfer from cytochrome to the chlorophyll reaction center in the photosynthetic bacterium Chromatium are properly accounted for in terms of the present theory.
Bacteriopheophytin, the magnesium-free base of bacteriochlorophyll, undergoes reversible one-electron reduction in organic solvents to yield an anionic free radical with characteristic optical and electron spin resonance spectra. The reduction potential of bacteriopheophytin, E1/2 approximately --0.55 V against a normal hydrogen electrode, compared to E1/2 approximately --0.85 V for bacteriochlorophyll, renders it a likely electron acceptor in the primary charge separation of photosynthesis. Comparison of these data with picosecond optical changes recently observed upon pulsed laser excitation of bacterial reaction centers leads us to propose that bacteriopheophytin is indeed a transient electron acceptor and that the primary charge separation of bacterial photosynthesis occurs between the bacteriochlorophyll complex P870 and bacteriopheophytin to yield the radicals of the oxidized chlorophyll dimer cation and reduced pheophytin anion.
Preparations of photosynthetic reaction centers from Rhodopseudomonas sphaeroides were excited with flashes lasting approximately 8 psec. Immediately after the excitation, there appeared a transient state which was characterized by new absorption bands near 500 and 680 nm, by a bleaching of bands near 540, 600, 760, and 870 nm, and by a blue shift of a band near 800 nm. The transient state decayed with an exponential decay time,t, of 246 plus or minus 16 psec after the flash. As the transient state decayed, the radical cation of the reaction center bacteriochlorophyll complex appeared. This indicates that the transient state is an intermediate in the photooxidation of the bacteriochlorophyll. The absorpiton spectrum of the transient state shows the state to be identical with a state (P-F) which has been detected previously in reaction centers that are prevented from completing the photooxidation, because of chemical reduction of the electron acceptor. Analysis of the spectrum suggests that the formation of P-F involves electron transfer from one bacteriochlorophyll molecule to another within the reaction center, or possibly from bacteriochlorophyll to the bacteriopheophytin of the complex. The initial absorbance changes after flash excitation also include a bleaching of an absorption band at 800 nm. The bleaching decays with tau approximately equal to 30 pse. The bleaching appers not to be a secondary effect, but rather to revael another early step in the primary photochemical reaction.
Reaction centers were found to bind two ubiquinones, both of which could be removed by o-phenanthroline and the detergent lauryldimethylamine oxide. One ubiquinone was more easily removed than the other. The low-temperature light-induced optical and electron paramagnetic resonance (EPR) changes were eliminated and restored upon removal and readdition of ubiquinone and were quantitatively correlated with the amount of tightly bound ubiquinone. We, therefore, conclude that this ubiquinone plays an obligatory role in the primary photochemistry. The easily removed ubiquinone is thought to be the secondary electron acceptor. The low-temperature charge recombination kinetics, as well as the optical and EPR spectra, were the same for untreated reaction centers and for those reconstituted with ubiquinone. This indicates that extraction and reconstitution were accomplished without altering the conformation of the active site. Reaction centers reconstituted with other quinones also showed restored photochemical activity, although they exhibited changes in their low-temperature recombination kinetics and light-induced (g = 1.8) EPR signal is interpreted in terms of a magnetically coupled ubiquinone--Fe2+ acceptor complex. A possible role of iron is to facilitate electron transfer between the primary and secondary ubiquinones.
A theory of electron transfer between two fixed sites by tunneling is developed. Vibronic coupling in the individual molecules produces an activation energy to transfer at high temperatures, and temperature-independent tunneling (when energetically allowed) at low temperature. The model is compared with known results on electron transfer in Chromatium and in Rhodopseudomonas spheroides. It quantitatively interprets these results, with parameters whose scale is verified by comparison with optical absorption spectra. According to this description, the separation between linking sites for electron transfer is 8-10 A in Chromatium, far smaller than earlier estimates.
The phenomenon of electron paramagnetic resonance was discovered by Zavoiskii (1944) in his studies of paramagnetic relaxation and resonance spectroscopy in the radiofrequency band (10 –100 MHz). He observed induced quantum transitions be tween Zeeman sublevels for parallel and perpendicular orientations of constant and high-frequency magnetic fields, and detected a dependence of the paramagnetic resonance absorption of high-frequency power on the ratio of the constant magnetic field strength to the frequency of the variable field.
This paper discusses some factors which limit the sensitivity of microwave paramagnetic resonance equipments. Several specific systems are analyzed and the results verified by measuring the signal-to-noise ratio with known amounts of a free radical. The two most promising systems, especially at low powers, employ either superheterodyne detection or barretter homodyne detection. A detailed description of a superhetrodyne spectrometer is given.
1. A reaction center-cytochrome c complex has been isolated from Chromatium vinosum which is capable of normal photochemistry and light-activated rapid cytochrome c553 and c555 oxidation, but which has no antenna bacteriochlorophyll. As is found in whole cells, ferrocytochrome c553 is oxidized irreversibly in milliseconds by light at 7 K.2. Room temperature redox potentiometry in combination with EPR analysis at 7 K, of cytochrome c553 and the reaction center bacteriochlorophyll dimer (BChl)2 absorbing at 883 nm yields identical results to those previously reported using optical analytical techniques at 77 K. It shows directly that two cytochrome c553 hemes are are equivalent with respect to the light induced (BChl)2⨥ At 7 K, only one heme can be rapidly oxidized in the light, commensurate with the electron capacity of the primary acceptor (quinone-iron) being unity.3. Prior chemical reduction of the quinone-iron followed by illumination at 200K, however, leads to the slow () oxidation of one cytochrome c553 heme, with what appears to be concommitant reduction of one of the two bacteriophytins (BPh) of the reaction center as shown by bleaching of the 760 nm band, a broad absorbance increase at approx. 650 nm and a bleaching at 543 nm. The 800 nm absorbing bacteriochlorophyll is also involved since there is also bleaching at 595 and 800 nm; at the latter wave-length the remaining unbleached band appears to shift significantly to the blue. No redox changes in the 883 absorbing bacteriochlorophyll dimer are seen during or after illumination under these conditions. The reduced part of the state represents what is considered to be the reduced form of the electron carrier (I) which acts as an intermediate between the bacteriochlorophyll dimer and quinoneiron. The state (oxidized ) relaxes in the dark at 200 K in approx. 20 min but below 77 K it is trapped on a days time scale.4. EPR analysis of the state trapped as described above reveals that one heme equivalent of cytochrome becomes oxidized for the generation of the state, a result in agreement with the optical data. Two prominent signals are associated with the trapped state in the g = 2 region, which can be easily resolved with temperature and microwave power saturation: one has a line width of 15 g and is centered at g = 2.003; the other, which is the major signal, is also a radical centered at g = 2.003 but is split by 60 G and behaves as though it were an organic free-radical spin-coupled with another paramagnetic center absorbing at higher magnetic field values; this high field partner could be the iron-quinone of the primary acceptor. The identity of two signals associated with I⨪ is consistent with the idea that the reduced intermediary carrier is not simply BPh⨪ but also involves a second radical, perhaps the 800 nm bacteriochlorophylls in the reduced state. As such, the single electron would be shared in some way, and it is probable that one of these centers will be very close to the paramagnetism of the iron-quinone. Alternatively, it is possible that the electron only occupies BPh⨪ (the optical changes associated with the 800 nm bacteriochlorophyll occurring on a secondary basis) and that some of the BPh⨪ population of the trapped state is not close enough to interact with the quinone-iron.5. Light-induced triplet state formation is dramatically diminished in material in which I as well as the quinone-iron is reduced before illumination. This supports the idea that with quinone-iron alone reduced before illumination, triplet formation requires light activated electron transfer from the bacteriochlorophyll dimer to I (not possible if I is already reduced) and that the triplet is formed by the return of the electron from I⨪ to (BChl)2⨥.6. Results indicate that although the two cytochrome c553 hemes may be equivalent at the point of activation, once one has become oxidized the other becomes less competent for oxidation by the (BChl)2⨥.
The photo-oxidation of the reaction center bacteriochlorophyll dimer or special pair was monitored at 1235 nm in Chromatium vinosum and at 1301 nm in Rhodopseudomonas viridis. In both species, the photo-oxidation was apparently complete within 10 ps after light excitation and proceeded unimpeded at low temperatures regardless of the prior state of reduction of the traditional primary electron acceptor, a quinone-iron complex. Thus the requirement for an intermediary electron carrier (I), previously established by picosecond measurements in Rps. sphaeroides (see ref. 4), is clearly a more general phenomenon.The intermediary carrier, which involves bacteriopheophytin, was examined from the standpoint of its role as the direct electron acceptor from the photo-excited reaction center bacteriochlorophyll dimer. To accomplish this, the extent of light induced bacteriochlorophyll dimer oxidation was measured directly by the picosecond response of the infrared bands and indirectly by EPR assay of the triplet/biradical, as a function of the state of reduction of the I/I⨪ couple (measured by EPR) prior to activation. Two independent methods of obtaining I in a stably reduced form were used: chemical equilibrium reduction, and photochemical reduction. In both cases, the results demonstrated that the intermediary carrier, which we designate I, alone governs the capability for reaction center bacteriochlorophyll photooxidation, and as such I appears to be the immediate and sole electron acceptor from the light excited reaction center bacteriochlorophyll dimer.
Experimental evidence for electron transfer, photosensitized by bacteriochlorophyll, from cytochrome c to a pigment complex P-760 (involving bacteriopheophytin-760 and also bacteriochlorophyll-800) in the reaction centers of Chromatium minutissimum has been described. This photoreaction occurs between 77 and 293 °K at a redox potential of the medium between −250 and −530 mV. Photoreduction of P-760 is accompanied by development of a wide absorption band at 650 nm and of an EPR signal with g = 2.0025±0.0005 and linewidth of 12.5±0.5 G, which are characteristic of the pigment radical anion.It is suggested that the photoreduction of P-760 occurs under the interaction of reduced cytochrome c with the reaction center state P+-890 · P−-760 which is induced by light. The existence of short-lived state P+-890 · P−-760 is indicated by the recombination luminescence with activation energy of 0.12 eV and . This luminescence is excited and emitted by bacteriochlorophyll and disappears when P-760 is reduced.At low redox potentials, the flash-induced absorbance changes related to the formation of the carotenoid triplet state with at 20 °C are observed. This state is not formed when P-760 is reduced at 293 and 160 °K. It is assumed that this state is formed from the reaction center state P+-890 · P−-760, which appears to be a primary product of light reaction in the bacterial reaction centers and which is probably identical with the state PF described in recent works.
1.1. Semiquinone anion minus quinone (Q̇−-Q) and neutral semiquinone minus quinone (Q̇H-Q) difference spectra have been measured for plastoquinone-9, ubiquinone-10 and ubiquinone-0, using pulse radiolysis in methanolic solution. The difference spectra were used to determine the absolute spectra of each semiquinone.2.2. The (Q̇−-Q) difference spectrum for plastoquinone-9 is similar to the difference spectrum detected at a time less than 20 ns after flash excitation of the photosynthetic light reaction Photosystem II in spinach chloroplasts [Stiehl, H. H. and Witt, H. T., (1968) Z. Naturforsch. 23b, 220–224; Witt, H. T. (1971) Q. Rev. Biophys. 4, 365–477].3.3. The ubiquinone Q̇−-Q spectra are similar to difference spectra found on illumination of reaction centre preparations from Rhodopseudomonas spheroides and Rhodospirillum rubrum [Slooten, L. (1972) Biochim. Biophys. Acta 275, 208–218; Clayton, R. K. and Straley, S. C. (1972) Biophys, J. 12, 1221–1234]. Ubiquinone has been suggested to be closely associated with the primary electron acceptor of bacterial photosynthesis.4.4. Rate constants were measured for formation of semiquinones by reaction of estaggered−staggeredCH3OH, ĊH2OH and ĊH2O− with quinone, and for the decays of the neutral semiquinones. The semiquinone anions were stable for seconds. The ubisemiquinone-6 anion reacts only slowly with oxygen, k ≈ 103 M−1 · s−1.5.5. The effect of substituents upon semiquinone physicochemical properties is discussed.
The spectroscopic properties of the intermediary electron carrier (I), which functions between the bacteriochlorophyll dimer, (BChl)2, and the primary acceptor quinone · iron, QFe, have been characterized in Rhodopseudomonas viridis. Optically the reduction of I is accompanied by a bleaching of bands at 545 and 790 nm and a broad absorbance increase around 680 nm which we attribute to the reduction of a bacteriopheophytin, together with apparent blue shifts of the bacteriochlorophyll bands at 830 and possibly at 960 nm. Low temperature electron paramagnetic resonance analysis also reveals complicated changes accompanying the reduction of I. In chromatophores I⨪ is revealed as a broad split signal centered close to g 2.003, which is consistent with I⨪ interacting, via exchange coupling and dipolar effects, with the primary acceptor Q⨪Fe. This is supported by experiments with reaction centers prepared with sodium dodecyl sulfate, which lack the Q⨪Fe g 1.82 signal, and also lack the broad split I⨪ signal; instead, I⨪ is revealed as an approximately 13 gauss wide free radical centered close to g 2.003. Reaction centers prepared using lauryl dimethylamine N-oxide retain most of their Q⨪Fe g 1.82 signal, and in this case I⨪ occurs as a mixture of the two EPR signals described above. However, the optical changes accompanying the reduction of I⨪ are very similar in the two reaction center preparations, so we conclude that there is no direct correlation between the two optical and the two EPR signals of I⨪. Perhaps the simplest explanation of the results is that the two EPR signals reflect the reduced bacteriopheophytin either interacting, or not interacting, with Q⨪Fe, while the optical changes reflect the reduction of bacteriophenophytin, together with secondary, perhaps electrochromic effects on the bacteriochlorophylls of the reaction center. However, we are unable to eliminate completely the possibility that there is also some electron sharing between the reduced bacteriopheophytin and bacteriochlorophyll.
The theory of indirect exchange in poor conductors is examined from
a new viewpoint in which the d (or f) shell electrons are placed
in wave functions assumed to be exact solutions of the problem of
a single d-electron in the presence of the full diamagnetic lattice.
Inclusion of d-electron interactions leads to three spin-dependent
effects which, in the usual order of their sizes, we call: superexchange
per se, which is always antiferromagnetic; direct exchange, always
ferromagnetic; and an indirect polarization effect analogous to nuclear
indirect exchange. Superexchange itself is shown to be closely related
to the poor conductivity, in agreement with experiment. By means
of crystal field theory the parameters determining superexchange
can be estimated, and in favorable cases (NiO, LaFeO3) the exchange
integrals can be evaluated with accuracy of several tens of percent.
Qualitative understanding of the whole picture of exchange in iron
group oxides and fluorides follows from these ideas.
We have shown that the rise and decay kinetics of the light-induced EPR signal are identical to the kinetics of the optical changes at 80 degrees K. This identity provides independent evidence that the EPR signal is due to the oxidized primary electron donor which is bacteriochlorophyll. The EPR and optical changes could be described by a model photochemical reaction scheme that takes into account spin-lattice relaxation. The optical decay rate was found to be temperature independent between 1.5 and 80 degrees K and to obey approximately first order kinetics. These results are consistent with the hypothesis that the charge recombination occurs via tunneling through a potential barrier. The decay constants at these temperatures were found to be the same for different bacterial species and strains. No differences were found between purified reaction centers of R. spheroides R-26 and whole cells. Reaction centers treated with sodium dodecylsulfate or urea were still photochemically active but showed a markedly different kinetic behavior. The decay constant may, therefore, serve as a probe to investigate the molecular environment of the primary reactants.
We have proposed that the "doublet" EPR spectra observed during catalysis by a number of coenzyme B12-requiring enzymes arises from a weak electrostatic exchange interaction between an organic free radical and low spin Co(II), B12r. By varying the magnitude of the exchange of coupling we have quite accurately simulated the published EPR spectra from the enzyme systems: diol dehydrase, glycerol dehydrase, ribonucleotide reductase, and ethanolamine ammon-ia lyase. A dipolar model was shown to be incompatible with the observed properties of these systems.
The origin of most of the electron paramagnetic resonances obtained at low temperature and low microwave power from heart tissue and subcellular fractions derived therefrom is now understood. A signal that emerges on partial reduction with characteristic lines at 3227 G (0.3227 tesla) and 3309 G (0.3309 tesla) (at 9.2 GHz) and disappears again on full reduction has remained unidentified. According to its behavior on oxidation-reduction, the substance giving rise to this signal has the properties of a two-electron acceptor. The signal is strongly dependent on temperature and can only be well resolved at less than 20 degrees K. It is readily elicited in submitochondrial particles by partial reduction, but has not been observed in submitochondrial particles from which ubiquinone has been removed by pentane extraction. When ubiquinone is reincorporated into extracted submitochondrial particles, the signal is again easily produced by partial reduction. Electron paramagnetic resonance spectra of partially reduced submitochondrial particles recorded at 34 GHz show lines centered about g approximately 2 with the same separation (approximately 82 G; approximately 0.0082 tesla) as do 9.2 GHz spectra, whereas no lines are detected with a separation of approximately 82 X 34/9.2 G (0.0082 X 34/9.2 tesla). We suggest, on the basis of these observations, that the unidentified signal arises from an interaction of ubisemiquinone and a second paramagnetic species. Three obvious choices exist concerning this second species: ubisemiquinone, flavin semiquinone, or an iorn-sulfur center. It is not possible without much additional information to decide between these possibilities. Since we have never observed the signal in the absence of the membrane-bound, high-potential type iron-sulfur protein, we have considered involvement of this species in the interaction. However, according to computer simulations of the observed electron paramagnetic resonance spectra, which yield best fits for semiquinone-semiquinone interaction, the possibility that ubi- or flavin semiquinone is the interaction partner appears more likely at this time. The interaction appears to be of the magnetic dipole-dipole type, but it is not certain whether there is also a contribution from spin exchange coupling. If it is assumed that the signal is due to magnetic dipole-dipole interaction, the distance of the partners is less than or equal to 7.7 A.
When reaction centers are illuminated by a series of single turnover flashes ubisemiquinone is formed and destroyed on alternate flashes. This oscillatory behaviour can be observed with both optical and electron spin resonance techniques. The oscillations are dependent upon the presence of excess ubiquinone in a manner which suggests that two molecules may act almost equivalently as metastable primary acceptors forming a two-electron gate between the one-electron primary photoact and a two-electron secondary acceptor pool.
The primary electron transfer processes in Rhodopseudomonas sphaeroides R-26 were studied as a function of temperature by means of picosecond spectroscopy. The first chemical step of the bacterial photosynthesis involves an electron transfer from the excited state of a bacteriochlorophyll a dimer, (BChl)2, to a bacteriopheophytin (BPh) to form the radical ion pair (BChl)2+. BPh-.. The upper limit for the formation time of this ion-pair was found to be 10 ps, at temperatures in the range 300-4.2 degree K. Similarly, the second chemical step, involving electron transfer from BPh-. to an ubiquinone-iron complex (QFe), was found to have a lifetime of approximately 150 ps, also independent of temperature in the same range. We interpret the absence of temperature dependence as indicating that process 2 proceeds via a tunneling mechanism. Utilizing our results in conjunction with electron tunneling theories, we calculate the distance between BPh-. and Q(Fe) to be 9--13 A. Our results also imply a closer proximity between (BChl)2 and BPh.
Picosecond and nanosecond spectroscopic techniques have been used to study the primary electron transfer processes in reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Following flash excitation, the first excited singlet state (P*) of the bacteriochlorophyll complex (P) transfers an electron to an intermediate acceptor (I) in less than 20 ps. The radical pair state P+I-) subsequently transfers an electron to another acceptor (X) in about 230 ps. There is an additional step of unknown significance exhibiting 35 ps kinetics. P+ subsequently extracts an electron from a cytochrome, with a time constant of about 270 ns. At low redox potential (X reduced before the flash), the state P+I- (or PF) lives approx. 15 ns. It decays, in part, into a longer lived state (PR), which appears to be a triplet state. State PR decays with an exponential time of approx. 55 microseconds. After continuous illumination at low redox potential (I and X both reduced), excitation with an 8-ps flash produces absorption changes reflecting the formation of the first excited singlet state, P*. Most of P* then decays with a time constant of 20 ps. The spectra of the absorbance changes associated with the conversion of P to P* or P+ support the view that P involves two or more interacting bacteriochlorophylls. The absorbance changes associated with the reduction of I to I- suggest that I is a bacteriopheophytin interacting strongly with one or more bacteriochlorophylls in the reaction center.
Purified photochemical reaction centers from three strains of Rhodopseudomonas sphaeroides and two of Rhodospirillium rubrum were reduced with Na2S2O4 so as to block their photochemical electron transfer reactions. They then were excited with flashes lasting 5-30 ns. In all cases, absorbance measurements showed that the flash caused the immediate formation of a transient state (PF) which had been detected previously in reaction centers from Rps. sphaeroides strain R26. Previous work has shown that state PF is an intermediate in the photochemical electron transfer reaction in the reaction centers of that particular strain, and the present work generalizes that conclusion. In the reaction centers from two strains that lack carotenoids (Rps. sphaeroides R26 and R. rubrum G9), the decay of PF yields a longer-lived state (PR) which is probably a triplet state of the bacteriochlorophyll of the reaction center. In the R26 preparation, the decay of PF was found to have a half-time of 10 +/- 2 ns. The decay kinetics rule out the identification of PF as the fluorescent excited singlet state of the reaction center. In the reaction centers from three strains that contain carotenoids (Rps sphaeroides 2.4.1 and Ga, and R. rubrum S1), state PR was not detected, and the decay of PF generated triplet states of carotenoids. The efficiency of the coupling between the decay of PF and the formation of the carotenoid triplet appeared to be close to 100% at room temperature, but somewhat lower at 77 degrees K. Taken with previous results, this suggests that the coupling is direct and does not require the intermediate formation of state PR. This conclusion would be consistent with the view that PF is a biradical which can be triplet in character.
A method is described for isolation of the Rhodopseudomonas viridis reaction center complex free of altered, 685 nm absorbing pigment. This improved preparation contains two c-type cytochromes in the ratio P-960: cytochrome c-558: cytochrome c-553 of 1:2:2 to 3. The near infrared spectral forms of the reduced preparation are located at 790, 832, 846, and 987 nm at 77 K; the oxidized complex absorbs at 790, 808, 829 and approx. 1310 nm. The 790 nm band is attributed to bacteriophaeophytin b and the other absorbances to bacteriochlorophyll b, The visible absorption bands may be assigned to these pigments and to the cytochromes present and, probably to a carotenoid. The presence of two bacteriochlorophyll b spectral forms in the P+-830 band suggests that exciton interactions occur among pigments in the oxidized, as well as the reduced, reaction center. Changes in the 790 and 544 nm bands upon illumination of the reaction center preparation at low redox potential may be indicative of a role for bacteriophaeophytin b in primary photochemical events.
In preparations of photochemical reaction centers from Rhodopseudomonas spheroides R-26, lowering the recox potential so as to reduce the primary electron acceptor prevents the photochemical transfer of an electron from bacteriochlorophyll to the acceptor. Measuring absorbance changes under these conditions, we found that a 20-ns actinic flash converts the reaction center to a new state, P-F, which then decays with a half-time that is between 1 and 10 ns at 295 degrees K. At 25 degrees K, the decay half-time is approx. 20 ns. The quantum yield of state P-F appears to be near 1.0, both at 295 and at 15 degrees K. State P-F could be an intermediate in the photochemical electron-transfer reaction which occurs when the acceptor is in the oxidized form. Following the decay of state P-F, we detected another state, P-R, with a decay half-time of 6 mus at 295 degrees K and 120 mus at 15 degrees K. The quantum yield of state P-R is approx. 0.1 at 295 degrees K, but rises to a value nearer 1.0 at 15 degrees K. The kinetics and quantum yields are consistent with the view that state P-R forms from P-F. State P-R seems likely to be a side-product, rather than an intermediate in the electron-transfer process. The decay kinetics indicate that state P-F cannot be identical with the lowest excited singlet state of the reaction center. One of the two states, P-F or P-R, probably is the lowest excited triplet state of the reaction center, but it remains unclear which one.
Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients.Two independent sets of measurements using a variety of methods show that a sample with A800 nm = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1.Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77.These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: ε802 nm = 288 (± 14) mM−1 · cm−1 and ε865 nm = 128 (± 6) mM−1 · cm−1. For reversible bleaching of the 865-nm band, Δεred - ox865nm = 112 (± 6) mM−1 · cm−1 (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.
Low-temperature e.p.r. (electron-paramagnetic-resonance) spectroscopy was used to detect electron-transport components in Chromatium chromatophores with e.p.r. signals in the g=2.00 region. High-potential iron protein (E(m8.0)=+325mV, where E(m8.0) is the midpoint potential at pH8) and a second component (g=1.90, E(m8.0)=+285mV) are oxidized in illuminated chromatophores. Two iron-sulphur proteins (g=1.94) with E(m8.0)=-290mV and E(m8.0)=-50mV are present. One (E(m8.0)=-50mV) is reduced on illumination. A component (g=1.82) with E(m8.0)=-135mV is photoreduced at 10 degrees K. The midpoint potential of this component is altered by o-phenanthroline and pH. The properties of this component suggest that it is the primary electron acceptor of a photochemical system. Another component (g=1.98) also has some of the properties of a primary electron acceptor, but its function cannot be completely defined. These results show that iron-sulphur proteins are present in the electron-transport system of Chromatium and indicate their role in electron transport.
A transient absorption spectrum has been measured in Rhodopseudomonas spheroides R26 reaction centers. Its salient features indicate that both the bacteriopheophytin and bacteriochlorophyll chromophores play a role in the excited state. Decay of this state yields a rise time for oxidation of the reaction center complex of about 150 picoseconds.
19) where Aeba, Aeca, Aeda are the differential extinction coefficients for forming I-Q-, IQ 2-, and I-Q ~-from IQ-. They were obtained from Eqns
- + A~4so
+ A~4SO @BULLET ~b~ [b] + ~d~ [d] (19) where Aeba, Aeca, Aeda are the differential extinction coefficients for forming I-Q-, IQ 2-, and I-Q ~-from IQ-. They were obtained from Eqns. 14--17 using
- Okamura