Vibronic coupling to electron transfer and the structure of the R. Viridis reaction center

This paper points out that the orientations of the porphyrins, bacteriochlorophyll and bacteriopheophytin, in the reaction centers of Rhodopseudomonas viridis, as shown by the new X-ray determined structure, have a peculiar orientation towards each other: electron donors are broadside toward the acceptors and acceptors are edgeon toward donors. Vibronic coupling which is the mechanism of converting free-energy loss in electron transport to vibrational energy is examined as a possible explanation. Preliminary calculations do not support this as an explanation of the orientations but suggest strongly that the non-heme iron atom has the function of promoting vibronic coupling in the electron transfer from bacteriopheophytin to menaquinone. It is further suggested that the system of electron transport from the special pair of bacteriochlorophyll to the bacteriopheophytin is arranged to keep virbonic coupling to a minimum to match the very small electronic free-energy loss in this region.Abbreviations BC Bacteriochlorophyll - BP Bacteriopheophytin - BC₂ Bacteriochlorophyll special pair, primary electron donor - Fe Non-heme iron atom - MQ Menaquinone, first quinone acceptor - UQ Ubiquinone, second quinone acceptor     

Early Bacteriopheophytin Reduction in Charge Separation in Reaction Centers of Rhodobacter sphaeroides

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on B_A and H_A via the appearance of the B_A and H_A anion bands. We observed an unexpectedly early rise of the H_A⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between B_A and H_A, and the effect of protein conformation on the electron transfer rate.     

Effect of deuteration and cryosolvents on the energy transduction in primary processes of photosynthesis.

Effects of cryosolvents and D2O/H2O substitution on the reaction centres (RCs) isolated from photosynthetic bacteria were studied with respect to the role of intra-protein hydrogen bonds in the primary photosynthetic electron transfer. As a result of such treatment of RCs, the charge separation rate between the photoactive bacteriochlorophyll (P2 dimer) and bacteriopheophytin and the rate of electron transfer to the primary quinone slowed down. The energy migration rate from bacteriopheophytin (BPheM), inactive in electron transport, to P2 decreased as well. Although cryosolvents can shift the redox potential of the photoactive pigment, there is no direct correlation between the P2 potential and the effects of these modifying agents on the photosynthetic process in RCs occurring with participation of P2. The removal of H subunit from the pigment-protein complex results in the pronounced weakening of the dimethyl sulfoxide modifying effects on the RC hydrogen bonds. The role of structural and dynamic state in the functioning of the photosynthetic bacterial RCs is analyzed. Relaxation processes in purple bacteria RCs accompanying the primary picosecond steps of energy transformation proceed with the participation of small proton-containing molecular groups in the immediate surroundings of electron transfer carriers. In this paper, we present results concerning mechanisms of primary photosynthetic steps, which were initiated by A. A. Krasnovsky and have been studied for several years at the Department of Biophysics. This paper is dedicated to the memory of our teacher Prof. A. A. Krasnovsky.     

Mutant reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> I(L177)H with strongly bound bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>: Structural properties and pigment-protein interactions

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H _A ^? . The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P_A and P_B in the P⁺ state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P_A and P_B macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P⁺Q^?/PQ spectra suggests the 17³-ester group of the bacteriochlorophyll P_A to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B_A and the bacteriopheophytin H_A. Structural changes are observed in the monomer bacteriochlorophyll B_B binding site in the inactive chromophore branch of the mutant RCs.     

Properties of mutant reaction centers of Rhodobacter sphaeroides with substitutions of histidine L153, the axial Mg2+ ligand of bacteriochlorophyll BA

Mutant reaction centers (RC) from Rhodobacter sphaeroides have been studied in which histidine L153, the axial ligand of the central Mg atom of bacteriochlorophyll B_A molecule, was substituted by cysteine, methionine, tyrosine, or leucine. None of the mutations resulted in conversion of the bacteriochlorophyll B_A to a bacteriopheophytin molecule. Isolated H(L153)C and H(L153)M RCs demonstrated spectral properties similar to those of the wild-type RC, indicating the ability of cysteine and methionine to serve as stable axial ligands of the Mg atom of bacteriochlorophyll B_A. Because of instability of mutant H(L153)L and H(L153)Y RCs, their properties were studied without isolation of these complexes from the photosynthetic membranes. The most prominent effect of the mutations was observed with substitution of histidine by tyrosine. According to the spectral data and the results of pigment analysis, the B_A molecule is missing in the H(L153)Y RC. Nevertheless, being associated with the photosynthetic membrane, this RC can accomplish photochemical charge separation with quantum yield of approximately 7% of that characteristic of the wild-type RC. Possible pathways of the primary electron transport in the H(L153)Y RC in absence of photochemically active chromophore are discussed.     

Investigation into the source of electron transfer asymmetry in bacterial reaction centers

We have investigated the primary photochemistry of two symmetry-related mutants of Rhodobacter sphaeroides in which the histidine residues associated with the central Mg2+ ions of the two bacteriochlorophylls of the dimeric primary electron donor (His-L173 and His-M202) have been changed to leucine, affording bacteriochlorophyll (BChl)/bacteriopheophytin (BPh) heterodimers. Reaction centers (RCs) from the two mutants, (L)H173L and (M)H202L, have remarkably similar spectral and kinetic properties, although they are quite different from those of wild-type RCs. In both mutants, as in wild-type RCs, electron transfer to BPhL and not to BPhM is observed. These results suggest that asymmetry in the charge distribution of the excited BChl dimer (P*) in wild-type RCs (due to differing contributions of the two opposing intradimer charge-transfer states) contributes only modestly to the directionality of electron transfer. The results also suggest that differential orbital overlap of the two BChls of P with the chromophores on the L and M polypeptides does not contribute substantially to preferential electron transfer to BPhL.     

Cooperative polymerization of photosynthetic pigments in formamide-water solution

The aggregation of bacteriochlorophyll a and bacteriopheophytin a into large oligomers with maximum optical absorption at 860 nm was studied in a 3:1 (vol/vol) formamide/water solution, using optical absorption spectroscopy and electron microscopy. The aggregation is cooperative and proceeds according to two equilibrium constants. Initially, two pigment molecules form a “seed” that absorbs at ≈860 nm. The equilibrium constant, K_a, governing this reaction equals 1.3 × 10³ M^-1 in the case of bacteriochlorophyll a (due to experimental limitations, K_a for bacteriopheophytin a could not be determined). The addition of monomers to aggregates consisting of two or more units is governed by an equilibrium constant, K_b, equal to 2.2 × 10⁶ M^-1 for bacteriochlorophyll a and ≈ 10⁹ M^-1 for bacteriopheophytin a. The enthalpy and entropy changes that drive the bacteriochlorophyll oligomer formation are -9.25 and ≈0.0 kcal/mol, respectively. Above a threshold concentration, the amount of oligomers remains constant but their length continues to increase. Each oligomer appears to consist of dimers that are associated by hydrophobic interactions among their alcohol residues, forming long strands. Single strands presumably coil into helices that are seen as cylinders. The bacteriochlorophyll a oligomers form cylinders with a constant diameter of 150 Å and an average length of 2,000 Å (at 1.5 × 10^-5 M bacteriochlorophyll a). These cylinders contain 200-250 bacteriochlorophyll a dimers. The bacteriopheophytin oligomers coil into wider cylinders (≈400 Å in diameter) which contain ≈600-700 bacteriopheophytin a dimers. In both cases, the separation between the dimers is ≈20 Å. At such distances, the dipolar interactions among adjacent dimers are negligible and do not affect the optical absorption of each individual pair. Therefore, the optical absorption of these pairs can be a tool for investigating the absorption pattern of photosynthetic pigments in vivo.     

Femtosecond spectroscopy of primary charge separation in modified reaction centers of Rhodobacter sphaeroides (R-26)

Femtosecond measurements of kinetics and spectra of absorbance changes (ΔA) were carried out with modified reaction centers (RCs) from Rhodobacter sphaeroides (R-26) from which nonactive bacteriochlorophyll BM (located in the M protein subunit) was removed. The band of BM at 800 nm in native RCs is shifted in femtosecond measurements and obscures the ΔA of active bacteriochlorophyll BL (L subunit). The spectrum of ΔA in modified RCs at 6 ps delay includes the bleachings of the bands of P (primary electron donor) at 870 nm, of BL at 805 nm and of HL (bacteriopheophytin located in the L subunit) at 755 nm showing the reduction of 0̃.5 mol BL and 0̃.5 mol HL per mol P⁺. These data confirm an earlier suggestion that BL participates as an electron acceptor in the light-induced primary charge separation and agree with recent X-ray analysis of Rhodopseudomonas viridis and R. sphaeroides RCs which shows a location of BL between P and HL.     

Structural and functional studies on the tetraheme cytochrome subunit and its electron donor proteins: the possible docking mechanisms during the electron transfer reaction

The photosynthetic reaction centers (RCs) classified as the group II possess a peripheral cytochrome (Cyt) subunit, which serves as the electron mediator to the special-pair. In the cycle of the photosynthetic electron transfer reactions, the Cyt subunit accepts electrons from soluble electron carrier proteins, and re-reduces the photo-oxidized special-pair of the bacteriochlorophyll. Physiologically, high-potential cytochromes such as the cytochrome c₂ and the high-potential iron–sulfur protein (HiPIP) function as the electron donors to the Cyt subunit. Most of the Cyt subunits possess four heme c groups, and it was unclear which heme group first accepts the electron from the electron donor. The most distal heme to the special-pair, the heme-1, has a lower redox potential than the electron donors, which makes it difficult to understand the electron transfer mechanism mediated by the Cyt subunit. Extensive mutagenesis combined with kinetic studies has made a great contribution to our understanding of the molecular interaction mechanisms, and has demonstrated the importance of the region close to the heme-1 in the electron transfer. Moreover, crystallographic studies have elucidated two high-resolution three-dimensional structures for the RCs containing the Cyt subunit, the Blastochloris viridis and Thermochromatium tepidum RCs, as well as the structures of their electron donors. An examination of the structural data also suggested that the binding sites for both the cytochrome c₂ and the HiPIP are located adjacent to the solvent-accessible edge of the heme-1. In addition, it is also indicated by the structural and biochemical data that the cytochrome c₂ and the HiPIP dock with the Cyt subunit by different mechanisms although the two electron donors utilize the same region for the interactions; cytochrome c₂ is recognized through electrostatic interactions while hydrophobic interactions are important in the HiPIP docking.     

Modulation of the Primary Electron Transfer Rate in Photosynthetic Reaction Centers by Reduction of a Secondary Acceptor

Photosynthetic application of picosecond spectroscopic techniques to bacterial reaction centers has led to a much greater understanding of the chemical nature of the initial steps of photosynthesis. Within 10 ps after excitation, a charge transfer complex is formed between the primary donor, a “special pair” of bacteriochlorophyll molecules, and a transient acceptor involving bacteriopheophytin. This complex subsequently decays in about 120 ps by donating the electron to a metastable acceptor, a tightly bound quinone.

Recent experiments with conventional optical and ESR techniques have shown that when reaction centers are illuminated by a series of single turnover flashes in the presence of excess electron donors and acceptors, a stable, anionic ubisemiquinone is formed on odd flashes and destroyed on even flashes, suggesting that the acceptor region contains a second quinone that acts as a two-electron gate between the reaction center and subsequent electron transport events involving the quinone pool.

Utilizing standard picosecond techniques, we have examined the decay of the charge transfer complex in reaction centers in the presence of the stable semiquinone, formed by flash illumination with a dye laser 10 s before excitation by a picosecond pulse. In this state the decay rate for the charge transfer complex is considerably slower than when no electron is present in the quinone acceptor region. This indicates fairly strong coupling between constituents of the reaction center-quinone acceptor complex and may provide a probe into the relative positions of the various components.

     

Hybrid complexes of photosynthetic reaction centers and quantum dots in various matrices: resistance to UV irradiation and heating

The effects of ultraviolet (UV) irradiation (up to 0.6 J/cm²) and heating (65 °C, 20 min) on the absorption spectra and electron transfer in dehydrated film samples of photosynthetic reaction centers (RCs) from purple bacterium Rhodobacter (Rb.) sphaeroides, as well as in hybrid structures consisting of RCs and quantum dots (QDs), have been studied. The samples were placed in organic matrices containing the stabilizers of protein structure—polyvinyl alcohol (PVA) and trehalose. UV irradiation led to partially irreversible oxidation of some RCs, as well as to transformation of some fraction of the bacteriochlorophyll (BChl) molecules into bacteriopheophytin (BPheo) molecules. In addition, UV irradiation causes degradation of some BChl molecules that is accompanied by formation of 3-acetyl-chlorophyll a molecules. Finally, UV irradiation destroys the RCs carotenoid molecules. The incorporation of RCs into organic matrices reduced pheophytinization. Trehalose was especially efficient in reducing the damage to the carotenoid and BChl molecules caused by UV irradiation. Hybrid films containing RC?+?QD were more stable to pheophytinization upon UV irradiation. However, the presence of QDs in films did not affect the processes of carotenoid destruction. The efficiency of the electronic excitation energy transfer from QD to P865 also did not change under UV irradiation. Heating led to dramatic destruction of the RCs structure and bacteriochlorins acquired the properties of unbound molecules. Trehalose provided strong protection against destruction of the RCs and hybrid (RC?+?QD) complexes.

     

X-ray structure analysis of a membrane protein complex. Electron density map at 3 A resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis

X-ray analysis of three-dimensional crystals of the photosynthetic reaction center from the purple bacterium Rhodopseudomonas viridis led to an electron density distribution at 3 A resolution calculated with phases from multiple isomorphous replacement. The protein subunits of the complex were identified. An atomic model of the prosthetic groups of the reaction center complex (4 bacteriochlorophyll b, 2 bacteriopheophytin b. 1 non-heme iron, 1 menaquinone, 4 heme groups) was built. The arrangement of the ring systems of the bacteriochlorophyll b and bacteriopheophytin b molecules shows a local 2-fold rotation symmetry; two bacteriochlorophyll b form a closely associated, non-covalently linked dimer ("special pair"). A different local 2-fold symmetry axis is observed for the heme groups of the cytochrome part.     

Wave packet motions coupled to electron transfer in reaction centers of Chloroflexus aurantiacus

Transient absorption difference spectroscopy with ~20 femtosecond (fs) resolution was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of Chloroflexus (C.) aurantiacus. In RCs, the composition of the B-branch chromophores is different with respect to that of purple bacterial RCs by occupying the B(B) binding site of accessory bacteriochlorophyll by bacteriopheophytin molecule (Phi(B)). It was found that the nuclear wave packet motion induced on the potential energy surface of the excited state of the primary electron donor P* by ~20 fs excitation leads to a coherent formation of the states $P;{+}\Phi_{\rm B};{-}$ and $P;{+}B_{\rm A};{-}$ (B(A) is a bacteriochlorophyll monomer in the A-branch of cofactors). The processes were studied by measuring coherent oscillations in kinetics of the absorbance changes at 900 nm and 940 nm (P* stimulated emission), at 750 nm and 785 nm (Phi(B) absorption bands), and at 1,020-1028 nm ($B_{\rm A};{-}$ absorption band). In RCs, the immediate bleaching of the P band at 880 nm and the appearance of the stimulated wave packet emission at 900 nm were accompanied (with a small delay of 10-20 fs) by electron transfer from P* to the B-branch with bleaching of the Phi(B) absorption band at 785 nm due to $\Phi_{\rm B};{-}$ formation. These data are consistent with recent measurements for the mutant HM182L Rb. sphaeroides RCs (Yakovlev et al., Biochim Biophys Acta 1757:369-379, 2006). Only at a delay of 120 fs was the electron transfer from P* to the A-branch observed with a development of the $B_{\rm A};{-}$ absorption band at 1028 nm. This development was in phase with the appearance of the P* stimulated emission at 940 nm. The data on the A-branch electron transfer in C. aurantiacus RCs are consistent with those observed in native RCs of Rb. sphaeroides. The mechanism of charge separation in RCs with the modified B-branch pigment composition is discussed in terms of coupling between the nuclear wave packet motion and electron transfer from P* to Phi(B) and B(A) primary acceptors in the B-branch and A-branch, respectively.     

Isolation and pigment composition of the reaction centers from purple photosynthetic bacterium Rhodopseudomonas palustris species

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.     

The photochemical reaction center of the bacteriochlorophyll b-containing organism Thiocapsa pfennigii

A photochemical reaction-center preparation has been made from a second bacteriochlorophyll b-containing organism, Thiocapsa pfennigii. The reaction-center unit is thought to be composed of one P-960, four bacteriochlorophyll, two bacteriopheophytin, one carotenoid molecules and polypeptides of M_r 40000, 37000, 34000, 27000 and 26000 probably plus quinones and metal atoms. The preparation also contains a low-potential cytochrome c-555 and a high-potential cytochrome c-557 bound to the reaction center in a 3–4:2–3:1 molar ratio with respect to P-960. The 40 kDa subunit is associated with the cytochromes, while the 37, 34 and 27 + 26 kDa subunits are proposed to be equivalent to the H, M and L polypeptides of bacteriochlorophyll a-containing reaction centers. The cytochromes are oxidized by P-960⁺. The three near-infrared absorption bands at 788, 840 and 968 nm are assigned to bacteriopheophytin, bacteriochlorophyll and the primary donor (P-960), respectively. The 778 nm peak resolves into two at 77 K; no further resolution of the other two peaks occurs. Illumination of the sodium dithionite-reduced reaction centers at 77 K by 960 nm-light results in P-960, transferring one electron from cytochrome c-555 mainly to a bacteriopheophytin molecule, absorbing at 781 nm. A similar treatment at room temperatures reduces most of the two bacteriopheophytin molecules. It is argued that both bacteriopheophytin molecules, possibly with some contribution from bacteriochlorophyll, form an intermediary electron-carrier complex between P-960 and a quinone in T. pfennigii. We could not substantiate that a bacteriochlorophyll molecule precedes the bacteriopheophytins in the electron transfer sequence. Although the biochemical characteristics of the reaction center are very similar to those of the other known bacterioclorophyll b-containing reaction center, that from Rhodopseudomonas viridis, their spectral characteristics are not. This has helped elucidate more about the function of each spectral form and led us to conclude that the 850 nm form in Rps. viridis is not the higher energy transition of the special pair of bacteriochlorophyll molecules forming P-960. Laser-flash-in-duced absorbance changes in T. pfennigii reaction-center preparation should now lead to a more complete understanding of the mechanism of the primary photochemical event.     

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)₂ and (BChl g′)₂ in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A ₀, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I.     

Relationship between altered structure and photochemistry in mutant reaction centers in which bacteriochlorophyll replaces the photoactive bacteriopheophytin

Qy-excitation resonance Raman (RR) spectra are reported for two mutant reaction centers (RCs) from Rhodobacter capsulatus in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated beta. The pigment change in both mutants is induced via introduction of a histidine residue near the photoactive cofactor. In one mutant, L(M212)H, the histidine is positioned over the core of the cofactor and serves as an axial ligand to the Mg+2 ion. In the other mutant, F(L121)H/F(L97)V, the histidine is positioned over ring V of the cofactor, which is nominally too distant to permit bonding to the Mg+2 ion. The salient observations are as follows: (1) The beta cofactor in F(L121)H/F(L97)V RCs is a five-coordinate BChl molecule. However, there is no evidence for the formation of a Mg-His bond. This bond is either much weaker than in the L(M212)H RCs or completely absent, the latter implying coordination by an alternative ligand. The different axial ligation for beta in the F(L121)H/F(L97)V versus L(M212)H RCs in turn leads to different conformations of the BChl macrocycles. (2) The C9-keto group of beta in F(L121)H/F(L97)V RCs is free of hydrogen bonding interactions, unlike the L(M212)H RCs in which the C9-keto of beta is hydrogen bonded to Glu L104. The interactions between other peripheral substituents of beta and the protein are also different in the F(L121)H/F(L97)V RCs versus L(M212)H RCs. Accordingly, the position and orientation of beta in the protein is different in the two beta-containing RCs. Nonetheless, previous studies have shown that the primary electron transfer reactions are very similar in the two mutants but differ in significant respects compared to wild-type RCs. Collectively, these observations indicate that changes in the conformation of a photoactive tetrapyrrole macrocycle or its interactions with the protein do not necessarily lead to significantly perturbed photochemistry and do not underlie the altered primary events in beta-type RCs.     

The effect of exchange of bacteriopheophytin a with plant pheophytin a on charge separation in Y(M210)W mutant reaction centers of Rhodobacter sphaeroides at low temperature

The bacteriopheophytin a molecules at the H(A) and H(B) binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H(A)(-), presumably by raising the free energy level of the P(+)B(A)(-) state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B(A). Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H(A), inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P(+)B(B)(-) state, where B(B) is the accessory bacteriochlorophyll in the "inactive" B-branch of the cofactors. The B(B) molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q(y) region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P(+)B(B)(-) state and absorption properties of the B(B) bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the B(B) binding pocket.     

Temperature dependence of the primary electron transfer reaction in pigment-modified bacterial reaction centers

The initial electron transfer steps in pigment modified reaction centers, where bacteriopheophytin is replaced by plant pheophytin (R26.Phe-a RCs) have been investigated over a wide temperature range by femtosecond time-resolved spectroscopy. The experimental data obtained in the maximum of the bacteriochlorophyll anion band at 1020 nm show the existence of a high and long-lived population of the primary acceptor P⁺B_A ^– even at 10 K. The data suggest a stepwise electron transfer mechanism with B_A as primary acceptor also in the low temperature domain. A detailed data analysis suggests that the pigment modification leads to a situation with almost isoenergetic primary and secondary acceptor levels, approximately 450 cm^–1 below P^*. A Gaussian distribution (with = 400 cm ^–1) of the G values has to be assumed to account for the strong dispersive character of the kinetics in this sample. Based on these assumptions, a model is presented that reproduces the observed kinetics, heterogeneity and temperature dependence.     

Orientation of reaction center and antenna chromophores in the photosynthetic membrane of rhodopseudomonas viridis

Whole cells of Rhodopseudomonas viridis were oriented in a magnetic field. The degree of orientation of the cells was determined by using a photoselection technique. In order to deduce the orientation of the antennae and chromophores of the reaction centers with respect to the membrane plane, we performed linear dichroism measurements of absolute spectra and light induced difference spectra linked to states P⁺I and PI^? on oriented cells. These measurements lead to the following conclusions:The antennae bacteriochlorophyll molecular plane is nearly perpendicular to the membrane. The Q_y and Q_x transitions moments of these molecules make respectively angles of 20 and 70°ith the membrane plane. The antenna carotenoid molecules make an angle of 45°ith the membrane.The primary electron donor possesses two transition moments centered respectively at 970 and 850 nm. The 970 nm transition moment is parallel to the membrane plane, the 850 nm transition is tilted out of the plane. Upon photooxidation of this primary electron donor, a monomer-like absorption band appears at 805 nm. Its transition makes an angle smaller than 25° with the membrane. The photooxidation of the dimer also induces an absorption band shift for the two other bacteriochlorophyll molecules of the reaction center. The absorption band shifts of the two bacteriochlorophyll molecules occur in opposite direction.One bacteriopheophytin molecule is photoreduced in state PI^?. This photoreduction induces an absorption band shift for only one bacteriochlorophyll molecule. Finally, the geometry of the dimeric primary donor seems to be affected by the presence of a negative charge in the reaction center.