Rhodopseudomonas Viridis Reaction Center Research Articles

Iron(II) carboxylate complexes based on a tetraimidazole ligand as models of the photosynthetic non-heme ferrous sites: synthesis, crystal structure, and Mössbauer and magnetic studies.

The preparations, X-ray structures, and detailed physical characterization are presented for new complexes involving an iron(II) center, a tetraimidazole ligand (TIM), and different carboxylates. [Fe(TIM)(C(6)H(5)CH(2)CO(2))](ClO(4)) (1) crystallizes in the Pbca space group with a = 10.8947(13), b = 20.343(2), and c = 22.833(3) A, Z = 8, and V = 5060.6(11) A(3). [Fe(TIM)(CH(3)CO(2))](ClO(4)) (2) crystallizes in the Ia space group with a = 17.117(2), b = 10.3358(12), and c = 25.658(3) A, beta = 90.301(13) degrees, Z = 8, and V = 4539.5(9) A(3). In both structures, the iron(II) is hexacoordinated to the four N(imidazole) donors of the TIM ligand and the two O donors of a bidentate carboxylate. The flexibility of the carboxylate bidentate coordination, symmetrical or more or less asymmetrical, associated with the steric demand of the TIM ligand results in a remarkable versatility of the Fe(II)N(4)O(2) coordination geometry. The diversity in carboxylate bidentate coordination modes has allowed us to clearly show the importance of the structural and electronic effects, through IR and Mössbauer spectroscopy, of this apparently tenuous carboxylate shift. Comparison of the structural and Mössbauer properties of these complexes with the non-heme ferrous site of photosynthetic systems (i) shows that the metric parameters of site 2b, including the symmetrically chelated bidentate carboxylate, are closer to those of the non-heme ferrous site in the bacterial reaction centers of Rhodopseudomonas viridis and R. sphaeroides and (ii) suggests that the ligand environment of the non-heme ferrous center of PS 2 is close to the axially distorted octahedral symmetry resulting from an asymmetrical bidentate coordination of the -CO(2) motif, as in complex 1.

Recombination reduction of photooxidized cytochrome c in reaction centers of Rhodopseudomonas viridis at low temperature.

The temperature dependence of dark reduction of photooxidized cytochrome c was studied in isolated preparations of Rhodopseudomonas viridis reaction centers. Within the range from room temperature to approximately 260 K this process was found to be mediated by thermal diffusion of exogenous donor molecules, whereas at lower temperatures photooxidized cytochrome is reduced as a result of indirect recombination with photoreduced primary quinone acceptor. Kinetic simulation allowed certain thermodynamic characteristics of this reaction to be calculated. To the first approximation, these characteristics correlate with the estimates obtained from the results of direct redox titration.

Structural Basis of the Drastically Increased Initial Electron Transfer Rate in the Reaction Center from a Rhodopseudomonas viridis Mutant Described at 2.00-Å Resolution

It has previously been shown that replacement of the residue His L168 with Phe (HL168F) in the Rhodopseudomonas viridis reaction center (RC) leads to an unprecedented drastic acceleration of the initial electron transfer rate. Here we describe the determination of the x-ray crystal structure at 2.00-A resolution of the HL168F RC. The electron density maps confirm that a hydrogen bond from the protein to the special pair is removed by this mutation. Compared with the wild-type RC, the acceptor of this hydrogen bond, the ring I acetyl group of the "special pair" bacteriochlorophyll, D(L), is rotated, and its acetyl oxygen is found 1.1 A closer to the bacteriochlorophyll-Mg(2+) of the other special pair bacteriochlorophyll, D(M). The rotation of this acetyl group and the increased interaction between the D(L) ring I acetyl oxygen and the D(M)-Mg(2+) provide the structural basis for the previously observed 80-mV decrease in the D(+)/D redox potential and the drastically increased rate of initial electron transfer to the accessory bacteriochlorophyll, B(A). The high quality of the electron density maps also allowed a reliable discussion of the mode of binding of the triazine herbicide terbutryn at the binding site of the secondary quinone, Q(B).

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].

Conformational heterogeneity of the bacteriopheophytin electron acceptor HA in reaction centers from Rhodopseudomonas viridis revealed by Fourier transform infrared spectroscopy and site-directed mutagenesis.

The light-induced Fourier transform infrared (FTIR) difference spectra corresponding to the photoreduction of either the HA bacteriopheophytin electron acceptor (HA-/HA spectrum) or the QA primary quinone (QA-/QA spectrum) in photosynthetic reaction centers (RCs) of Rhodopseudomonas viridis are reported. These spectra have been compared for wild-type (WT) RCs and for two site-directed mutants in which the proposed interactions between the carbonyls on ring V of HA and the RC protein have been altered. In the mutant EQ(L104), the putative hydrogen bond between the protein and the 9-keto C=O of HA should be affected by changing Glu L104 to a Gln. In the mutant WF(M250), the van der Waals interactions between Trp M250 and the 10a-ester C=O of HA should be modified. The characteristic effects of both mutations on the FTIR spectra support the proposed interactions and allow the IR modes of the 9-keto and 10a-ester C=O of HA and HA- to be assigned. Comparison of the HA-/HA and QA-/QA spectra leads us to conclude that the QA-/QA IR signals in the spectral range above 1700 cm-1 are largely dominated by contributions from the electrostatic response of the 10a-ester C=O mode of HA upon QA photoreduction. A heterogeneity in the conformation of the 10a-ester C=O mode of HA in WT RCs, leading to three distinct populations of HA, appears to be related to differences in the hydrogen-bonding interactions between the carbonyls of ring V of HA and the RC protein. The possibility that this structural heterogeneity is related to the observed multiexponential kinetics of electron transfer and the implications for primary processes are discussed. The effect of 1H/2H exchange on the QA-/QA spectra of the WT and mutant RCs shows that neither Glu L104 nor any other exchangeable carboxylic residue changes appreciably its protonation state upon QA reduction.

Modeling the Bacterial Photosynthetic Reaction Center. 2. A Combined Quantum Mechanical/Molecular Mechanical Study of the Structure of the Cofactors in the Reaction Centers of Purple Bacteria

Ab initio and other computational studies of bacterial reaction center cofactors are usually performed using the observed (low-resolution) X-ray structures. Unfortunately, these geometries are necessarily approximate and this can have dramatic influences on calculated properties. For example, the calculated energies of the four bacteriochlorophylls in Rhodobacter sphaeroides vary by over 160 kcal mol-1. To overcome this problem, a combined quantum mechanical/molecular mechanical (QM/MM) method is employed to optimize the structure of the special pair and other cofactors in the photosynthetic reaction centers of Rhodopseudomonas viridis and Rhodobacter sphaeroides, while a purely MM model is used to refine the structure of the remaining protein environment. Specifically, the QM/MM optimizations are performed using a semiempirical AM1-based formalism. After relaxation, the energies of the bacteriochlorophylls differ by only typical conformer relative energies, ca. 5 kcal mol-1. Another example of improved c...

Charge Separation in Photosynthetic Reaction Centers

Structural influences on the direction of electron transfer in the charge separation process of the photosynthetic reaction centers of Rhodopseudomonas viridis and Rhodobacter sphaeroides are studied using quantum chemical models to calculate the electronic factor. Our results support the sequential mechanism for the primary charge separation. Using crystallographic coordinates refined in 1994, we find a larger coupling between the special pair D and the accessory bacteriochlorophyll BA of the A branch than between D and BB of the inactive B branch. We have been able to localize the coupling to the acetyl group of ring I of DB and the methyl group of ring III of BA. The corresponding contact between DA and BB has less coupling, apparently due to a distorting hydrogen bond between the acetyl group on DA and the imidazole side group of HisL168. The coupling between BA and bacteriopheophytin ΦA is accomplished by two methyl groups, directly connected to the conjugated π system of the chromophore.

Efficient exchange of the primary quinone acceptor Q(A) in isolated reaction centers of Rhodopseudomonas viridis.

A key step in the conversion of solar energy into chemical energy by photosynthetic reaction centers (RCs) occurs at the level of the two quinones, Q(A) and Q(B), where electron transfer couples to proton transfer. A great deal of our understanding of the mechanisms of these coupled reactions relies on the seminal work of Okamura et al. [Okamura, M. Y., Isaacson, R. A., & Feher, G. (1975) Proc. Natl. Acad. Sci. USA 88, 3491-3495], who were able to extract with detergents the firmly bound ubiquinone Q(A) from the RC of Rhodobacter sphaeroides and reconstitute the site with extraneous quinones. Up to now a comparable protocol was lacking for the RC of Rhodopseudomonas viridis despite the fact that its Q(A) site, which contains 2-methyl-3-nonaprenyl-1,4-naphthoquinone (menaquinone-9), has provided the best x-ray structure available. Fourier transform infrared difference spectroscopy, together with the use of isotopically labeled quinones, can probe the interaction of Q(A) with the RC protein. We establish that a simple incubation procedure of isolated RCs of Rp. viridis with an excess of extraneous quinone allows the menaquinone-9 in the Q(A) site to be almost quantitatively replaced either by vitamin K(1), a close analogue of menaquinone-9, or by ubiquinone. To our knowledge, this is the first report of quinone exchange in bacterial photosynthesis. The Fourier transform infrared data on the quinone and semiquinone vibrations show a close similarity in the bonding interactions of vitamin K(1) with the protein at the Q(A) site of Rp. viridis and Rb. sphaeroides, whereas for ubiquinone these interactions are significantly different. The results are interpreted in terms of slightly inequivalent quinone-protein interactions by comparison with the crystallographic data available for the Q(A) site of the two RCs.

Primary charge separation. The primary processes of bacterial photosynthesis — ultrafast reactions for the optimum use of light energy

AbstractFemtosecond spectroscopy is used to study the molecular mechanisms of the primary electron transfer. Data on native and mutated reaction centers of Rhodopseudomonas viridis show, that primary electron transfer is an ultrafast stepwise reaction. The electron is transferred via a chain of pigments: In a first reaction step the electron is transported from the special pair to the accessory bacteriochlorophyll with a time constant of ≈ 3 ps. A second, faster reaction carries the electron with 0.65 ps to a bacteriopheophytin. Experiments on mutated reaction centers with strongly modified reaction times yield additional information on energetics, reorganisation energies and electronic coupling of the reaction center. A consistent theoretical treatment of the data shows that standard non‐adiabatic theory describes well the primary electron transfer process. It also reveals that the realised reaction parameters optimise the reaction centers for highest possible quantum yield.

Electron Transfer from the Tetraheme Cytochrome to the Special Pair in the Rhodopseudomonas viridis Reaction Center: Effect of Mutations of Tyrosine L162

The structure of the photosynthetic reaction center (RC) from Rhodopseudomonas viridis is known to high resolution. It contains a firmly bound tetraheme cytochrome from which electrons are donated to a special pair (P) of bacteriochlorophylls, which is photooxidized upon absorption of light. Tyrosine at position 162 of the L-subunit of the reaction center (L 162 Y) is a highly conserved residue positioned halfway between P and the proximal heme group (c-559) of the cytochrome. By specific mutagenesis this residue was exchanged against the amino acids phenylalanine (F), glycine (G), methionine (M), leucine (L), tryptophan (W), threonine (T), and histidine (H). All mutants were expressed in Rps. viridis using a recently established transformation system [Laussermair & Oesterhelt (1992) EMBO J. 11, 777-783]. They were shown biochemically to synthesize all four subunits of the RC (cytochrome, subunits L, M, and H) and to assemble them correctly into the membrane. The structures of two mutants (L 162 F and L 162 T) were determined and found not to differ significantly from the wild-type structure. All mutants grew photosynthetically. The absorption spectrum of all the mutants is the same as in WT, but the redox potential of P and of c-559 was changed by the mutations. The kinetics of electron transfer from the heme group to the special pair were measured in chromatophores by flash absorption. As found earlier in the wild type (Y) several exponential components were needed to fit the data.(ABSTRACT TRUNCATED AT 250 WORDS)

The Nature of the Near-Infrared Electronic Absorption at 1250 nm in the Spectra of the Radical Cations of the Special Pairs in the Photosynthetic Reaction Centers of Rhodobacter sphaeroides and Rhodopseudomonas Viridis

The Nature of the Near-Infrared Electronic Absorption at 1250 nm in the Spectra of the Radical Cations of the Special Pairs in the Photosynthetic Reaction Centers of Rhodobacter sphaeroides and Rhodopseudomonas Viridis

The peculiarities of the electron transfer from cytochrome to the cation-radical of bacteriochlorophyll dimer in reaction centers from Rps. viridis

The kinetics of tunnel electron transitions in reaction centers of photosynthetic bacteria Rhodopseudomonas viridis are characterized by paradoxical peculiarities. (1) The time of the high-temperature (300 K) transfer of an electron to the cation-radical of bacteriochlorophyll BC1 + 2 from the neighbour heme C 380 is almost twice as long as that from the almost twice as distant heme C 20. (2) At low temperature (77 K) the majority (≈ 80%) of nearby hemes C 380 is unable to reduce BCl 2 + while all remote hemes C 20 participate effectively in such reductions as though they disconnect C 380 hemes. Both these peculiarities find their explanation on the basis of the mechanism of two-electron relay tunneling: C 380→ e−BCl 2 +, C 20→ e−C 380 +.

Time-resolved spectroscopy of the excited electronic state of reaction centers of Rhodopseudomonas viridis

The spectral properties of the excited electronic state of the reaction centers of Rhodopseudomonas (Rps.) viridis are studied by dichroic transient absorption spectroscopy with sub-picosecond time resolution. The theoretical analysis of the experimental results allows the assignment of the transient absorption from two dimer bands of the special pair and show its excitonic coupling to other pigments.

Temperature-dependent electron spin polarization of the triplet state of the primary donor in Rhodopseudomonas viridis

The electron spin polarization (ESP) of the triplet of the primary donor ( 3P) in reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis shows an anisotropic temperature dependence (Van Wijk, F.G.H. and Schaafsma, T.J. (1988) Biochim. Biophys. Acta 936, 236). The reported inversion of the initial electron spin polarization (IESP) for the canonical Y-direction of 3P at 100 K has been explained by means of coherent S-T ± mixing in the radical pair, due to a fast relaxing electron spin on the iron-quinone acceptor complex X (Hore, P.J., Hunter, D.A., Van Wijk, F.G.H., Schaafsma, T.J. and Hoff, A.J. (1988) Biochim. Biophys. Acta 936, 249). Using direct-detection EPR, we show that at 100 K the IESP in randomly oriented samples is not inverted for the canonical Y-direction of 3P. Furthermore, in single crystals the IESP at 100 K is shown to be almost zero for the complete YZ-plane of 3P. Since X ·− shows a strong g-anisotropy, the model of Hore et al., in which polarization-inversion only occurs when the effective g-value of X ·− is around g = 2.00, is inadequate to explain the temperature-dependent changes of the IESP. Therefore, we conclude that anisotropic fast spin-lattice relaxation in the radical pair triplet state is the origin of the temperature dependence of the ESP. The inversion for the canonical Y-direction under continuous illumination is the result of the interplay of spin-lattice relaxation in 3P and its triplet decay rates, in combination with changes in the IESP.

Fourier transform infrared spectroscopy and electrochemistry of the primary electron donor in Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers: Vibrational modes of the pigments in situ and evidence for protein and water modes affected by P+ formation

Protein electrochemistry in an ultra-thin-layer electrochemical cell suitable for UV/vis and IR spectroscopy has been used to characterize the vibrational modes of the primary electron donors of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers in their neutral and cation radical states (P and P+, respectively). The P-->P+ redox transitions could be well separated from redox reactions of other cofactors according to their redox midpoint potential. The IR difference bands of the primary electron donor bacteriochlorophylls all titrate in unison and exhibit the correct midpoint potential. Comparison of the difference spectra with those of isolated bacteriochlorophylls a and b in organic solvents of different polarity and proton activity [Mäntele, W., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472] leads to similar conclusions on the binding and interaction of the pigments within the protein matrix as previously obtained from light-induced P+Q-/PQ difference spectra. Equilibration of the reaction centers in D2O leads to few but distinct shifts of bands and changes of band intensities at 1662, 1634, and 1526 cm-1 (Rhodobacter sphaeroides) and 1694, 1664, 1648, 1630, and 1532 cm-1 (Rhodopseudomonas viridis) as well as to smaller deviations at other wavenumbers. The H-->D-sensitive band at 1662 cm-1 is interpreted in terms of a histidine NH2+ bending mode. A second H/D-sensitive difference band around 1648 cm-1 in the Rhodopseudomonas viridis reaction center may be associated with the peptide C = O of one of the amino acids surrounding P [eventually of the histidine(s) ligating the Mg] which is affected by P+ formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron transfer from the tetraheme cytochrome to the special pair in isolated reaction centers of Rhodopseudomonas viridis

Kinetics of electron transfer from the bound tetraheme cytochrome c to the primary donor (P) have been measured in isolated reaction centers of the purple bacterium Rhodopseudomonas viridis by time-resolved flash absorption spectroscopy. The influence of two major parameters has been studied: temperature (7-305 K) and the redox state of the cytochrome. Most experiments were done with one heme (c-559), two hemes (c-559 and c-556), or three hemes (c-559, c-556, and c-552) poised in a reduced state before the flash. Measurements were done at 1283 nm in the absorption band of P+, and in the region of cytochrome alpha-bands. At room temperature, c-559 donates an electron to P+ with a half-time of 115, 190, or 230 ns (with three, two, or one heme reduced, respectively) and is then eventually rereduced by c-556 (t1/2 = 1.7 microseconds) or by c-552 (in less than 40 ns). The kinetics also include a minor microsecond phase of P+ reduction. At decreasing temperatures, the polyphasic character of P+ rereduction is accentuated. Fast phases (115 ns-10 microseconds) are slightly slowed down, following Arrhenius behavior with a weak activation energy (3.6-8.6 kJ.mol-1), until they become temperature-independent. Their extent decreases rather sharply, at temperatures which vary according to the redox poising: 250, 210, or 80 K when one, two, or three hemes are reduced, respectively. In the last case, P+ can still be reduced at low temperature, apparently directly by c-552 (t1/2 = 1.1 ms, nearly temperature-independent).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of pressure on charge separation in photosynthetic bacterial reaction centers of Rhodopseudomonas viridis

Reaction centers of Rhodopseudomonas viridis were studied as a function of pressure using picosecond difference absorption spectroscopy. High pressure increased the rate of electron transfer from P +H − LQ A to P +H LQ − A, measured by the decay of the transient optical absorption of H − L in the 650–700 nm wavelength range, from (181 ± 17 ps) −1 at 0.1 MPa to (127 ± 16 ps) −1 at 120 MPa. At 240 MPa the decay became biexponential with a fast component (198 ± 66 ps) −1 of 33% amplitude and a slow component (7.8 ± 1.4 ns) −1 of 67% amplitude. At 350 MPa the fast component was undetectable and only a slow exponential decay of (4.1 ± 0.3 ns) −1 was observed. The slow component is attributed to the charge recombination process P +H − LQ A → PH LQ A.

Second-site mutation at M43 (Asn?Asp) compensates for the loss of two acidic residues in the QB site of the reaction center

Two acidic residues, L212Glu and L213Asp, in the QB binding sites of the photosynthetic reaction centers of Rhodobacter capsulatus and Rhodobacter sphaeroides are thought to play central roles in the transfer of protons to the quinone anion(s) generated by photoinduced electron transfer. We constructed the site-specific double mutant L212Ala-L213Ala in R. capsulatus, that is incapable of growth under photosynthetic conditions. A photocompetent derivative of that strain has been isolated that carries the original L212Ala-L213Ala double mutation and a second-site suppressor mutation at residue M43 (Asn→Asp), outside of the QB binding site, that is solely responsible for restoring the photosynthetic phenotype. The Asp,Asn combination of residues at the L213 and M43 positions is conserved in the five species of photosynthetic bacteria whose reaction center sequences are known. In R. capsulatus and R. sphaeroides, the pair is L213Asp-M43Asn. But, the reaction centers of Rhodopseudomonas viridis, Rhodospirillum rubrum and Chloroflexus aurantiacus reverse the combination to L213Asn-M43Asp. In this respect, the QB site of the suppressor strain resembles that of the latter three species in that it couples an uncharged residue at L213 with an acidic residue at M43. These reaction centers, in which L213 is an amide, must employ an alternative proton transfer pathway. The observation that the M43Asn→Asp mutation in R. capsulatus compensates for the loss of both acidic residues at L212 and L213 suggests that M43Asp is involved in a new proton transfer route in this species that resembles the one normally used in reaction centers of Rps. virddis, Rsp. rubrum and C. aurantiacus.

Effect of temperature on the kinetics of electron transfer from the tetraheme cytochrome to the primary donor in Rhodopseudomonas viridis

The kinetics of electron transfer from the third highest potential heme (c-522, E m = +20 mV) to the primary donor (P-960) have been measured by flash absorption spectroscopy in isolated reaction centers of Rhodopseudomonas viridis between 300 K and 7 K. The data are analyzed on the basis of three exponential components with a very fast phase (? 1 2 = 120 ns) dominating at high temperature and a very slow one (? 1 2 = 1.2 ms) at low temperature. This multiphasic behavior is interpreted in terms of the existence of three states with a temperature-dependent population and a very limited effect of the temperature on the kinetics for each state.

Light-induced charge separation in Rhodopseudomonas viridis reaction centers monitored by Fourier-Transform Infrared difference spectroscopy: the quinone vibrations

Static FTIR light-induced difference spectra have been recorded for reaction centers from Rhodopseudomonas viridis in the following charge-separated states: P+QA(-)-PQA, P+QB(-)-PQB, I(-)-I, I-QA(-)-IQA, and I-QA(2-)-IQA. A comparison of the I(-)-I difference spectra with the I-QA(-)-IQA difference spectra reveals new bands which can be assigned to QA- vibrations; these vibrations are also observed in the P+QA(-)-PQA and P+QB(-)-PQB difference spectra. Through an analysis of all of the static difference spectra, the electron-transfer pathway can be monitored in the infrared from the primary donor, P, to the secondary acceptor, QB, via the intermediate acceptor, I, and the primary acceptor, QA. The difference spectra are dominated by absorbance changes of prosthetic groups, with very few identifiable contributions from amino acids and little overall structural change in the protein backbone, involving only one or two residues for the various charge-separated states. Oxidation of the primary donor in the reaction center shows the characteristic absorbance changes of the 9-keto and 10-ester carbonyl groups observed upon oxidation of bacteriochlorophyll b in a non-hydrogen-bonded environment [Ballschmiter, K. H., & Katz, J. J. (1969) J. Am. Chem. Soc. 91, 2661-2677]. Reduction of the quinones in the reaction center yields absorbance changes of the carbonyls observed during reduction of quinones in a hydrogen-bonded environment [Bauscher, M., Nabedryk, E., Bagley, K., Breton, J., & Mäntele, W. (1990) FEBS Lett. 261, 191-195].(ABSTRACT TRUNCATED AT 250 WORDS)