RESUMEN
Using absorption spectroscopy in the visible/near-IR and mid-IR regions, spectral and photochemical properties of isolated reaction centers (RCs) from Rhodobacter sphaeroides R-26 were studied in dried films on the inorganic support surface (quartz or CaF2 plates) under vacuum dehydration conditions (10-2 or 7·10-5 mm Hg). Three detergents, N,N-dimethyldodecylamine N-oxide (LDAO), Triton X-100 (TX100), and n-dodecyl-ß-D-maltoside (DM), were tested for their ability to stabilize the RC-detergent complexes in the vacuum-dried state. It was shown that in the presence of LDAO, RC complexes underwent destruction in vacuum. In contrast, DM provided an environment that minimized irreversible disruptive changes in the RCs in vacuum. The effects of vacuum dehydration on the RC-DM films included a small increase in the content of α-helices in the RC protein, a short-wavelength reversible shift in the optical transitions of pigments, and minor changes in the electronic structure of the P+ dimer. The films retained their photochemical activity upon excitation with high-intensity light (200 mW/cm2). TX100 also helped to maintain spectral and functional properties of the RCs in vacuum; however, in this case, the stabilizing effect was less pronounced than in the presence of DM, especially, at high detergent concentrations. The results are discussed within the framework of a model suggesting that the detergent-protein interactions and the properties of detergent micelles play a dominant role in maintaining the structure of the RCs upon vacuum dehydration of the RC complexes. The obtained data can be useful for developing hybrid photoconverting systems based on bacterial RCs.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Vacio , Procesos Fotoquímicos , Rhodobacter sphaeroides/aislamiento & purificación , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de SuperficieRESUMEN
In our recent X-ray study, we demonstrated that substitution of the natural leucine residue M196 with histidine in the reaction center (RC) from Rhodobacter (Rba.) sphaeroides leads to formation of a close contact between the genetically introduced histidine and the primary electron donor P (bacteriochlorophylls (BChls) PA and PB dimer) creating a novel pigment-protein interaction that is not observed in native RCs. In the present work, the possible nature of this novel interaction and its effects on the electronic properties of P and the photochemical charge separation in isolated mutant RCs L(M196)H are investigated at room temperature using steady-state absorption spectroscopy, light-induced difference FTIR spectroscopy, and femtosecond transient absorption spectroscopy. The results are compared with the data obtained for the RCs from Rba. sphaeroides pseudo-wild type strain. It is shown that the L(M196)H mutation results in a decrease in intensity and broadening of the long-wavelength Qy absorption band of P at ~865 nm. Due to the mutation, there is also weakening of the electronic coupling between BChls in the radical cation P+ and increase in the positive charge localization on the PA molecule. Despite the significant perturbations of the electronic structure of P, the mutant RCs retain high electron transfer rates and quantum yield of the P+QA- state (QA is the primary quinone acceptor), which is close to the one observed in the native RCs. Comparison of our results with the literature data suggests that the imidazole group of histidine M196 forms a π-hydrogen bond with the π-electron system of the PB molecule in the P dimer. It is likely that the specific (T-shaped) spatial organization of the π-hydrogen interaction and its potential heterogeneity in relation to the bonding energy is, at least partially, the reason that this type of interaction between the protein and the pigment and quinone cofactors is not realized in the native RCs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Histidina/metabolismo , Leucina/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Transporte de Electrón , Histidina/genética , Cinética , Leucina/genética , Mutagénesis Sitio-Dirigida , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Studying pigment-protein interactions in the photosynthetic reaction centers (RCs) is important for the understanding of detailed mechanisms of the photochemical process. This paper describes spectral and photochemical characteristics, pigment composition, and stability of the Rhodobacter sphaeroides RCs with the I(L177)Y and I(M206)Y amino acid substitutions. The obtained data are compared with the properties of I(L177)H, I(L177)D, and I(M206)H RCs reported previously. It is shown that the I(L177)Y and I(M206)Y mutations cause a similar shift of the QYP band in the absorption spectra of the mutant RCs and do not affect the distribution of the electron spin density within the photo-oxidized P+ dimer. The differences in the position and amplitude of the QYB band in the I(L177)Y and I(M206)Y RCs were determined. The results indicate the possibility of new pigment-protein interactions in the vicinity of monomeric bacteriochlorophylls in the A and B chains, which might be of interest for future research.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Mutagénesis Sitio-Dirigida , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Estructura Terciaria de Proteína , Rhodobacter sphaeroides/metabolismo , EspectrofotometríaRESUMEN
This review focuses on recent experimental data obtained by site-directed mutagenesis of the reaction center in purple nonsulfur bacteria. The role of axial ligation of (bacterio)chlorophylls in the regulation of spectral and redox properties of these pigments, as well as correlation between the structure of chromophores and nature of their ligands, are discussed. Cofactor ligation in various types of reaction centers is compared, and possible reasons for observed differences are examined in the light of modern ideas on the evolution of photosynthesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacterioclorofilas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteobacteria/metabolismo , Proteínas Bacterianas/genética , Bacterioclorofilas/química , Evolución Molecular , Ligandos , Mutagénesis Sitio-Dirigida , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Rhodobacter sphaeroides/metabolismoRESUMEN
An algorithm to extract kinetics of the ion radical bands from the strong absorption background in the transient absorption spectra of the Rhodobacter sphaeroides reaction centers upon femtosecond excitation of the primary electron donor is suggested. The rising kinetics of the transient absorption band at 1020 nm and the bleaching kinetics of the 545-nm band constructed using the proposed method are adequately fitted by the kinetic equations for sequential electron transfer from the excited primary donor to the BA (monomeric bacteriochlorophyll) molecule, and then to the HA (bacteriopheophytin serving as an electron acceptor) molecule with the rate constants of 3.5 ± 0.2 and 0.8 ± 0.1 ps, respectively. The kinetics of the bacteriochlorophyll absorption band at 600 nm shows both the ultrafast bleaching of the P870 dimer and slower bleaching of the BA monomer due to its transition to the anion radical. The plotted kinetics of the ion radical bands is in agreement with the concentration profiles of the charge-separated states produced by the global target analysis of experimental data using the model of sequential electron transfer in the reaction centers.
Asunto(s)
Algoritmos , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/química , CinéticaRESUMEN
In green photosynthetic bacteria, the chlorosomal bacteriochlorophyll molecules are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Despite numerous investigations, a consensus regarding the spatial structure of chlorosomal antennae has not yet been reached. For the first time, we demonstrated by coherent femtosecond spectroscopy at cryogenic temperature that the very low-frequency (~101â¯cm-1) vibrations of bacteriochlorophyll c pigments in isolated Chloroflexus aurantiacus chlorosomes are sensitive to their oligomerisation extent which depends on the light intensity during the growth of the cell cultures. We explained this sensitivity in terms of the coupling of delocalised vibration modes of BChl c molecules aggregated into chains within their antenna unit building blocks. These findings, together with previously obtained spectroscopy and microscopy data, confirmed that the unit building blocks functioning within Chloroflexus aurantiacus chlorosomal antenna are built up from the rather short (2-5 BChl c pigments) quasi-linear chains. The approach presented here seems to be perspective since it directly reveals structural and dynamical properties of the oligomeric systems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacterioclorofilas/metabolismo , Chloroflexus/metabolismo , Proteínas Bacterianas/química , Bacterioclorofilas/química , Cinética , Luz , Polimerizacion , Temperatura , VibraciónRESUMEN
Energy relaxation was studied with difference femtosecond spectroscopy in reaction centers of the YM210L mutant of the purple photosynthetic bacterium Rhodobacter sphaeroides at low temperature (90 K). A dynamical long-wavelength shift of stimulated emission of the excited state of the bacteriochlorophyll dimer P was found, which starts simultaneously with P* formation and is accompanied by a change in the spectral shape of this emission. The characteristic value of this shift was about 30 nm, and the characteristic time about 200 fs. Difference kinetics ΔA measured at fixed wavelengths demonstrate the femtosecond shift of the P* stimulated emission appearing as a dependence of these kinetics on wavelength. We found that the reported long-wavelength shift can be explained in terms of electron-vibrational relaxation of the P* excited state with time constants of vibrational and electronic relaxation of 100 and 50 fs, respectively. Alternative mechanisms of the dynamical shift of the P* stimulated emission spectrum are also discussed in terms of energy redistribution between vibrational modes or coherent excitation of the modes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas Bacterianas/genética , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Dimerización , Cinética , Láseres de Estado Sólido , Mutagénesis Sitio-Dirigida , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Rhodobacter sphaeroides/efectos de la radiación , EspectrofotometríaRESUMEN
In the absorption spectrum of Rhodobacter sphaeroides reaction centers, a minor absorption band was found with a maximum at 1053 nm. The amplitude of this band is ~10,000 times less and its half-width is comparable to that of the long-wavelength absorption band of the primary electron donor P870. When the primary electron donor is excited by femtosecond light pulses at 870 nm, the absorption band at 1053 nm is increased manifold during the earliest stages of charge separation. The growth of this absorption band in difference absorption spectra precedes the appearance of stimulated emission at 935 nm and the appearance of the absorption band of anion-radical BA- at 1020 nm, reported earlier by several researchers. When reaction centers are illuminated with 1064 nm light, the absorption spectrum undergoes changes indicating reduction of the primary electron acceptor QA, with the primary electron donor P870 remaining neutral. These photoinduced absorption changes reflect the formation of the long-lived radical state PBAHAQA-.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Fotosíntesis/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Transporte de Electrón/fisiologíaRESUMEN
A model of heme-quinone redox interaction has been developed for cytochrome b559 in photosystem II. The quinone QC in the singly protonated form may function as an interacting quinone. The electrostatic effect between the charges on the heme iron of the cytochrome and QCH leads to appearance of three forms of the cytochrome with different redox potentials. A simple and effective mechanism of redox regulation of the electron transfer pathways in photosystem II is proposed.
Asunto(s)
Grupo Citocromo b/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Beta vulgaris/enzimología , Beta vulgaris/metabolismo , Cianobacterias/enzimología , Cianobacterias/metabolismo , Grupo Citocromo b/química , Cinética , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/química , TermodinámicaRESUMEN
In the bacterial photosynthetic reaction center (RC), asymmetric protein environment of the bacteriochlorophyll (BChl) dimer largely determines the photophysical and photochemical properties of the primary electron donor. Previously, we noticed significant differences in properties of Rhodobacter sphaeroides RCs with identical mutations in symmetry-related positions - I(M206)H and I(L177)H. The substitution I(L177)H resulted in covalent binding of BChl PA with the L-subunit, as well as in 6-coordination of BChl BB, whereas in RC I(M206)H no such changes of pigment-protein interactions were found. In addition, the yield of RC I(M206)H after its isolation from membranes was significantly lower than the yield of RC I(L177)H. This study shows that replacement of amino acid residues in the M203-M206 positions near BChls PB and BA by symmetry-related residues from the L-subunit near BChls PA and BB leads to further decrease in RC amount in the membranes associated obviously with poor assembly of the complex. Introduction of a new hydrogen bond between BChl PB and its protein environment by means of the F(M197)H mutation stabilized the mutant RC but did not affect its low yield. We suggest that the mutation I(M206)H and substitution of amino acid residues in M203-M205 positions could disturb glycolipid binding on the RC surface near BChl BA that is important for stable assembly of the complex in the membrane.
Asunto(s)
Bacterioclorofilas/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Rhodobacter sphaeroides/genética , Sustitución de Aminoácidos , Bacterioclorofilas/metabolismo , Dimerización , Histidina/genética , Enlace de Hidrógeno , Mutagénesis Sitio-Dirigida , Mutación , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismoRESUMEN
Mid-infrared (4500-1150 cm(-1)) absorbance changes induced by continuous illumination of Mn-depleted core complexes of photosystem II (PSII) from spinach in the presence of exogenous electron acceptors (potassium ferricyanide and silicomolybdate) were studied by FTIR difference spectroscopy in the temperature range 100-265 K. The FTIR difference spectrum for photooxidation of the chlorophyll dimer P680 was determined from the set of signals associated with oxidation of secondary electron donors (ß-carotene, chlorophyll) and reduction of the primary quinone QA. On the basis of analysis of the temperature dependence of the P680(+)/P680 FTIR spectrum, it was concluded that frequencies of 13(1)-keto-C=O stretching modes of neutral chlorophyll molecules PD1 and PD2, which constitute P680, are similar to each other, being located at ~1700 cm(-1). This together with considerable difference between the stretching mode frequencies of keto groups of PD1(+) and PD2(+) cations (1724 and 1709 cm(-1), respectively) is in agreement with a literature model (Okubo et al. (2007) Biochemistry, 46, 4390-4397) suggesting that the positive charge in the P680(+) dimer is mainly localized on one of the two chlorophyll molecules. A partial delocalization of the charge between the PD1 and PD2 molecules in P680(+) is supported by the presence of a characteristic electronic intervalence band at ~3000 cm(-1). It is shown that a bleaching band at 1680 cm(-1) in the P680(+)/P680 FTIR spectrum does not belong to P680. A possible origin of this band is discussed, taking into account the temperature dependence (100-265 K) of light-induced absorbance changes of PSII core complexes in the visible spectral region from 620 to 720 nm.
Asunto(s)
Clorofila/metabolismo , Luz , Manganeso , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Temperatura , Clorofila/química , Oxidación-Reducción/efectos de la radiación , Espectroscopía Infrarroja por Transformada de FourierAsunto(s)
Proteínas Bacterianas/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Fenómenos Electromagnéticos , Cinética , Modelos Moleculares , Mutación , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Rhodobacter sphaeroides , Espectrometría Raman , VibraciónRESUMEN
Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700-710 nm at 278 K. It was found that the initial charge separation between P680* and ChlD1 (Chl-670) takes place with a time constant of ~1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680*((1-δ)) (P680(δ+)ChlD1(δ-)), where δ ~ 0.5. The subsequent electron transfer from P680(δ+)ChlD1(δ-) to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (ChlD1(δ-)) and bleaching of the PheoD1 bands at 420, 545, and 680 nm with development of the Pheo(-) band at 460 nm. Further electron transfer to QA occurs within 250 ps in accordance with earlier data. The spectra of P680(+) and Pheo(-) formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ~1 and ~13 ps due to the formation of P680(δ+)ChlD1(δ-) and P680(+)PheoD1(-), respectively.
Asunto(s)
Radicales Libres/química , Complejo de Proteína del Fotosistema II/química , Clorofila/química , Transporte de Electrón , Iones/química , Cinética , Feofitinas/química , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
Under physiological conditions (278 K) femtosecond pump-probe laser spectroscopy with 20-fs time resolution was applied to study primary charge separation in spinach photosystem II (PSII) core complexes excited at 710 nm. It was shown that initial formation of anion radical band of pheophytin molecule (Pheoâ») at 460 nm is observed with rise time of ~11ps. The kinetics of the observed rise was ascribed to charge separation between Chl (chlorophyll a) dimer, primary electron donor in PSII (P680*) and Pheo located in D1 protein subunit (PheoD1) absorbing at 420 nm, 545 nm and 680 nm with formation of the ion-radical pair P680âºPheoDIâ». The subsequent electron transfer from Pheo(D1)â» to primary plastoquinone electron acceptor (Q(A)) was accompanied by relaxation of the 460-nm band and occurred within ~250 ps in good agreement with previous measurements in Photosystem II-enriched particles and bacterial reaction centers. The subtraction of the P680⺠spectrum measured at 455 ps delay from the spectra at 23 ps or 44 ps delay reveals the spectrum of Pheo(DI)â», which is very similar to that measured earlier by accumulation method. The spectrum of Pheo(DI)â» formation includes a bleaching (or red shift) of the 670 nm band indicating that Chl-670 is close to Pheo(D1). According to previous measurements in the femtosecond-picosecond time range this Chl-670 was ascribed to Chl(D1) [Shelaev, Gostev, Vishnev, Shkuropatov, Ptushenko, Mamedov, Sarkisov, Nadtochenko, Semenov and Shuvalov, J. Photochemistry and Photobiology, B: Biology 104 (2011) 45-50]. Stimulated emission at 685 nm was found to have two decaying components with time constants of ~1ps and ~14ps. These components appear to reflect formation of P680âºChl(D1)â» and P680âºPheo(D1)â», respectively, as found earlier. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.
Asunto(s)
Complejo de Proteína del Fotosistema II/química , Clorofila/química , Clorofila A , Radicales LibresRESUMEN
Primary charge separation in reaction centers (RCs) of bacterial photosynthesis is modeled in this work. An incoherent population dynamics of RCs states is formulated by kinetic equations. It is assumed that charge separation is accompanied by regular motion of the system along additional coordinates. This motion modulates an energetics of the reactions, and this modulation causes femtosecond oscillations in the population of the states. The best qualitative and quantitative accordance with experimental data on native, modified and mutant RCs of Rba. sphaeroides is achieved in the five states model that includes two excited states P(*)905BAHA and P(*)940BAHA and three charge separated states I, P(+)BA(-)HA and P(+)BAHA(-) (P is a primary electron donor, bacteriochlorophyll dimer, BA and HA are electron acceptors, monomeric bacteriochlorophyll and bacteriopheophytin in active A-branch respectively). The excited states emit at 905 and 940 nm and have approximately the same energy and high interaction rate. The intermediate state I is populated earlier than the P(+)BA(-)HA state and has energy close to the energy of the excited states, a high rate of population and depopulation and spectral identity to the BA(-). A sum of the I and P(+)BA(-)HA populations fits the experimental kinetics of the BA(-) absorption band at 1020 nm. The model explains an oscillatory phenomenon in the kinetics of the P(*) stimulated emission and of the BA(-) absorption. In the schemes without the I state, accordance with the experiment is achieved at unreal parameter values or is not achieved at all. A qualitative agreement of the model with the experiment can be achieved at a wide range of parameter values. The nature of the states I and P(*)940BAHA is discussed in terms of partial charge separation between P and BA and inside P respectively.
Asunto(s)
Electrones , Modelos Biológicos , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Cinética , Rhodobacter sphaeroides/metabolismo , TermodinámicaRESUMEN
The electron and proton transport mediated by protein-bound cofactors in photosynthesis have been investigated by various methods in order to determine the energetics, the dynamics and the pathway of this process. In purple bacteria, primary photosynthetic charge separation and the build-up of a proton gradient across the periplasmic membrane are catalyzed by the photosynthetic reaction centre (RC). Here, the purification, crystallization and preliminary X-ray analysis of wild-type and L(M196)H-mutant RCs of Rhodobacter sphaeroides are presented, enabling study of the influence of the protein environment of the primary electron donor on the spectral properties and photochemical activity of the RC.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mutación/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Rhodobacter sphaeroides , Proteínas Bacterianas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Proteínas del Complejo del Centro de Reacción Fotosintética/aislamiento & purificación , Rhodobacter sphaeroides/genéticaRESUMEN
The reaction of the irreversible chemical reduction of the 13(1)-keto C=O group of pheophytin a (Pheo a) with sodium borohydride in reaction centers (RCs) of functionally active spinach photosystem II (PS II) core complexes was studied. Stable, chromatographically purified PS II core complex preparations with altered chromophore composition are obtained in which ~25% of Pheo a molecules are modified to 13(1)-deoxo-13(1)-hydroxy-Pheo a. Some of the chlorophyll a molecules in the complexes were also irreversibly reduced with borohydride to 13(1)-deoxo-13(1)-hydroxy-chlorophyll a. Based on the results of comparative study of spectral, biochemical, and photochemical properties of NaBH4-treated and control preparations, it was concluded that: (i) the borohydride treatment did not result in significant dissociation of the PS II core complex protein ensemble; (ii) the modified complexes retained the ability to photoaccumulate the radical anion of the pheophytin electron acceptor in the presence of exogenous electron donor; (iii) only the photochemically inactive pheophytin PheoD2 is subjected to the borohydride treatment; (iv) the Qx optical transition of the PheoD2 molecule in the RC of PS II core complexes is located at 543 nm; (v) in the Qy spectral region, PheoD2 probably absorbs at ~680 nm.
Asunto(s)
Borohidruros/química , Borohidruros/farmacología , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Spinacia oleracea/enzimología , Relación Estructura-ActividadRESUMEN
Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll B(A) at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P(+)H(A)(-) state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P(+)B(A)(-)H(A) and P(+)B(A)H(A)(-) states. The data give grounds for assuming that the value of the energy difference between the states P*, P(+)B(A)(-)H(A), and P(+)B(A)H(A)(-) at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll B(A) is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P(+)B(A)(-) state with respect to P*.