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Spontaneous emission from reaction centers of photosynthetic bacteria has been recorded with a time resolution of 50 fs. Excitation was made directly into both the special-pair band (850 nm) and the Qx band of bacteriochlorophylls (608 am). Rhodobacter sphaeroides R26, Rhodobacter capsuleas wild type, and four mutants of Rb. capsulatus were studied. In all cases the fluorescence decay was not single exponential and was well fit as a sum of two exponential decay components. The short components are in excellent agreement with the single component detected by measurements of stimulated emission. The origin of the nonexponential decay is discussed in terms of heterogeneity, the kinetic scheme, and the possibility of slow vibrational relaxation.The mechanism of the initial electron transfer step in the reaction center (RC) of photosynthetic bacteria has been the subject of intense study over the past 10 years. This initial step is ultrafast, occurring in about 3 ps at room temperature (1). As the understanding of the RC improves the need arises for more precise kinetic data. In particular, questions arise as to the exponentiality of the observed kinetic signals (2-8), the possibility of differing behavior at different wavelengths (3, 4), the existence of oscillatory components (5), and the existence of spectral shifts (3, 6) accompanying the excitation and subsequent electron transfer processes.The primary method used for ultrafast studies of the primary charge separation step has been time-resolved absorption spectroscopy, generally with low-repetition-rate (10-30 Hz) relatively high-power (excitation pulse energies in the range 1-30 pJ) laser systems. In addition to the limited dynamic range and signal/noise ratios of such measurements, precise determination of kinetics requires that accurate account be taken of all the competing absorptions and bleachings at the detection wavelength. In measurements of the decay of the excited state of the special pair (P*) by stimulated emission, most workers have made measurements at or near the isosbestic point in the spectrum consisting of ground state (P) bleaching and absorption of the radical cation of P (P+) and P*. However, such a procedure makes it difficult to observe longer decay components in the stimulated emission and to look for the presence of spectral evolution or wavelength-dependent kinetics. Zinth and coworkers (7) could not rule out the presence of a 10-to 20-ps component within their experimental accuracy. More recently Vos et al. (5), after significantly improving their signal/noise ratio, reported that the stimulated emission in Rhodobacter sphaeroides R26 (R26) did not decay exponentially but was well described by two decay times (2.9 and 12 ps) with relative amplitudes of 65% and 35%. This observation is very significant for kinetic analyses of absorption changes in other portions of the spectrum, in particular for discussion of whether the primary process should be described by a one-step superexchange or two-step sequential mechanism (2-15).An...

Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor Q,,. In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the QB binding site, all of which lack the protonatable amino acids Glu-L212 and Asp-L213. These strains are the photosynthetically incompetent site-specific mutant Glu-L212/Asp-L213 --Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from Q8 (class 3 strain, Ala-Ala + Arg-M231 -* Leu; class 4 strain, Ala-Ala + Asn-M43 --Asp). At from primary quinone QA to QB. The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of QB and to increase an apparent "functional" pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long-range electrostatic effects.Photosynthetic organisms convert light excitation energy into chemical free energy. This is accomplished at the level of the photochemical reaction centers (RCs), which span the photosynthetic membranes. The RC from the purple bacterium Rhodopseudomonas viridis was the first membrane protein for which successful crystallization has led to the determination of its three-dimensional structure (1). The RC structure from Rhodobacter (Rb.) sphaeroides was more recently determined (2-4). These complexes are constituted by three proteins, L, M, and H, whose molecular masses range between 30 and 35 kDa. The cofactors involved in the primary electron transfer processes are noncovalently bound to the L and M proteins. A transmembrane charge separation is initiated between a primary electron donor, P (situated near the periplasmic side of the membrane), and a system of two quinones, QA and QB, located near the cytoplasmic side ofthe membrane. QA, which is a one-electron acceptor, is found in a relatively hydrophobic environment of the M protein, at variance to QB, which functions as a two-electron acceptor and is bound in a more polar region of the L protein. Absorption of two photons by the system results in the transfer of two electrons to QB, the uptake of two protons by the protein, and the formation of the quinol molecule QBH2. This loosely bound species leaves the RC and is replaced by an oxidized quinone from the pool present in the membrane, following which electron and proton transfers may be reinitiated.The direct involvement of a few amino acids near QB in proton donation to Q2B-has recen...

Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy. These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB. In the wild-type Rb. sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site. These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant. The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds. In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively. The pH dependences of K2, the QA-QB <--> QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

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