The temperature dependence of fluorescence on the picosecond to nanosecond time scale from the reaction centers of Rhodobacter sphaeroides strain R-26 and two mutants with elevated P/P+ midpoint potentials has been measured with picosecond time resolution. In all three samples, the kinetics of the fluorescence decay is complex and can only be well described with four or more exponential decay terms spanning the picosecond to nanosecond time range. Multiexponential fits are needed at all temperatures between 295 and 20 K. The complex decay kinetics are explained in terms of a dynamic solvation model in which the charge-separated state is stabilized after formation by protein conformational changes. Many of these motions have not had time to occur on the time scale of initial electron transfer and/or are frozen out at low temperature. This results in a time- and temperature-dependent enthalpy change between the excited singlet state and the charge-separated state that is the dominant term in the free energy difference between these states. Long-lived fluorescence is still observed even at 20 K, particularly for the high-potential mutants. This implies that the driving force for electron transfer on the nanosecond time scale at low temperature is less than 200 cm-1 (25 meV) in R-26 reaction centers and even smaller on the picosecond time scale or in the high-potential mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

The mutation, H(M182)L, in the Rhodobacter sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll on the inactive side (B-side) of the RC with a bacteriopheophytin (the new cofactor is referred to as φB). In φB containing RCs, P* stimulated emission decays with an accelerated time constant of 2.6 ± 0.1 ps at room temperature compared to 3.1 ± 0.2 ps in WT RCs. Analysis of the time-resolved spectra implies that two states are being formed during the initial reaction in the mutant:  the usual P+HA - state, as seen in WT, and a new state, P+φB -. P+φB - is formed during the decay of P* and recombines to the ground state with a lifetime of 200 ± 20 ps. The yield of the P+φB - state is 35 ± 5% at room temperature, while the remaining 65 ± 5% of the initial electron-transfer results in P+HA -. There does not appear to be any further electron transfer from φB - to HB. Apparently, in the H(M182)L mutant, the state P+φB - is lower in free energy than the P+HB - state.

Femtosecond transient absorption spectroscopy has been used to investigate the excitation wavelength dependence of energy transfer and initial charge separation processes in reaction centers of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides (R-26) at room temperature. The Q Y transition bands of the bacteriopheophytins (H), bacteriochlorophyll monomers (B), and special pair (P) were selectively excited with pulses of 150 fs duration and 5 nm spectral bandwidth. Absorbance changes were analyzed over the entire wavelength region from 700 to 1000 nm. From this analysis we concluded the following: (1) As seen by others, energy transfer between H, B, and P is extremely fast, occurring on the 100-300 fs time scale.(2) The spectral evolution of the system is excitation wavelength dependent for picoseconds after excitation, implying that vibrational relaxation is not complete on the time scale of either energy transfer or charge separation and suggesting that the pathway of charge separation may be excitation wavelength dependent.(3) The absorbance change spectra of the initial excited states of B and H are not consistent with intensity borrowing between these bands, reopening the question of what gives rise to the complex absorbance changes normally associated with the H A -state. (4) The 10-20 ps component of the stimulated emission decay is excitation wavelength dependent and spectrally different from the dominant 2-3 ps decay of the stimulated emission. This component is unlikely to represent a static conformational heterogeneity in the reaction center charge separation rate. These conclusions lead to the proposal of the following model for energy and electron transfer in the reaction center. Energy transfer in this system is very fast because it is mediated by electron exchange interactions between cofactors (implying relatively strong electronic coupling for electron transfer) and because there is little nuclear displacement between donor and acceptor potential surfaces during energy transfer. Electron transfer is slower than energy transfer because the nuclear displacement is larger, and the rate is limited by movement along the reaction coordinate. Thus, initial electron transfer occurs in the near adiabatic limit before vibrational relaxation is complete. This model would explain many issues which have been difficult to resolve using standard electron transfer models including the difficulty in identifying the P + B A -intermediate and the insensitivity of the initial electron transfer rate to temperature and driving force.

Structural changes in chromatophores of Rhodobacter sphaeroides reaction center mutants associated with the substitution of amino acid residues near the primary electron donor P have been investigated by light-induced FTIR difference spectroscopy. The single-site mutations Leu-L131 to His and Leu-M160 to His and the corresponding double mutation were designed to introduce a proton-donating residue that could form a hydrogen bond with the keto carbonyl of ring V of each bacteriochlorophyll (PL and PM) of the dimer. The presence of large positive bands at approximately 1550, 1480, and 1295 cm-1, as well as at 2600-2800 cm-1 in the light-induced P+QA-/PQA FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone QA, demonstrates that the BChl dimer state of P+ is preserved in the LH(L131), LH(M160), and LH(M160)+LH(L131) mutants, although frequency shifts and amplitude changes can be observed, notably for LH(M160). Compared to wild type, these changes are thought to reflect a different charge repartition over the two BChls in P+. Large frequency downshifts in the 9-keto C=O stretching region of the P+QA-/PQA FTIR difference spectra of chromatophores are observed in the mutant samples relative to wild type. For the LH(M160) mutant, a large differential signal at 1678/1664 cm-1 is assigned to a shift, upon photooxidation, of the 9-keto C=O of PM hydrogen-bonded to His-M160, while that at 1718/1696 cm-1 corresponds to the free 9-keto C=O of PL.(ABSTRACT TRUNCATED AT 250 WORDS)

A 51 bp section of the Rhodobacter capsulatus photosynthetic reaction center M subunit gene (nucleotides M562-M612 of the pufM structural sequence) encoding amino acids M187-M203 was replaced by the homologous region of the L subunit gene. This resulted in the symmetrization of much of the amino acid environment of the reaction center initial electron donor, P. This is the first in a series of large-scale symmetry mutations and is referred to as sym1. The sym1 mutant was able to grow photosynthetically, indicating that reaction center function was largely intact. Isolated reaction centers showed an approximately 10-nm blue shift in the QY band of P. The standard free energy change between P* and P+BphA- determined from analysis of the long-lived fluorescence from quinone-reduced reaction centers decreased from about -120 meV in the wild-type to about -75 meV in the sym1 mutant. A 65-70% quantum yield of electron transfer from P* to P+QA- was observed, most of the yield loss occurring between P* and P+BphA-. The decay of the stimulated emission from P* was about 3-fold slower in this mutant than in the wild-type. Time-resolved spectral analysis of the charge-separated intermediates formed in sym1 reaction centers indicated that the major product was P+BphA-. A model-dependent analysis of the observed rates and electron-transfer yields gave the following microscopic rate constants for sym1 reaction centers (wild-type values under the same conditions are given in parentheses): [formula: see text] Analysis of the sym1 mutant, mutants near P made by other groups, and interspecies variation of amino acids in the vicinity of P suggests that the protein asymmetry in the environment of the initial electron donor is important for optimizing the rate and yield of electron transfer, but is not strictly required for overall reaction center function.