KINETICS AND FREE-ENERGY GAPS OF ELECTRON-TRANSFER REACTIONS IN RHODOBACTER-SPHAEROIDES REACTION CENTERS

The rates of the light-driven, electron-transfer reactions in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are examined in mutant strains in which tyrosine (M)210 is replaced by phenylalanine, isoleucine, or tryptophan. The spectra of the absorbance changes between 700 and 975 nm, following excitation by 0.6-ps pulses at 605 nm, are analyzed globally by singular value decomposition. The spectra measured at room temperature are interpreted in terms of a model in which the excited bacteriochlorophyll dimer (P*) transfers an electron to a bacteriopheophytin (H(L)) with time constants of 3.5 +/- 0.3, 10.5 +/- 1.0, 16 +/- 2, and 41 +/- 4 ps in wild-type RCs and the Phe, Ile, and Trp mutants, respectively, and an electron then moves from H(L)- to a quinone (Q(A)) with a time constant of 0.16 ns in wild-type RCs, 0.24 ns in the Phe mutant, and 0.20 ns in the Ile and Trp mutants. The first step speeds up with decreasing temperature in wild-type RCs, remains virtually unchanged in the Phe mutant, and slows down in the Ile and Trp mutants. At 80 K, the signals in the 850-975-nm region include an apparent shift of the stimulated emission or absorption spectrum of P*, with a time constant of 5 ps in the Ile mutant and 13 ps in the Trp mutant. Most of the electron transfer to H(L) occurs with time constants of 55 and 155 ps in the Ile and Trp mutants, respectively, and probably occurs from the relaxed form of P*. Electron transfer from the initial state cannot be ruled out, however. Relaxations of P* are not resolved in wild-type RCs or the Phe mutant. The midpoint potential (E(m)) of the P/P+ redox couple is measured by an electrochemical technique; the E(m), values are 500 +/- 5, 530 +/- 6, 533 +/- 3, and 552 +/- 10 mV for the wild-type and the Phe, Ile, and Trp mutant RCs, respectively. These values are corroborated by chemical titrations. The free energy change (DELTAG-degrees) associated with formation of the P+HL- radical pair from P* also is determined by measuring the amplitude of fluorescence on the nanosecond time scale after blocking electron transfer from H(L)- to Q(A). The free energy of P+H(L)- is elevated by an amount comparable to that calculated from the increase in the E(m) of P in the Ile mutant and by about 16 meV more than this in the Phe and Trp mutants. Nonadiabatic electron-transfer theory is used to relate the rate constant of the formation of P+H(L)- to DELTAG-degrees. The altered temperature dependence of the reaction in the mutants cannot be explained adequately on the assumption that the mutations only alter the overall DELTAG-degrees, but it can be accounted for by assuming that they also increase the free energy of an additional state (P+B(L)-) that serves as both a kinetic and a virtual intermediate. The requisite increases in the free energy of P+B(L)- are greater than the measured changes in the free energy of P+H(L)-. The P+Q(A)- --> PQ(A) back-reaction speeds up with decreasing temperature in all four strains. At room temperature, this reaction has time constants of 0.105 +/- 0.01, 0.100 +/- 0.005, 0.13 +/- 0.01, and 0.045 +/- 0.005 s in the wild-type and the Phe, Ile, and Trp mutant RCs, respectively. The large acceleration in the Trp mutant cannot be explained simply in terms of the change in DELTAG-degrees for this reaction.