RELATIONSHIP BETWEEN RATE AND FREE-ENERGY DIFFERENCE FOR ELECTRON-TRANSFER FROM CYTOCHROME C(2) TO THE REACTION-CENTER IN RHODOBACTER-SPHAEROIDES

The rate of electron transfer from cytochrome c(2) to the bacteriochlo rophyll dimer of the reaction center from the photosynthetic bacterium Rhodobacter sphaeroides has been investigated using time-resolved opt ical spectroscopy. Measurements were performed on a series of mutant r eaction centers in which the midpoint potentials of the bacteriochloro phyll dimer vary over a range of 350 mV. Dramatic changes in the chara cteristic time of electron transfer were observed, with the measured v alues ranging from 7730 to 80 ns compared to 960 ns for wild type. The binding constants (0.15 to 0.25 mu M(-1)) and the second-order rate c onstants for the slow component (5.5 x 10(8) to 9.4 x 10(8) M(-1) s(-1 )) for the mutants are similar to the corresponding values for wild ty pe (0.35 mu M(-1) and 11 x 10(8) M(-1) s(-1)), indicating that the bin ding of the cytochrome to the reaction center is not changed in the mu tants. In the mutants with the fastest rates, an additional minor comp onent was resolved that is probably due to formation of a reaction cen ter-cytochrome complex in an unfavorable configuration with a binding constant an order of magnitude weaker than the major component. The al tered midpoint potentials in the mutants result in values for the free energy difference for this electron transfer reaction ranging from -6 5 to -420 meV compared to -160 meV for wild type. The relationship bet ween the rate and free energy difference was well fit by a Marcus equa tion using a reorganization energy of 500 meV. Based upon this fit, a distance of 9-14 Angstrom was predicted for the edge to edge separatio n between the heme and the bacteriochlorophyll dimer. Since the reorga nization energy is over 300 meV greater than the free energy differenc e for wild type, the rate of electron transfer from the cytochrome to the reaction center is not optimized. The mutants allow an experimenta l study of the consequences of altered free energy differences in inte rprotein electron transfer, giving insight into the factors that deter mine the rates of electron transfer in biological systems.