The rate of electron transfer from cytochrome c2 to the bacteriochlorophyll dimer of the reaction center from the photosynthetic bacterium Rhodobacter sphaeroides has been investigated using timeresolved optical spectroscopy. Measurements were performed on a series of mutant reaction centers in which the midpoint potentials of the bacteriochlorophyll dimer vary over a range of 350 mV. Dramatic changes in the characteristic time of electron transfer were observed, with the measured values ranging from 7730 to 80 ns compared to 960 ns for wild type. The binding constants (0.15 to 0.25 μ M-1) and the second-order rate constants for the slow component (5.5 × 108 to 9.4 × 108 M-1 s-1) for the mutants are similar to the corresponding values for wild type (0.35 μ M-1 and 11 × 108 M-1 s-1), indicating that the binding of the cytochrome to the reaction center is not changed in the mutants. In the mutants with the fastest rates, an additional minor component was resolved that is probably due to formation of a reaction center-cytochrome complex in an unfavorable configuration with a binding constant an order of magnitude weaker than the major component. The altered midpoint potentials in the mutants result in values for the free energy difference for this electron transfer reaction ranging from -65 to -420 meV compared to -160 meV for wild type. The relationship between the rate and free energy difference was well fit by a Marcus equation using a reorganization energy of 500 meV. Based upon this fit, a distance of 9–14 Å was predicted for the edge to edge separation between the heme and the bacteriochlorophyll dimer. Since the reorganization energy is over 300 meV greater than the free energy difference for wild type, the rate of electron transfer from the cytochrome to the reaction center is not optimized. The mutants allow an experimental study of the consequences of altered free energy differences in interprotein electron transfer, giving insight into the factors that determine the rates of electron transfer in biological systems.
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