Reaction of Cytochrome-C2 with Photosynthetic Reaction Centers from Rhodopseudomonas-Viridis
by Knaff, David B.; Willie, Anne N.; Long, Joan E.; Kriaucionas, Aidas; Durham, Bill; Millett, Francis Spencer
The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30-mu-M, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554. The photooxidation of horse cytochrome c by Rps. viridis reaction centers was much slower than that of Rps. viridis cytochrome c2, and obeyed second-order kinetics with a rate constant of 1.7 x 10(5) M-1 s-1. The reaction of cytochrome c2 with reaction centers in membranes prepared from Rps. viridis cells was also studied, with results similar to those reported above using purified reaction centers.
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