A pre-steady-state kinetic study of electron transfer in the isolated cytochrome bf complex from spinach

Abstract

The stopped-flow and rapid-freeze-EPR methods were used to study the reaction of the solubilized cytochrome bf complex from spinach with duroquinol and plastocyanin. Both cytochrome f and cytochrome b-563 show biphasic kinetics with duroquinol. The initial phase of reduction of the cytochromes shows second-order kinetics with a common rate constant of 1.3 · 10 5 M −1 · s −1. The rapid phase is followed by further slow reduction of cytochrome f and reoxidation of cytochrome b-563. The Rieske iron-sulfur center is reduced together with cytochrome f but remains at a higher level of oxidation during the reaction. The reaction of the partially reduced complex with plastocyanin indicates that the Rieske center and cytochrome f equilibrate rapidly. The quinone antagonists DBMIB and DNP-INT both inhibit the reduction of the cytochromes. The results are consistent with a mechanism where the semiquinone resulting from the reduction of the Rieske center reduces a low-potential b-563 as has been suggested for a Q-cycle model for the cytochrome bf complex.