Assignment and charge translocation stoichiometries of the major electrogenic phases in the reaction of cytochrome c oxidase with dioxygen

The reaction of cytochrome c oxidase with dioxygen has been studied by mean s of time-resolved measurements of electrical membrane potential (Delta psi ). Microsecond time resolution was achieved by starting with the GO-inhibit ed enzyme, which was photolyzed after addition of oxygen. The time course o f the reaction could be fitted by using a five-step sequential reaction as a. model. The first two phases of the reaction, which correspond in time to binding of oxygen followed by formation of the P (peroxy) intermediate, as observed spectroscopically, are not associated with net charge displacemen t across the membrane. After this lag, Delta psi develops in three phases, which correspond in time to the conversion of P to the F (ferryl) intermedi ate, in a single phase, and conversion of F to O (the fully oxidized enzyme ), in two phases. The amplitude of Delta psi was approximately equal for th e P --> F and F --> portions of the reaction. When the oxygen reaction is s tarted with incompletely reduced enzyme, it will halt at the P or F state. When the reaction was allowed to proceed to the F state, but no further, on ly the fast phase of Delta psi formation was observed, whereas no Delta psi was generated if the reaction was halted at P. This finding places the ass ignments of phases in the electrometric data on a firmer basis-they are no longer based solely on temporal correspondence with phases in the spectrosc opic data. To define the number of charges transferred across the membrane during the reaction, some kind of calibration is needed. For this purpose, another type of reaction-electron transfer following CO photolysis in the a bsence of oxygen ("backflow")-was studied. Parallel spectroscopic and elect rometric measurements showed that the fast electron transfer from the low-s pin heme to Cu-A in the backflow process results in approximately ii times smaller amplitude of Delta psi as compared with Delta psi generated in the reaction of the reduced enzyme with oxygen (the polarity is also reversed). If it is assumed that transfer of an electron from the low-spin heme to Cu -A amounts to movement of a unit charge across half of the membrane dielect ric, charge translocation in the reaction of the reduced enzyme with oxygen amounts to approximately 5.5 unit charges-the value predicted if all four protons pumped during the catalytic cycle are translocated during the oxida tive part of the reaction.