Mechanistic studies of the enzyme

A single turnover in the reaction of cytochrome c oxidase involves (1) reduction of the four metal centers by four equivalents of reduced cytochrome c, (2) binding of dioxygen to the partially or fully reduced enzyme, (3) transfer of four electrons to dioxygen, coupled with (4) protonation by four equivalents of protons to produce two equivalents of water, all without the leakage of any substantial amount of potentially harmful partially reduced dioxygen byproducts such as superoxide or hydrogen peroxide. 44 - 46 At low temperatures, the reaction can be slowed down, so that the individual steps in the dioxygen reduction can be observed. Such experiments are carried out using the fully reduced enzyme to which CO has been bound. Binding of CO to the Fell heme center in reduced cytochrome c oxidase inhibits the enzyme and makes it unreactive to dioxygen. The CO-inhibited derivative can then be mixed with dioxygen and the mixture cooled. Photolysis of metal-CO complexes almost always leads to dissociation of CO, and CO-inhibited cytochrome c oxidase is no exception. Photolytic dissociation of CO frees the Fell heme, thereby initiating the reaction with dioxygen, which can then be followed spectroscopically. 44-46 Dioxygen reacts very rapidly with the fully reduced enzyme to produce a species that appears to be the dioxygen adduct of cytochrome a3 (Reaction 5.48). Such a species is presumed to be similar to other mononuclear oxyheme derivatives. The dioxygen ligand in this species is then rapidly reduced to peroxide by the nearby CUB, forming what is believed to be a binuclear /-L-peroxo species (Reaction 5.49). These steps represent a two-electron reduction of dioxygen to the peroxide level, and are entirely analogous to the model reactions discussed above (Reactions 5.36 to 5.46), except that the binuclear intermediates contain one copper and one heme iron. The /-L-peroxo FellI - (02 2 -) - CUll species is then reduced by a third electron, resulting in cleavage of the 0-0 bond (Reaction 5.50). One of the oxygen atoms remains with iron in the form of a ferryl complex, i. e., an Fe IV oxo, and the other is protonated and bound to copper in the form of a CuII aquo complex. 65 Reduction by another electron leads to hydroxo complexes of both the Fe III heme and the CuII centers (Reaction 5.51).65 Protonation then causes dissociation of two water molecules from the oxidized cytochrome a3-CuB center (Reaction 5.52).

Several important questions remain to be resolved in cytochrome c oxidase research. One is the nature of the ligand bridge that links cytochrome a3 and CUB in the oxidized enzyme. Several hypotheses have been advanced (imidazolate, thiolate sulfur, and various oxygen ligands), but then discarded or disputed, and there is consequently no general agreement concerning its identity. However, EXAFS measurements of metal-metal separation and the strength of the magnetic coupling between the two metal centers provide evidence that a single atom bridges the two metals. 45 ,46 Another issue, which is of great importance, is to find out how the energy released in the reduction of dioxygen is coupled to the synthesis of ATP. It is known that this occurs by coupling the electron-transfer steps to a proton-pumping process, but the molecular mechanism is unknown.46 Future research should provide some interesting insights into the mechanism of this still mysterious process.