Role of the PR intermediate in the reaction of cytochrome c oxidase with O2.

The first discernible intermediate when fully reduced cytochrome c oxidase reacts with O2 is a dioxygen adduct (compound A) of the binuclear heme iron-copper center. The subsequent decay of compound A is associated with transfer of an electron from the low-spin heme a to this center. This reaction eventually produces the ferryl state (F) of this center, but whether an intermediate state may be observed between A and F has been the subject of some controversy. Here we show, using both optical and EPR spectroscopy, that such an intermediate (P(R)) indeed exists and that it exhibits spectroscopic properties quite distinct from F. The optical spectrum of P(R) is similar or identical to the spectrum of the P(M) intermediate that is formed after compound A when two-electron-reduced enzyme reacts with O2. An unusual EPR spectrum with features of a CuB(II) ion that interacts magnetically with a nearby paramagnet [cf. Hansson, O., Karlsson, B., Aasa, R., Vänngård, T., and Malmström, B.G (1982) EMBO J. 1, 1295-1297; Blair, D. F., Witt, S. N., and Chan, S. I. (1985) J. Am. Chem. Soc. 107, 7389-7399] can be uniquely assigned to the P(R) intermediate, not being found in either the P(M) or F intermediate. The binuclear center in the P(R) state may be assigned as having an Fe(a3)(IV)=O CuB(II) structure, as in both the P(M) and F states. The spectroscopic differences between these three intermediates are evaluated. The P(R) state has a key role as an initiator of proton translocation by the enzyme, and the thermodynamic and electrostatic bases for this are discussed.