Spectroscopic characterization of compound C and related derivatives of cytochrome oxidase (magnetic circular dichroism spectroscopy/resonance Raman spectroscopy/cytochrome a3/intermediate spin state)

Magnetic circular dichroism and resonance Ra- man spectra have been obtained for three "oxygenated" deriva- tives of cytochrome c oxidase (ferrocytochrome c:oxygen oxido- reductase, EC 1.9.3.1). The spectra are interpreted and four alternatives for the states of cytochrome a3 and Cuu are evaluated. The reduction of oxygen to water by cytochrome oxidase (fer- rocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) apparently requires a minimal functional unit composed of two molecules of heme a and two copper atoms. One heme, cytochrome a, and one copper (alternatively called Cud (detectable by electron paramagnetic resonance, EPR) or CUA) seem to exist as single, structurally isolated, metal centers. Cytochrome a appears to be a low-spin (1. s.) hemoprotein, as is typical of electron transfer cytochromes. Despite considerable effort at characterization, Cud is structurally undefined. There is a body of data (1) which suggests that the second heme, cytochrome a3, and second cop- per ion (Cuu, CUB, Cua) exist as a binuclear center, and there have been a number of proposals for the structure of this bin- uclear center (2, 3). The reaction of oxygen is believed to occur with reduced cytochrome a3 apparently in the five-coordinate high-spin (h.s.) ferrous state (4), leaving a coordination site available for ligation by the oxygen molecule. One approach in the elucidation of the mechanism of oxygen reduction by this enzyme has employed low-temperature trap- ping techniques to stabilize putative intermediates in the over- all reoxidation process. Chance and his colleagues (5, 6) have developed and refined this method and contributed substan- tially to current insights into this process. By using these meth- ods, a number of spectral intermediates have been observed during the reoxidation of four-electron fully reduced and two- electron partially reduced (mixed-valence) cytochrome oxidase, viz: