1؉ -CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu B between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.

Time-resolved resonance Raman spectroscopy has been used to study the reduction of dioxygen by the mitochondrial enzyme, cytochrome oxidase. In agreement with earlier reports, indicates that the peroxy species we detect occurs upon proton uptake from bulk solution; whether this species bridges to CUB remains uncertain. For the ferryl, v(Fe4W+O) is at 790 cm-'. In our time-resolved spectra, the 358 cm-' mode appears prior to the 790 cm-' vibration. By using kinetic parameters deduced from the time-resolved Raman work and from a variety of time-resolved optical studies from other laboratories, we have assigned rate constants to several steps in the linear reaction sequence proposed by G. T. Babcock and M. Wikstrom [(1992) Nature (London) 356, 301-309]. Simulations of this kinetic scheme provide insight into the temporal behavior of key intermediates in the 02 reduction process. A striking aspect of the reaction time course is that rapid 02-binding and trapping chemistry is followed by a progressive slowing down of succeeding steps in the process, which allows the various transient species to build up to concentrations sufficient for their detection by our time-resolved techniques. Our analysis indicates that this behavior reflects a mechanism in which conditions that allow efficient dioxygen bond cleavage are not inherent to the active site but are only established as the reaction proceeds. This catalytic strategy provides an effective means by which to couple the free energy available in late intermediates in the reduction reaction to the proton-pumping function of the enzyme.Cytochrome c oxidase couples the one-electron oxidation of cytochrome c to the four-electron reduction of molecular oxygen and links these electron transfers to proton translocation across the inner mitochondrial membrane (for reviews, see refs. 1-5). The mammalian enzyme contains four redox-active metal centers, two heme a-bound irons and two copper ions. One of the two copper centers, CUA, is the site of ferrous cytochrome c oxidation (6, 7). Electron injection is followed by rapid equilibration of the reducing equivalents between CUA and the low-spin heme center, cytochrome a (2, 8, 9). The latter species serves as the electron-queueing point for controlled electron transfer to the binuclear cytochrome a3/CuB site, where the dioxygen reduction reaction takes place. The regulated electron transfer from cytochrome a forms part of the basis for a model in which events in the binuclear center drive proton translocation (5).Due to the unusual ligand-binding kinetic properties of its binuclear center, cytochrome oxidase is unique among oxygen-activating heme proteins in being susceptible to a detailed kinetic analysis of its reaction with dioxygen. The Gibson-Greenwood flow-flash technique was originally developed for this purpose (10), and the method has since been adapted to a variety of spectroscopic techniques (11-15). Of these, the resonance Raman approach provides considerable structural insight, and it has been applied extens...

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Reaction pathways in the enzymatic formation and cleavage of the N-N and N-O bonds, respectively, are difficult to verify without the structure of the intermediates, but we now have such information on the heme a(3)(2+)-NO species formed in the reaction of ba(3)-oxidase with NO from resonance Raman spectroscopy. We have identified the His-heme a(3)(2+)-NO/Cu(B)(1+) species by its characteristic Fe-NO and N-O stretching frequencies at 539 and 1620 cm(-)(1), respectively. The Fe-NO and N-O frequencies in ba(3)-oxidase are 21 and 7 cm(-)(1) lower and higher, respectively, than those observed in Mb-NO. From these results and earlier Raman and FTIR measurements, we demonstrate that the protein environment of the proximal His384 that is part of the Q-proton pathway controls the strength of the Fe-His384 bond upon ligand (CO vs NO) binding. We also show by time-resolved FTIR spectroscopy that Cu(B)(1+) has a much lower affinity for NO than for CO. We suggest that the reduction of NO to N(2)O by ba(3)-oxidase proceeds by the fast binding of the first NO molecule to heme a(3) with high-affinity, and the second NO molecule binds to Cu(B) with low-affinity, producing the temporal co-presence of two NO molecules in the heme-copper center. The low-affinity of Cu(B) for NO binding also explains the NO reductase activity of the ba(3)-oxidase as opposed to other heme-copper oxidases. With the identification of the His-heme a(3)(2+)-NO/Cu(B)(1+) species, the structure of the binuclear heme a(3)-Cu(B)(1+) center in the initial step of the NO reduction mechanism is known.