Role of the Two Metals in the Active Sites of Heme Copper Oxidases-A Study of NO Reduction in Cytochrome Oxidase.

The superfamily of heme copper oxidases reduces molecular oxygen or nitric oxide, and the active sites comprise a high-spin heme group ( or ) and a non-heme metal (Cu or Fe). The C family of cytochrome oxidases, with the high-spin heme and Cu in the active site, is a subfamily of the heme copper oxidases that can reduce both molecular oxygen, which is the main substrate, and nitric oxide. The mechanism for NO reduction in oxidase is studied here using hybrid density functional theory and compared to other cytochrome oxidases (A and B families), with a high-spin heme and Cu in the active site, and to cytochrome dependent NO reductase, with a high-spin heme and a non-heme Fe in the active site. It is found that the reaction mechanism and the detailed reaction energetics of the oxidases are not similar to those of cytochrome dependent NO reductase, which has the same type of high-spin heme group but a different non-heme metal. This is in contrast to earlier expectations. Instead, the NO reduction mechanism in oxidases is very similar to that in the other cytochrome oxidases, with the same non-heme metal, Cu, and is independent of the type of high-spin heme group. The conclusion is that the type of non-heme metal (Cu or Fe) in the active site of the heme copper oxidases is more important for the reaction mechanisms than the type of high-spin heme, at least for the NO reduction reaction. The reason is that the proton-coupled reduction potentials of the active site cofactors determine the energetics for the NO reduction reaction, and they depend to a larger extent on the non-heme metal. Observed differences in NO reduction reactivity among the various cytochrome oxidases may be explained by differences outside the BNC, affecting the rate of proton transfer, rather than in the BNC itself.