Insights into the mechanism of nitric oxide reductase from a Fe -depleted variant.

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c-dependent NO reductase (cNOR). cNOR contains four redox-active cofactors: three hemes and a nonheme iron (Fe ). Heme b and Fe constitute the active site, but the specific mechanism of NO-binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks Fe to investigate the role of Fe during catalysis. We show that in the Fe -less cNOR, binding of both NO and O to heme b still occurs but further reduction is impaired, although to a lesser degree for O than for NO. Implications for the catalytic mechanisms of cNOR are discussed.