Insights into the mechanism of nitric oxide reductase from a Fe B ‐depleted variant

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c ‐dependent NO reductase ( c NOR ). c NOR contains four redox‐active cofactors: three hemes and a nonheme iron (Fe B ). Heme b 3 and Fe B 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 c NOR variant that lacks Fe B to investigate the role of Fe B during catalysis. We show that in the Fe B ‐less c NOR , binding of both NO and O 2 to heme b 3 still occurs but further reduction is impaired, although to a lesser degree for O 2 than for NO . Implications for the catalytic mechanisms of c NOR are discussed.