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 (FeB). Heme b3 and FeB 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 FeB to investigate the role of FeB during catalysis. We show that in the FeB‐less cNOR, binding of both NO and O2 to heme b3 still occurs but further reduction is impaired, although to a lesser degree for O2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed.