Clade II nitrous oxide respiration of Wolinella succinogenes depends on the NosG, ‐C1, ‐C2, ‐H electron transport module, NosB and a Rieske/cytochrome bc complex

Summary Microbial reduction of nitrous oxide (N 2 O) is an environmentally significant process in the biogeochemical nitrogen cycle. However, it has been recognized only recently that the gene encoding N 2 O reductase ( nosZ ) is organized in varying genetic contexts, thereby defining clade I (or ‘typical’) and clade II (or ‘atypical’) N 2 O reductases and nos gene clusters. This study addresses the enzymology of the clade II Nos system from Wolinella succinogenes , a nitrate‐ammonifying and N 2 O‐respiring Epsilonproteobacterium that contains a cytochrome c N 2 O reductase ( c NosZ). The characterization of single non‐polar nos gene deletion mutants demonstrated that the NosG, ‐C1, ‐C2, ‐H and ‐B proteins were essential for N 2 O respiration. Moreover, cells of a W. succinogenes mutant lacking a putative menaquinol‐oxidizing Rieske/cytochrome bc complex (QcrABC) were found to be incapable of N 2 O (and also nitrate) respiration. Proton motive menaquinol oxidation by N 2 O is suggested, supported by the finding that the molar yield for W. succinogenes cells grown by N 2 O respiration using formate as electron donor exceeded that of fumarate respiration by about 30%. The results demand revision of the electron transport chain model of clade II N 2 O respiration and challenge the assumption that NosGH(NapGH)‐type iron‐sulfur proteins are menaquinol‐reactive.