Summary: The nitrite oxidizing Alphaproteobacterium , Nitrobacter winogradskyi , primarily conserves energy from the oxidation of nitrite (NO ₂ ⁻ ) to nitrate (NO ₃ ⁻ ) through aerobic respiration. Almost 20 years ago, NO‐dependent NADH formation was reported to occur in both aerobic and anaerobic cell suspensions of N. winogradskyi strain ‘agilis’, suggesting that NO oxidation might contribute to energy conservation by Nitrobacter . Recently, the N. winogradskyi Nb‐255 genome was found to contain a gene (Nwin_2648) that encodes a putative copper‐containing nitrite reductase (NirK), which may reduce NO ₂ ⁻ to NO. In this study, the putative nirK was found to be maximally transcribed under low O ₂ (between zero and 4% O ₂ ) in the presence of NO ₂ ⁻ . Transcription of nirK was not detected under anaerobic conditions in the absence of NO ₂ ⁻ or in the presence of NO ₃ ⁻ and pyruvate. Although net production of NO could not be detected from either aerobically grown or anaerobically incubated cells, exogenous NO was consumed by viable cells and concomitantly inhibited NO ₂ ⁻ ‐dependent O ₂ uptake in a reversible, concentration dependent manner. Both NO ₂ ⁻ ‐dependent O ₂ uptake and NO consumption were inhibited by 1 mM cyanide suggesting involvement of cytochrome oxidase with NO consumption. Abiotic consumption of NO was measured, yet, both the rates and kinetics of NO transformation in buffer alone, or by heat killed, or cyanide‐treated cells differed from those of viable cells. In light of this new information, a modified model is proposed to explain how NirK and NO manage electron flux in Nitrobacter .

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