The NOR (nitric oxide reductase) from Paracoccus denitrificans catalyses the two-electron reduction of NO to N2O (2NO+2H++2e→N2O+H2O). The NOR is a divergent member of the superfamily of haem-copper oxidases, oxygen-reducing enzymes which couple the reduction of oxygen with translocation of protons across the membrane. In contrast, reduction of NO catalysed by NOR is non-electrogenic which, since electrons are supplied from the periplasmic side of the membrane, implies that the protons needed for NO reduction are also taken from the periplasm. Thus NOR must contain a proton-transfer pathway leading from the periplasmic side of the membrane into the catalytic site. The proton pathway has not been identified, and the mechanism and timing of proton transfer during NO reduction is unknown. To address these questions, we have studied the reaction between NOR and the chemically less reactive oxidant O2 [Flock, Watmough and Ädelroth (2005) Biochemistry 44, 10711–10719]. When fully reduced NOR reacts with O2, proton-coupled electron transfer occurs in a reaction that is rate-limited by internal proton transfer from a group with a pKa of 6.6. This group is presumably an amino acid residue close to the active site that acts as a proton donor also during NO reduction. The results are discussed in the framework of a structural model that identifies possible candidates for the proton donor as well as for the proton-transfer pathway.

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