A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Abstract Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N 2 O (2NO + 2e −  + 2H +  → N 2 O + H 2 O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O 2 -reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O 2 , we demonstrate that protons are indeed consumed from the ‘outside’. First, multiple turnover reduction of O 2 resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O 2 shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O 2 as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O 2 show electron transfer coupled to proton uptake from outside the NOR-liposomes with a τ  = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711–10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.