A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N2O (2NO + 2e(-) + 2H(+) -> N2O + H2O) as part of the denitriffication 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 tau = 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. (c) 2007 Elsevier B.V. All rights reserved.