title: The nitric-oxide reductase from Paracoccus denitrificans uses a single specific proton pathway.
abstract: The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.
authors.affiliation: From the Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Josy
authors.initials: J
authors.lastname: Ter Beek
authors.affiliation: From the Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Nils
authors.initials: N
authors.lastname: Krause
authors.affiliation: From the Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Joachim
authors.initials: J
authors.lastname: Reimann
authors.affiliation: From the Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Lachmann
authors.affiliation: From the Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address: piaa@dbb.su.se.
authors.forename: Pia
authors.initials: P
authors.lastname: Ädelroth
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport / Q000502:physiology; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011522:Protons
chemical_list: D001426:Bacterial Proteins; D011522:Protons; D009569:Nitric Oxide; D010088:Oxidoreductases; C034747:nitric-oxide reductase; D010100:Oxygen
keywords: Bioenergetics/Electron Transfer Complex; Electron Transfer; Enzyme Kinetics; Flow-Flash; Heme-Copper Oxidase; Kinetic Isotope Effect; Membrane Biophysics; Nitric Oxide; Proton Transport