title: Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: enzymatic activity and active site structure.
abstract: Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
authors.affiliation: RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan; Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan.
authors.forename: Erina
authors.initials: E
authors.lastname: Terasaka
authors.affiliation: RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan; Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Okada
authors.affiliation: RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan; Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Sato
authors.affiliation: Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Sako
authors.affiliation: RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan; Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan. Electronic address: yshiro@riken.jp.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Shiro
authors.affiliation: RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan. Electronic address: ttosha@spring8.or.jp.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Tosha
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006873:Hydroquinones / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D006873:Hydroquinones; D010088:Oxidoreductases; C034747:nitric-oxide reductase
keywords: Cytochrome c oxidase; Heme; Nitric oxide reductase; Non-heme metal; Proton transfer; Resonance Raman