title: Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase.
abstract: We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser(719), NarG-His(1163), and NarG-His(1184)); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His(1092) and NarG-His(1098)). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of -88 and -36 mV, respectively). Ala variants of His(1092) and His(1098) also elicit large ΔEm values of -143 and -101 mV, respectively. An Arg variant of His(1092) elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.
authors: From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors: Sheng-Yi
authors: SY
authors: Wu
authors: From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors: Richard A
authors: RA
authors: Rothery
authors: From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada joel.weiner@UAlberta.ca.
authors: Joel H
authors: JH
authors: Weiner
mesh_terms: D020134:Catalytic Domain; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D008982:Molybdenum / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011622:Pterins / Q000378:metabolism*
chemical_list: D011622:Pterins; C490076:pyranopterin; D008982:Molybdenum; D050901:Nitrate Reductase
keywords: EPR; charge-transfer; electrochemistry; electron paramagnetic resonance (EPR); electron transfer; molybdenum; nitrate reductase; redox tuning; reductase