title: Viscosity effects on eukaryotic nitrate reductase activity.
abstract: Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1-3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.
authors.affiliation: The Nitrate Elimination Company, Inc., Lake Linden, Michigan 49945, USA.
authors.forename: Guillaume G
authors.initials: GG
authors.lastname: Barbier
authors.forename: Wilbur H
authors.initials: WH
authors.lastname: Campbell
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D001665:Binding Sites; D002021:Buffers; D002384:Catalysis; D003579:Cytochrome Reductases / Q000737:chemistry; D045303:Cytochromes b / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D005292:Ferricyanides / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010269:Paraquat / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000494:pharmacology; D014783:Viscosity; D003313:Zea mays / Q000378:metabolism
chemical_list: D002021:Buffers; D005292:Ferricyanides; D005656:Fungal Proteins; D011522:Protons; D011994:Recombinant Proteins; C007931:hexacyanoferrate III; D013395:Sucrose; D045303:Cytochromes b; D003579:Cytochrome Reductases; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D005990:Glycerol; D010269:Paraquat