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J. Biol. Chem., Vol. 280, Issue 28, 26049-26054, July 15, 2005

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Viscosity Effects on Eukaryotic Nitrate Reductase Activity^*

Guillaume G. Barbier and Wilbur H. Campbell

From the The Nitrate Elimination Company, Inc., Lake Linden, Michigan 49945 and Department of Biological Sciences, Michigan Technological University, Houghton, Michigan, 49931

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.

Received for publication, August 23, 2004 , and in revised form, May 13, 2005.

^* This work was supported in part by a SBIR grant (Contract R44GM56598) (to NECi). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Plant Biology, Michigan State University, East Lansing, MI 48824.

To whom correspondence should be addressed. Tel.: 906-296-1000; Fax: 906-296-8003; E-mail: bill{at}nitrate.com.

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