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.

Molecular Mechanism of 14-3-3 Protein-mediated Inhibition of Plant Nitrate Reductase

“…6). This model is consistent with the observation that increasing solution viscosity significantly decreases steadystate turnover of both the holo-enzyme and the Mo-heme fragment (39). Once 14-3-3 has bound to phosphorylated NR and the open conformation is stabilized, electron transfer from the heme to the molybdenum center slows dramatically, resulting in the observed decrease in activity.…”

Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction

“…The substrate sensitivity of ε Nar suggests an enzymatic mechanism in which the rate of electron transfer is partially rate‐determining. In this respect, electron transfer is cited to constitute a rate‐determining step of catalysis by eukaryotic reductases (Skipper et al ; Barbier and Campbell ) which may have analogous kinetic mechanisms to Nar . However, substrate sensitivity of ε Nar was not observed among assays fuelled by methyl or benzyl viologen—save for a single assay at lower temperature.…”

Dynamic Model for Enzyme Action

“…2) Enzyme activity can be influenced by the viscosity of the solvent [26]. A physical study demonstrates that local protein dynamics are independent of the solvent and that global protein conformational change is sensitive to it [27].…”