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.
Highlights
Nitrate reductase (NaR,1 EC 1.7.1.1–3) is a homodimer with each monomer consisting of a ϳ100-kDa polypeptide and cofactors FAD, heme-iron, and molybdenum-molybdopterin (MoMPT) with a stoichiometry of 1:1:1 [1, 2]
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)
NAD(P)H-dependent ferricyanide reduction is catalyzed by NaR with a kcat of ϳ1000 sϪ1 and reduced methyl viologen (MV) nitrate reduction is catalyzed by NaR with a kcat ϳ800 sϪ1
Summary
NaR, eukaryotic NAD(P)H:nitrate reductase; At, A. thaliana; Cyt b, cytochrome b domain of NaR; CbR, cytochrome b reductase fragment of NaR; IET, internal electron/proton transfer; Mo-MPT, molybdenum-molybdopterin; MOPS, 3-(N-morpholino)propanesulfonic acid; MoR, Mo-MPT reductase fragment of NaR; MV, methyl viologen; S-NaR1, simplified nitrate reductase; SOX, mammalian sulfite oxidase; YNaR1, yeast NAD(P)H:NaR; ZmNaR, Z. mays NaR; AtNR, A. thaliana NaR; ZmCbR, Z. mays Cyt b reductase fragment; ZmMoR, corn molybdenum reductase; SoMoR, Spinacea oleracea molybdenum reductase. By analogy, if IET is the rate-limiting event in NaR catalysis and involves large conformational movement of Cyt b domain relative to the other larger modules of the enzyme to which it is tethered via Hinges 1 and 2 (Fig. 1), solution viscosity may have a significant impact on NaR-catalytic rates and help to demonstrate the rate-limiting nature of IET in NaR catalysis Monomeric polyhydroxylated molecules such as glycerol and sucrose, called microviscosogens [15, 16], have been used in many studies of protein conformational changes [17,18,19,20,21]. A large negative impact of viscosity was observed in the other assays performed, leading to the conclusion that a large conformational change involving the gating of Cyt b is most likely to be the limiting step in catalysis by NaR
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
