Conformation-dependent hydride transfer in neuronal nitric oxide synthase reductase domain.
Calmodulin (CaM) activates the constitutive isoforms of mammalian nitric oxide synthase by triggering electron transfer from the reductase domain FMN to the heme. This enables the enzymes to be regulated by Ca(2+) concentration. CaM exerts most of its effects on the reductase domain; these include activation of electron transfer to electron acceptors, and an increase in the apparent rate of flavin reduction by the substrate NADPH. It has been shown that the former is caused by a transition from a conformationally locked form of the enzyme to an open form as a result of CaM binding, improving FMN accessibility, but the latter effect has not been explained satisfactorily. Here, we report the effect of ionic strength and isotopic substitution on flavin reduction. We found a remarkable correlation between the rate of steady-state turnover of the reductase domain and the rate of flavin reduction over a range of different ionic strengths. The reduction of the enzyme by NADPH was biphasic, and the amplitudes of the phases determined through global analysis of stopped-flow data correlated with the proportions of enzyme known to exist in the open and closed conformations. The different conformations of the enzyme molecule appeared to have different rates of reaction with NADPH. Thus, proximity of FMN inhibits hydride transfer to the FAD. In the CaM-free enzyme, slow conformational motion (opening and closing) limits turnover. It is now clear that this motion also controls hydride transfer during steady-state turnover, by limiting the rate at which NADPH can access the FAD.
Welland
Andrew
A
School of Chemistry, University of Edinburgh, Edinburgh, UK.
Daff
Simon
S
D000818:Animals; D055162:Biocatalysis; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002417:Cattle; D005486:Flavin Mononucleotide / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000737:chemistry; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011522:Protons; D051381:Rats; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D011522:Protons; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D052248:Nitric Oxide Synthase Type I