Kay C J, Barber M J, Solomonson L P, Kau D, Cannons A C, Hipkin C R
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612.
Biochem J. 1990 Dec 1;272(2):545-8. doi: 10.1042/bj2720545.
Visible spectra of oxidized and reduced Candida nitratophila assimilatory NAD(P)H:nitrate reductase yielded absorbance maxima of 413 nm and 423 nm, and 525 nm and 555 nm respectively, characteristic of a b5-type cytochrome. E.p.r. spectra of the partially reduced enzyme revealed a single Mo(V) species (g1 = 1.9957, g2 = 1.9664 and g3 = 1.9658) exhibiting superhyperfine coupling to a single proton [A(1H)av. = 1.4 mT]. Oxidation-reduction midpoint potentials (E'0) (25 degrees C, pH 7) for the haem and Mo-pterin prosthetic groups were determined by visible and e.p.r. potentiometric titrations and yielded values of E'0 = -174 mV (n = 1) for the haem and E'0 = -3 mV and E'0 = -27 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples respectively. Comparison of initial rates of the NADH-oxidizing and nitrate-reducing partial activities at various ionic strengths indicated electron transfer from reduced haem to Mo was rate-limiting during turnover. These results suggest a close similarity between Candida nitratophila and Chlorella vulgaris nitrate reductases.
氧化型和还原型嗜硝酸盐假丝酵母同化性NAD(P)H：硝酸还原酶的可见光谱分别在413 nm和423 nm，以及525 nm和555 nm处产生最大吸光度，这是b5型细胞色素的特征。部分还原酶的电子顺磁共振光谱显示出单一的Mo(V)物种（g1 = 1.9957，g2 = 1.9664，g3 = 1.9658），与单个质子表现出超精细偶合[A(1H)av. = 1.4 mT]。通过可见光谱和电子顺磁共振电位滴定法测定了血红素和钼蝶呤辅基的氧化还原中点电位（E'0）（25℃，pH 7），血红素的E'0值为-174 mV（n = 1），Mo(VI)/Mo(V)和Mo(V)/Mo(IV)偶联的E'0值分别为-3 mV和-27 mV。在不同离子强度下对NADH氧化和硝酸盐还原部分活性的初始速率进行比较表明，在周转过程中，从还原型血红素到钼的电子转移是限速步骤。这些结果表明嗜硝酸盐假丝酵母和普通小球藻硝酸还原酶之间有密切的相似性。
