Grivennikova V G, Gavrikova E V, Timoshin A A, Vinogradov A D
Department of Biochemistry, School of Biology, Moscow State University, Russia.
Biochim Biophys Acta. 1993 Jan 8;1140(3):282-92. doi: 10.1016/0005-2728(93)90067-p.
A simple system for aerobic assay of the quinol-fumarate reductase reaction catalyzed by purified soluble bovine heart succinate-ubiquinone reductase in the presence of NADH, NAD(P)H-quinone reductase (DT-diaphorase) and an appropriate quinone is described. The reaction is inhibited by carboxin, suggesting that the same quinone/quinol binding site is involved in electron transfer from succinate to ubiquinone and from ubiquinol to fumarate. The kinetic properties of the reaction in both directions and comparative affinities of the substrate binding sites of the enzyme to substrates (products) and competitive inhibitors are reported. Considerable difference in affinity of the substrates binding site to oxaloacetate was demonstrated when the enzyme was assayed in the direct and reverse directions. These results were taken to indicate that the oxidized dicarboxylate-free enzyme is an intermediate during the steady-state succinate-ubiquinone reductase reaction, whereas the reduced dicarboxylate-free enzyme is an intermediate of the steady-state ubiquinol-fumarate reductase reaction. No difference in the reactivity of the substrate-protected cysteine and arginine residues was found when the pseudo-first-order rate constants for N-ethylmaleimide and phenylglyoxal inhibition were determined for oxidized and quinol-reduced enzyme. Quinol-fumarate reductase activity was reconstituted from the soluble succinate dehydrogenase and low-molecular-mass ubiquinone reactivity conferring protein(s). No reduction of cytochrome b was observed in the presence of quinol generating system, whereas S-3 low temperature EPR-detectable iron-sulfur center was completely reduced by quinol under equilibrium (without fumarate) or steady-state (in the presence of fumarate). No significant reduction of ferredoxin type iron-sulfur centers was detected during the steady-state quinol-fumarate oxidoreductase reaction. The data obtained eliminate participation of cytochrome b in the quinol-fumarate reductase reaction and show that the rate limiting step of the overall reaction lies between iron-sulfur center S-3 and lower midpoint potential redox components of the enzyme.
本文描述了一个简单的系统，用于在烟酰胺腺嘌呤二核苷酸（NADH）、烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）-醌还原酶（DT-黄递酶）和合适的醌存在的情况下，对纯化的可溶性牛心琥珀酸-泛醌还原酶催化的醌-富马酸还原酶反应进行需氧分析。该反应受羧菌灵抑制，这表明相同的醌/醌醇结合位点参与了从琥珀酸到泛醌以及从泛醇到富马酸的电子转移。报告了该反应在两个方向上的动力学性质以及该酶底物结合位点对底物（产物）和竞争性抑制剂的比较亲和力。当在正向和反向测定该酶时，发现底物结合位点对草酰乙酸的亲和力存在显著差异。这些结果表明，氧化态无二羧酸酶是稳态琥珀酸-泛醌还原酶反应的中间体，而还原态无二羧酸酶是稳态泛醇-富马酸还原酶反应的中间体。当测定氧化态和泛醇还原态酶对N-乙基马来酰亚胺和苯乙二醛抑制的伪一级速率常数时，未发现底物保护的半胱氨酸和精氨酸残基的反应性存在差异。泛醇-富马酸还原酶活性由可溶性琥珀酸脱氢酶和赋予低分子量泛醌反应性的蛋白质重构而成。在存在泛醇生成系统的情况下，未观察到细胞色素b的还原，而在平衡状态（无富马酸）或稳态（存在富马酸）下，S-3低温电子顺磁共振（EPR）可检测的铁硫中心被泛醇完全还原。在稳态泛醇-富马酸氧化还原酶反应过程中，未检测到铁氧化还原蛋白型铁硫中心的显著还原。所获得的数据排除了细胞色素b参与泛醇-富马酸还原酶反应，并表明整个反应的限速步骤位于铁硫中心S-3和该酶较低中点电位的氧化还原组分之间。
