An Electron-bifurcating Caffeyl-CoA Reductase
An Electron-bifurcating Caffeyl-CoA Reductase
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DOI：
10.1074/jbc.m112.444919
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发表时间：
2013-04-19
影响因子：
4.8
通讯作者：
Mueller, Volker
中科院分区：
文献类型：
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作者：
Bertsch, Johannes;Parthasarathy, Anutthaman;Mueller, Volker
低电位电子载体铁氧还蛋白（E-0'接近-500 mV）用于为产乙酸细菌伍氏醋酸杆菌中唯一的生物能偶联位点（钠动力铁氧还蛋白：NAD（+）氧化还原酶（Rnf））提供燃料。因为铁氧还蛋白与生理电子供体的还原是高度吸能的，所以它必须与放能反应耦合。一个候选者是NADH依赖性咖啡酰辅酶A还原。我们从A中纯化了一个复合物。woodii含有咖啡酰辅酶A还原酶和电子转移黄素蛋白。该酶含有三个由carCDE基因编码的亚基，并且预测除了FAD之外还具有两个[4Fe-4S]簇作为辅因子，这与4摩尔FAD、9摩尔铁和9摩尔酸不稳定硫的实验测定一致。该酶复合物催化还原甲基紫精的咖啡酰辅酶A依赖性氧化。以NADH为供体，它催化咖啡酰辅酶A还原，但该反应被加入铁氧还蛋白高度刺激。光谱分析表明，铁氧还蛋白和咖啡酰辅酶A同时减少，并确定了1.3：1的化学计量。显然，A. woodii使用黄素依赖性电子分叉的新机制，通过将其与咖啡酰-CoA的放能NADH依赖性还原偶联，来驱动以NADH作为还原剂的内能铁氧还蛋白还原。
A low potential electron carrier ferredoxin (E-0' approximate to -500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin: NAD(+) oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA.