Four Cys residues in heterodimeric 2-oxoacid:ferredoxin oxidoreductase are required for CoA-dependent oxidative decarboxylation but not for a non-oxidative decarboxylation
Four Cys residues in heterodimeric 2-oxoacid:ferredoxin oxidoreductase are required for CoA-dependent oxidative decarboxylation but not for a non-oxidative decarboxylation
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异二聚体 2-含氧酸：铁氧还蛋白氧化还原酶中的四个 Cys 残基是 CoA 依赖性氧化脱羧所需的，但非氧化脱羧不需要
DOI：
10.1016/j.bbapap.2014.01.015
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
and T. Wakagi
中科院分区：
文献类型：
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作者：
Z. Yan;S. Fushinobu;and T. Wakagi
来自托科代硫化叶菌 (StOFOR) 的异二聚体 2-含氧酸：铁氧还蛋白氧化还原酶 (OFOR) 仅具有一个 [4Fe–4S]2 + 簇，由 4 个半胱氨酸残基（C12、C15、C46 和 C197）连接。该酶没有其他半胱氨酸。为了阐明这些 Cys 残基在 2-含氧酸氧化脱羧过程中保持铁硫簇的作用，将这些 Cys 残基中的一个或两个用 Ala 取代以产生 C12A、C15A、C46A、C197A 和 C12/15A 突变体。所有突变体都显示出铁硫簇的丢失，除了 C197A 突变体保留了一些未识别类型的铁硫簇。在 OFOR 中添加丙酮酸后，野生型酶在 320 nm 处显示出发色团，并显示出与羟乙基-ThDP 自由基相对应的稳定大 EPR 信号，而突变型酶则没有显示出任何自由基中间体的形成或乙酰辅酶 A 的产生，这表明完整的 [4Fe-4S] 簇对于这些过程是必需的。在没有电子受体的情况下，添加 CoA 后，野生型 OFOR 中的稳定自由基中间体迅速分解。据报道，丙酮酸的非氧化脱羧作用产生乙醛，其他 OFOR 需要 CoA，但 StOFOR 催化丙酮酸产生乙醛，不依赖于 CoA，无论铁硫簇是否完整 [4Fe-4S] 类型。提出了单簇 StOFOR 的综合反应方案。
Heterodimeric 2-oxoacid:ferredoxin oxidoreductase (OFOR) from Sulfolobus tokodaii (StOFOR) has only one [4Fe–4S]2 +cluster, ligated by 4 Cys residues, C12, C15, C46, and C197. The enzyme has no other Cys. To elucidate the role of these Cys residues in holding of the iron–sulfur cluster in the course of oxidative decarboxylation of a 2-oxoacid, one or two of these Cys residues was/were substituted with Ala to yield C12A, C15A, C46A, C197A and C12/15A mutants. All the mutants showed the loss of iron–sulfur cluster, except the C197A one which retained some unidentified type of iron–sulfur cluster. On addition of pyruvate to OFOR, the wild type enzyme exhibited a chromophore at 320 nm and a stable large EPR signal corresponding to a hydroxyethyl-ThDP radical, while the mutant enzymes did not show formation of any radical intermediate or production of acetyl-CoA, suggesting that the intact [4Fe–4S] cluster is necessary for these processes. The stable radical intermediate in wild type OFOR was rapidly decomposed upon addition of CoA in the absence of an electron acceptor. Non-oxidative decarboxylation of pyruvate, yielding acetaldehyde, has been reported to require CoA for other OFORs, but StOFOR catalyzed acetaldehyde production from pyruvate independent of CoA, regardless of whether the iron–sulfur cluster is intact [4Fe–4S] type or not. A comprehensive reaction scheme for StOFOR with a single cluster was proposed.