[2Fe-2S] to [4Fe-4S] cluster conversion in Escherichia coli biotin synthase
[2Fe-2S] to [4Fe-4S] cluster conversion in Escherichia coli biotin synthase
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DOI：
10.1021/bi9706430
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发表时间：
1997-09-30
期刊：
影响因子：
2.9
通讯作者：
Johnson, MK
中科院分区：
文献类型：
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作者：
Duin, EC;Lafferty, ME;Johnson, MK
利用紫外-可见吸收光谱、变温磁圆二色谱（VTMCD）、电子顺磁共振（EPR）和共振拉曼光谱研究了重组大肠杆菌生物素合酶中Fe-S簇的类型和性质。结果证实了在有氧纯化的样品中，在同二聚体的每个亚基中存在一个S = 0 [2Fe-2S]（2+）簇，并且Fe-S伸缩频率表明不完全的半胱氨酰-S配位。然而，吸收和共振拉曼研究表明，在60%（v/v）乙二醇或甘油存在下，用连二亚硫酸盐进行厌氧还原导致两个[2Fe-2S]（2+）簇合物近化学计量转化为一个具有完全半胱氨酰-S配位的S = 0 [4Fe-4S]（2+）簇合物。化学计量和在不添加铁或硫化物的情况下实现还原簇转化的能力表明，[4Fe-4S]（2+）簇通过[2Fe-2S]（2+）簇的还原二聚在亚基界面处形成。EPR和VTMCD研究表明，在用60%（v/v）甘油处理的样品中，超过50%的Fe以[4Fe-4S]（+）簇的形式存在。[4Fe-4S]（+）团簇为混合自旋体系，基态为S = 1/2（g = 2.044，1.944，1.914）和S = 3/2（g = 5.6共振）。亚基桥接[4Fe-4S]（2+，+）簇，其可以在纯化期间经历氧化降解为[2Fe-2S]（2+）簇，被认为是需要S-腺苷甲硫氨酸的Fe-S酶的共同特征，并且通过涉及C-H或C-C键的均裂的自由基机制起作用，即，生物素合酶、厌氧核糖核苷酸还原酶、丙酮酸甲酸裂解酶、赖氨酸2，3-氨基变位酶和硫辛酸合酶。[4Fe-4S]（2+，+）簇最可能的作用在于通过直接或间接促进S-腺苷甲硫氨酸的还原性单电子裂解形成甲硫氨酸和5 ′-脱氧腺苷自由基来启动自由基机制。进一步表明氧化簇转化为[2Fe-2S]（2+）簇可能在这些自由基酶中起生理作用。通过提供响应于氧化应激而调节酶活性的方法，而没有不可逆的簇降解。
The type and properties of the Fe-S cluster in recombinant Escherichia coli biotin synthase have been investigated in as-prepared and dithionite-reduced samples using the combination of UV-visible absorption and variable-temperature magnetic circular dichroism (VTMCD), EPR, and resonance Raman spectroscopies. The results confirm the presence of one S = 0 [2Fe-2S](2+) cluster in each subunit of the homodimer in aerobically purified samples, and the Fe-S stretching frequencies suggest incomplete cysteinyl-S coordination. However, absorption and resonance Raman studies show that anaerobic reduction with dithionite in the presence of 60% (v/v) ethylene glycol or glycerol results in near-stoichiometric conversion of two [2Fe-2S](2+) clusters to form one S = 0 [4Fe-4S](2+) cluster with complete cysteinyl-S coordination. The stoichiometry and ability to effect reductive cluster conversion without the addition of iron or sulfide suggest that the [4Fe-4S](2+) cluster is formed at the subunit interface via reductive dimerization of [2Fe-2S](2+) clusters. EPR and VTMCD studies indicate that more than 50% of the Fe is present as [4Fe-4S](+) clusters in samples treated with 60% (v/v) glycerol after prolonged dithionite reduction. The [4Fe-4S](+) cluster exists as a mixed spin system with S = 1/2 (g = 2.044, 1.944, 1.914) and S = 3/2 (g = 5.6 resonance) ground states. Subunit-bridging [4Fe-4S](2+,+) clusters, that can undergo oxidative degradation to [2Fe-2S](2+) clusters during purification, are proposed to be a common feature of Fe-S enzymes that require S-adenosylmethionine and function by radical mechanisms involving the homolytic cleavage of C-H or C-C bonds, i.e., biotin synthase, anaerobic ribonucleotide reductase, pyruvate formate lyase, lysine 2,3-aminomutase, and lipoic acid synthase. The most likely role for the [4Fe-4S](2+,+) cluster lies in initiating the radical mechanism by directly or indirectly facilitating reductive one-electron cleavage of S-adenosylmethionine to form methionine and the 5'-deoxyadenosyl radical, It is further suggested that oxidative cluster conversion to [2Fe-2S](2+) clusters may play a physiological role in these radical enzymes, by providing a method of regulating enzyme activity in response to oxidative stress, without irreversible cluster degradation.