An anchoring role for FeS clusters: Chelation of the amino acid moiety of S-adenosylmethionine to the unique iron site of the [4Fe-4S] cluster of pyruvate formate-lyase activating enzyme
An anchoring role for FeS clusters: Chelation of the amino acid moiety of S-adenosylmethionine to the unique iron site of the [4Fe-4S] cluster of pyruvate formate-lyase activating enzyme
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DOI：
10.1021/ja027078v
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发表时间：
2002-09-25
影响因子：
15
通讯作者：
Hoffman, BM
中科院分区：
文献类型：
--
作者：
Walsby, CJ;Ortillo, D;Hoffman, BM
丙酮酸甲酸裂解酶激活酶（PFL-AE）通过催化活性[4Fe−4S]+簇与S-腺苷甲硫氨酸（S-Met）的相互作用在丙酮酸甲酸裂解酶上产生催化必需的甘氨酰自由基。与其他Fe−S/β-Met酶家族的成员一样，PFL-AE被认为通过产生β-Met衍生的5 '-脱氧腺苷自由基中间体发挥作用;然而，产生这种自由基的机制步骤仍有待阐明。虽然所有的Fe−S/Met酶家族的成员似乎都在[4Fe−4S]簇中有一个独特的铁位点，但基于保守的三个半胱氨酸簇结合基序的存在，这个独特位点的作用一直是难以捉摸的。在这里，我们利用35 GHz脉冲电子核双共振（ENDOR）研究PFL-AE的[4Fe−4S]+簇与同位素标记的蛋氨酸（表示为[1+/蛋氨酸]）的复合物，以表明独特的铁用于锚蛋氨酸催化。用~（17）O标记的Met在簇合物上的偶联频率为A = 12.2 MHz，与簇合物上的铁离子直接配位一致。这是支持13 C-ENDOR与羧基碳标记的13 C，它显示了0.71 MHz的超精细耦合。在氨基上富集15 N的蛋氨酸产生A（15 N）= 5.8MHz的光谱，与氨基直接配位到簇的独特铁一致。总之，结果表明，[4Fe−4S]簇的独特铁通过与甲硫氨酸片段的氨基和羧基形成经典的N/O螯合物来固定蛋氨酸。
Pyruvate formate-lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical on pyruvate formate-lyase via the interaction of the catalytically active [4Fe−4S]+cluster with S-adenosylmethionine (AdoMet). Like other members of the Fe−S/AdoMet family of enzymes, PFL-AE is thought to function via generation of an AdoMet-derived 5‘-deoxyadenosyl radical intermediate; however, the mechanistic steps by which this radical is generated remain to be elucidated. While all of the members of the Fe−S/AdoMet family of enzymes appear to have a unique iron site in the [4Fe−4S] cluster, based on the presence of a conserved three-cysteine cluster binding motif, the role of this unique site has been elusive. Here we utilize 35-GHz pulsed electron nuclear double resonance (ENDOR) studies of the [4Fe−4S]+cluster of PFL-AE in complex with isotopically labeled AdoMet (denoted [1+/AdoMet]) to show that the unique iron serves to anchor the AdoMet for catalysis. AdoMet labeled with17O at the carboxylate shows a coupling ofA= 12.2 MHz, consistent with direct coordination of the carboxylate to the unique iron of the cluster. This is supported by13C-ENDOR with the carboxylato carbon labeled with13C, which shows a hyperfine coupling of 0.71 MHz. AdoMet enriched with15N at the amino position gives rise to a spectrum withA(15N) = 5.8 MHz, consistent with direct coordination of the amino group to a unique iron of the cluster. Together, the results demonstrate that the unique iron of the [4Fe−4S] cluster anchors AdoMet by forming a classical N/O chelate with the amino and carboxylato groups of the methionine fragment.