Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase
Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase
复制标题
DOI：
10.1021/bi035666v
复制
发表时间：
2004-02-24
期刊：
影响因子：
2.9
通讯作者：
Huynh, BH
中科院分区：
文献类型：
--
作者：
Jameson, GNL;Cosper, MM;Huynh, BH
生物素合成酶（Biotin synthase, BioB）在s -腺苷蛋氨酸（SAM）依赖性反应中，通过在脱硫生物素的C6和C9之间插入一个硫原子，将脱硫生物素转化为生物素。从大肠杆菌中纯化的重组BioB是一个同源二聚体分子，每个单体含有一个[2Fe-2S]（2+）簇。它在体外无活性，不添加外源铁。用Fe2+和S2-对纯化后的含[2Fe-2S]的BioB进行厌氧重组，产生一种每个单体（[2Fe-2S]/[4Fe-4S] BioB）大约含有一个[2Fe-2S]（2+）和一个[4Fe-4S]（2+）簇的BioB形式。在不添加铁的情况下，[2Fe-2S]/[4Fe-4S] BioB具有活性，每个单体可产生约0.7当量的生物素。为了更好地确定Fe-S团簇在生物素b反应中的作用，利用穆斯堡尔光谱和电子顺磁共振（EPR）光谱监测了脱硫生物素转化为生物素过程中Fe-S团簇的状态。结果表明，[4Fe-4S]（2+）簇在反应过程中是稳定的，并以SAM结合的形式存在，支持了目前的共识，即[4Fe-4S]簇的功能作用是结合SAM并促进SAM的还原裂解以产生催化必需的5'-脱氧腺苷基自由基。结果还表明，大约2/3的[2Fe-2S]簇在翻转实验结束时（25℃下24 h）被降解。在翻转过程中观察到与[2Fe-2S]（+）团簇具有一致光谱性质的瞬态物质，表明[2Fe-2S]（2+）团簇的降解是由团簇的还原引起的。这种在生物素形成过程中观察到的[2Fe-2S]簇的降解与Jarrett及其同事提出的[2Fe-2S]簇的牺牲s提供功能相一致（Ugulava et al. (2001) Biochemistry 40, 8352-8358）。有趣的是，[2Fe-2S]（2+）簇的降解被发现与生物素的形成不平行。[2Fe-2S]（2+）簇的初始衰变速率比生物素的初始形成速率快约1个数量级，这表明如果[2Fe-2S]簇是生物素合成的直接S供体，那么将S插入到去硫生物素中并不是限速步骤。或者，[2Fe-2S]簇可能不是直接的S供体。相反，[2Fe-2S]簇的降解可能产生蛋白质结合的多硫化物或过硫化物，作为生物素生产的直接S供体。
Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe2+ and S2- produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, Mossbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately 2/3 of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degreesC). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel biotin formation. The initial decay rate of the [2Fe-2S](2+) cluster is about 1 order of magnitude faster than the initial formation rate of biotin, indicating that if the [2Fe-2S] cluster is the immediate S donor for biotin synthesis, insertion of S into dethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe-2S] cluster may not be the immediate S donor. Instead, degradation of the [2Fe-2S] cluster may generate a protein-bound polysulfide or persulfide that serves as the immediate S donor for biotin production.