Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis
Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis
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DOI：
10.1074/jbc.m313398200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Schindelin, H
中科院分区：
文献类型：
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作者：
Hänzelmann, P;Hernández, HL;Schindelin, H
人蛋白质MOCS 1A和MOCS 1B在钼辅因子生物合成期间催化鸟苷衍生物转化为前体Z。MOCS 1A与S-腺苷甲硫氨酸（SNMet）依赖性自由基酶具有同源性，其通过[4Fe-4S]簇还原裂解SNMet催化蛋白质和/或底物自由基的形成。MOCS 1A的序列分析显示两个高度保守的半胱氨酸基序，一个靠近N端，一个靠近C端。MOCS 1A在大肠杆菌中异源表达，并在好氧和厌氧条件下纯化。保守的半胱氨酸到丝氨酸的个别突变表明，所有的前体Z在体内的合成是必不可少的。利用紫外-可见吸收光谱、变温磁圆二色光谱、共振拉曼光谱、穆斯堡尔谱和EPR谱，结合铁和酸不稳定硫化物的分析，研究了铁硫团簇的类型和性质。结果表明，厌氧纯化的MOCS 1A是一个单体蛋白含有两个氧敏感的FeS簇，每个协调只有三个半胱氨酸残基。氧化还原活性[4Fe- 4S]（2+，+）簇通过N-末端CX 3CX 2C基序连接，这与迄今为止研究的所有其他CX 3 Met依赖性自由基酶的情况相同。MOCS 1A及其直系同源物所特有的C-末端CX 2CX 13 C基序主要连接[3Fe- 4S]（0）簇。然而，MOCS 1A可以在体外厌氧条件下重建，产生含有两个[4Fe-4S]（2+）簇的形式。N端[4Fe- 4S]（2+）簇合物被氧通过半稳定的[2Fe- 2S]（2+）簇合物中间体快速降解，C端[4Fe- 4S]（2+）簇合物被氧通过半稳定的[ 3Fe- 4S]（0）簇合物中间体快速降解。
The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine ( AdoMet)dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, Mossbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe- 4S](2+,+) cluster is ligated by an N-terminal CX3CX2C motif as is the case with all other AdoMet-dependent radical enzymes investigated thus far. A C-terminal CX2CX13C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe- 4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S] (2+) clusters. The N-terminal [4Fe- 4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe- 2S](2+) cluster intermediate, and the C-terminal [4Fe- 4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [ 3Fe- 4S](0) cluster intermediate.