Evidence that the Streptomyces developmental protein WhiD, a member of the WhiB family, binds a [4Fe-4S] cluster
Evidence that the Streptomyces developmental protein WhiD, a member of the WhiB family, binds a [4Fe-4S] cluster
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DOI：
10.1074/jbc.m412622200
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发表时间：
2005-03-04
影响因子：
4.8
通讯作者：
Buttner, MJ
中科院分区：
文献类型：
--
作者：
Jakimowicz, P;Cheesman, MR;Buttner, MJ
WhiD是革兰氏阳性菌天蓝色链霉菌孢子形成后期所必需的。WhiD是WhiB样转录因子家族的成员，该家族存在于放线菌中，但不存在于其他生物中。这个蛋白质家族有四个几乎不变的半胱氨酸，这表明这些残基可能作为金属辅因子的配体。过表达的WhiD，从大肠杆菌中纯化，含有亚化学计量量的铁，并具有[2Fe-2S]簇的吸收光谱特征。Fe-S团簇在厌氧条件下重构后，WhiD含有约4个铁原子/单体和约4个硫离子，并具有[4Fe-4S]团簇的吸收光谱特征。重构的WhiD没有给出制备的电子顺磁共振信号，但是在用连二亚硫酸盐还原后，给出了电子顺磁共振信号（g类似于2.06，1.94），这与[4Fe-4S]（2+）簇单电子还原成电子自旋为S = 1/2的[4Fe-4S]（1+）状态一致。厌氧重组的[4Fe-4S]簇是氧敏感的。在暴露于空气中时，在410和505 nm处的吸收首先增加，然后随时间显示出稳定的下降，直到蛋白质在近UV/可见光区无色。这些变化与氧诱导的从[4Fe-4S]到[2Fe-2S]簇的变化一致，随后簇从蛋白质中完全丢失。四个保守的半胱氨酸残基Cys-23、-53、-56和-62中的每一个都是WhiD体内功能所必需的。
WhiD is required for the late stages of sporulation in the Gram-positive bacterium Streptomyces coelicolor. WhiD is a member of the WhiB-like family of putative transcription factors that are present throughout the actinomycetes but absent from other organisms. This family of proteins has four near-invariant cysteines, suggesting that these residues might act as ligands for a metal cofactor. Overexpressed WhiD, purified from Escherichia coli, contained substoichiometric amounts of iron and had an absorption spectrum characteristic of a [2Fe-2S] cluster. After Fe-S cluster reconstitution under anaerobic conditions, WhiD contained similar to4 iron atoms/monomer and similar amounts of sulfide ion and gave an absorption spectrum characteristic of a [4Fe-4S] cluster. Reconstituted WhiD gave no electron paramagnetic resonance signal as prepared but, after reduction with dithionite, gave an electron paramagnetic resonance signal (g similar to2.06, 1.94) consistent with a one-electron reduction of a [4Fe-4S](2+) cluster to a [4Fe-4S](1+) state with electron spin of S = 1/2. The anaerobically reconstituted [4Fe-4S] cluster was oxygen sensitive. Upon exposure to air, absorption at 410 and 505 nm first increased and then showed a steady decrease with time until the protein was colorless in the near UV/visible region. These changes are consistent with an oxygen-induced change from a [4Fe-4S] to a [2Fe-2S] cluster, followed by complete loss of cluster from the protein. Each of the four conserved cysteine residues, Cys-23, -53, -56, and -62, was essential for WhiD function in vivo.