COORDINATION OF SELENIUM TO MOLYBDENUM IN FORMATE DEHYDROGENASE-H FROM ESCHERICHIA-COLI
COORDINATION OF SELENIUM TO MOLYBDENUM IN FORMATE DEHYDROGENASE-H FROM ESCHERICHIA-COLI
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DOI：
10.1073/pnas.91.16.7708
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发表时间：
1994-08-02
影响因子：
11.1
通讯作者：
STADTMAN, TC
中科院分区：
文献类型：
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作者：
GLADYSHEV, VN;KHANGULOV, SV;STADTMAN, TC
来自大肠杆菌的甲酸脱氢酶H含有多个氧化还原中心，包括钼酸盐辅助因子、铁硫中心和硒代半胱氨酸残基（多肽链中的SeCys-140），这对催化活性至关重要。本研究表明，在天然酶中加入甲酸盐可诱导Mo(V)物种的典型信号。该信号是由电子顺磁共振（EPR）光谱检测。以Se-77取代天然同位素丰度Se，导致该信号发生转变，表明Se与Mo直接配位。140位半胱氨酸取代硒代半胱氨酸残基的突变酶催化活性降低，并表现出不同的EPR信号。由于野生型酶的硒含量测定表明每摩尔硒含量约为1克原子，因此我们得出结论，蛋白质中SeCys-140残基的硒原子与钼直接配位。甲酸脱氢酶H中硒半胱氨酸残基两侧的氨基酸序列与其他几种原核钼依赖酶中的保守序列相似。在大多数这些酶中，半胱氨酸残基，或在少数情况下，丝氨酸或硒代半胱氨酸残基，出现在甲酸脱氢酶H的SeCys-140对应的位置。与这些酶中的甲酸脱氢酶H类似，Mo的配体中至少有一个应该由蛋白质的氨基酸残基提供。这个配体可以是硒代半胱氨酸残基的硒，半胱氨酸残基的硫，或者，在丝氨酸残基的情况下，氧。
Formate dehydrogenase H from Escherichia coli contains multiple redox centers, which include a molybdopterin cofactor, an iron-sulfur center, and a selenocysteine residue (SeCys-140 in the polypeptide chain) that is essential for catalytic activity. Here we show that addition of formate to the native enzyme induces a signal typical of Mo(V) species. This signal is detected by electron paramagnetic resonance (EPR) spectroscopy. Substitution of Se-77 for natural isotope abundance Se leads to transformation of this signal, indicating a direct coordination of Se with Mo. Mutant enzyme with cysteine substituted at position 140 for the selenocysteine residue has decreased catalytic activity and exhibits a different EPR signal. Since determination of the Se content of wild-type enzyme indicates approximate to 1 gram atom per mel, we conclude that it is the Se atom of the SeCys-140 residue in the protein that is coordinated directly with Mo. The amino acid sequence flanking the selenocysteine residue in formate dehydrogenase H is similar to a conserved sequence found in several other prokaryotic molybdopterin-dependent enzymes. In most of these other enzymes a cysteine residue, or in a few cases a serine or a selenocysteine residue, occurs in the position corresponding to SeCys-140 of formate dehydrogenase H. By analogy with formate dehydrogenase H in these other enzymes, at least one of the ligands to Mo should be provided by an amino acid residue of the protein. This ligand could be the Se of a selenocysteine residue, sulfur of a cysteine residue, or, in the case of a serine residue, oxygen.