Evidence for a proton transfer network and a required persulfide-bond-forming cysteine residue in Ni-containing carbon monoxide dehydrogenases
Evidence for a proton transfer network and a required persulfide-bond-forming cysteine residue in Ni-containing carbon monoxide dehydrogenases
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DOI：
10.1021/bi036062u
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发表时间：
2004-05-18
期刊：
影响因子：
2.9
通讯作者：
Lindahl, PA
中科院分区：
文献类型：
--
作者：
Kim, EJ;Feng, J;Lindahl, PA
来自Moorella thermoacetica的一氧化碳脱氢酶在称为C簇的镍-铁-硫活性位点催化CO可逆氧化为CO2。提出的质子转移途径和最近发现的半胱氨酸残基的突变体的特征在于形成与C-簇的过硫键。四个被取代的组氨酸残基被单独突变为丙氨酸。His 116和His 122对催化作用是必需的，而His 113和His 119减弱催化作用但不是必需的。显著的活性被双突变体“拯救”，其中His 116被Ala取代，并且His也在位置115处引入。在双突变体中也挽救了活性，其中His 122被Ala取代，并且His同时在位置121或位置123处引入。还通过在位置116处用Cys替换His来拯救活性。C簇附近保守的Lys 587突变减弱了活性，但并没有消除它。在Lys 587和His 113均变为Ala的双突变体中，活性几乎消失。保守的Asn 284的突变也减弱活性。这些效应表明存在负责质子转移的氨基酸残基网络，而不是单一的线性途径。形成过硫化物的Cys 316的Ser突变体基本上是无活性的，并且没有显示来自C簇的电子顺磁共振信号。电子吸收和金属分析表明，该突变体中不存在C簇。过硫键似乎是必不可少的组装或C-簇的稳定性，并可能引发催化活性所需的C-簇的氧化还原化学。
Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur active site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semiconserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. The activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or position 123. Activity was also rescued by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no electron paramagnetic resonance signals originating from the C-cluster. Electronic absorption and metal analysis suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or the stability of the C-cluster, and possibly for eliciting the redox chemistry of the C-cluster required for catalytic activity.