Oxidation-State-Dependent Binding Properties of the Active Site in a Mo-Containing Formate Dehydrogenase.
Oxidation-State-Dependent Binding Properties of the Active Site in a Mo-Containing Formate Dehydrogenase.
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DOI：
10.1021/jacs.7b03958
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发表时间：
2017-07-26
影响因子：
15
通讯作者：
Hirst J
中科院分区：
文献类型：
--
作者：
Robinson WE;Bassegoda A;Reisner E;Hirst J
大肠杆菌含钼甲酸脱氢酶H （EcFDH-H）是研究二氧化碳可逆还原生成甲酸的一个强有力的模型系统。然而，FDH的催化机制目前仍有争议，在转换过程中，主要的Mo配位球是否保持饱和，或者其中一个配体是否解离以允许直接结合底物，都存在争议。在这里，我们描述了活性位点的氧化态依赖性变化如何改变其抑制剂的结合特性。利用蛋白质膜电化学，我们发现EcFDH-H对甲酸盐的氧化被N3 -、OCN -、SCN -、NO2 -和NO3 -强烈地抑制，而对CO2的还原仅被微弱地抑制，没有竞争性。在催化过程中，Mo中心在正式的Mo(VI)=S和Mo(IV) -SH态之间循环，通过模拟在不同电位、底物和抑制剂浓度下记录的计时电流数据，我们证明了选择性抑制剂与Mo(VI)=S态结合可以抑制甲酸盐氧化和CO2还原。抑制剂结合亲和力对Mo氧化态和抑制剂电子给体强度的强烈依赖表明，抑制剂（和底物）直接结合到Mo中心。考虑到我们的数据对甲酸盐氧化和二氧化碳还原机制的影响，我们建议抑制剂在硒代半胱氨酸配体解离后与Mo结合，以产生一个空的配位位点进行催化和关闭。
Molybdenum-containing formate dehydrogenase H from Escherichia coli (EcFDH-H) is a powerful model system for studies of the reversible reduction of CO2 to formate. However, the mechanism of FDH catalysis is currently under debate, and whether the primary Mo coordination sphere remains saturated or one of the ligands dissociates to allow direct substrate binding during turnover is disputed. Herein, we describe how oxidation-state-dependent changes at the active site alter its inhibitor binding properties. Using protein film electrochemistry, we show that formate oxidation by EcFDH-H is inhibited strongly and competitively by N3–, OCN–, SCN–, NO2–, and NO3–, whereas CO2 reduction is inhibited only weakly and not competitively. During catalysis, the Mo center cycles between the formal Mo(VI)=S and Mo(IV)—SH states, and by modeling chronoamperometry data recorded at different potentials and substrate and inhibitor concentrations, we demonstrate that both formate oxidation and CO2 reduction are inhibited by selective inhibitor binding to the Mo(VI)=S state. The strong dependence of inhibitor-binding affinity on both Mo oxidation state and inhibitor electron-donor strength indicates that inhibitors (and substrates) bind directly to the Mo center. We propose that inhibitors bind to the Mo following dissociation of a selenocysteine ligand to create a vacant coordination site for catalysis and close by considering the implications of our data for the mechanisms of formate oxidation and CO2 reduction.
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DOI：
10.1073/pnas.91.16.7708
发表时间：
1994-08-02
影响因子：
11.1
作者：
GLADYSHEV, VN;KHANGULOV, SV;STADTMAN, TC
通讯作者：
STADTMAN, TC
影响因子：
62.1
作者：
Hille, Russ;Hall, James;Basu, Partha
通讯作者：
Basu, Partha
影响因子：
56.9
作者：
Boyington, JC;Gladyshev, VN;Sun, PD
通讯作者：
Sun, PD
影响因子：
5.4
作者：
Hartmann, Tobias;Leimkuehler, Silke
通讯作者：
Leimkuehler, Silke
影响因子：
2.9
作者：
BARBER, MJ;MAY, HD;FERRY, JG
通讯作者：
FERRY, JG