Investigations on the Role of Proton-Coupled Electron Transfer in Hydrogen Activation by [FeFe]-Hydrogenase
Investigations on the Role of Proton-Coupled Electron Transfer in Hydrogen Activation by [FeFe]-Hydrogenase
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DOI：
10.1021/ja508629m
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发表时间：
2014-10-29
影响因子：
15
通讯作者：
King, Paul W.
中科院分区：
文献类型：
--
作者：
Mulder, David W.;Ratzloff, Michael W.;King, Paul W.
质子耦合电子转移（PCET）是氧化还原反应能量转换的核心过程。[FeFe]-氢化酶通过独特的金属辅因子H-簇催化分子H2的可逆活化，H-簇通过周围蛋白质环境进行微调以进行快速PCET转换。氢团簇的电子跃迁和结构跃迁与质子转移（PT）步骤的相关性在实验上还没有得到很好的解决。在这里，我们探讨如何修改保守的PT网络通过半胱氨酸？丝氨酸取代位置169近端的H-集群莱茵衣藻[FeFe]-氢化酶（CrHydA 1）的影响H-集群使用电子顺磁共振（EPR）和傅里叶变换红外（FTIR）光谱。尽管催化活性大幅下降，EPR和FTIR光谱揭示不同的H-簇催化状态下的还原和氧化条件。在H-2或连二亚硫酸钠还原处理下，EPR谱显示与还原的[4Fe-S-4]（H+）亚簇一致的信号。FTIR光谱显示上移？CO模式的能量与[2Fe]（H）子簇的氧化态增加一致，这是由DFT分析证实。与野生型CrHydA（1）的情况相反，与Hred和Hsred状态相关的光谱在Cys Ser变体中较少，表明-SH与-OH的交换改变了H-簇在稳态条件下催化循环的不同还原状态之间的平衡方式。
Proton-coupled electron transfer (PCET) is a fundamental process at the core of oxidation-reduction reactions for energy conversion. The [FeFe]-hydrogenases catalyze the reversible activation of molecular H2 through a unique metallocofactor, the H-cluster, which is finely tuned by the surrounding protein environment to undergo fast PCET transitions. The correlation of electronic and structural transitions at the H-cluster with proton-transfer (PT) steps has not been well-resolved experimentally. Here, we explore how modification of the conserved PT network via a Cys ? Ser substitution at position 169 proximal to the H-cluster of Chlamydomonas reinhardtii [FeFe]-hydrogenase (CrHydA1) affects the H-cluster using electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopy. Despite a substantial decrease in catalytic activity, the EPR and FTIR spectra reveal different H-cluster catalytic states under reducing and oxidizing conditions. Under H-2 or sodium dithionite reductive treatments, the EPR spectra show signals that are consistent with a reduced [4Fe-S-4](H+) subcluster. The FTIR spectra showed upshifts of ?CO modes to energies that are consistent with an increase in oxidation state of the [2Fe](H) subcluster, which was corroborated by DFT analysis. In contrast to the case for wild-type CrHydA(1), spectra associated with Hred and Hsred states are less populated in the Cys Ser variant, demonstrating that the exchange of -SH with -OH alters how the H-cluster equilibrates among different reduced states of the catalytic cycle under steady-state conditions.