Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O(2)-tolerant NAD(+)-reducing [NiFe] hydrogenase.
Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O(2)-tolerant NAD(+)-reducing [NiFe] hydrogenase.
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DOI：
10.1039/c4sc02982h
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发表时间：
2015
期刊：
影响因子：
8.4
通讯作者：
Cramer SP
中科院分区：
文献类型：
--
作者：
Lauterbach L;Wang H;Horch M;Gee LB;Yoda Y;Tanaka Y;Zebger I;Lenz O;Cramer SP
核共振振动光谱用于表征复杂的多辅因子酶中的所有含铁辅因子。氢化酶是复杂的金属酶，其催化分子氢可逆地分裂成质子和电子，基本上没有过电位。来自真养罗尔斯通氏菌的NAD+还原可溶性氢化酶（SH）即使在通常有毒的分子氧存在下也能够进行H2转化。基本反应的分子细节在很大程度上是未知的，主要是因为对酶中存在的各种金属辅因子的结构和功能的了解有限。在这里，所有含铁的辅因子的SH进行了研究57 Fe特定的核共振振动光谱（NRVS）。我们的数据提供了一个[2Fe2S]中心和四个[4Fe4S]簇的实验证据，这与氨基酸序列组成一致。当SH与NADH孵育时，仅[2Fe2S]簇和四个[4Fe4S]簇中的一个被还原。这一发现解释了大量的FeS簇和少量的FeS簇相关的信号之间的差异，如通过电子顺磁共振光谱分析的几个NAD+还原氢化酶检测到的。首次利用NRVS通过选择性13 C标记CO配体，区分了来自[NiFe]活性中心的Fe-CO和Fe-CN模式。该策略还揭示了主导单个Fe-CO模式的分子坐标。本方法探讨了复杂的振动签名的Fe-S簇和氢化酶活性位点，从而表明NRVS是一个强大的工具，用于阐明复杂的生物催化剂含有多个辅因子。
Nuclear resonance vibrational spectroscopy is used to characterize all Fe-containing cofactors in a complex multicofactor enzyme. Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.
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影响因子：
16.6
作者：
Kamali, Saeed;Wang, Hongxin;Cramer, Stephen P.
通讯作者：
Cramer, Stephen P.
影响因子：
15
作者：
Horch, Marius;Schoknecht, Janna;Zebger, Ingo
通讯作者：
Zebger, Ingo
影响因子：
15
作者：
Mitra D;George SJ;Guo Y;Kamali S;Keable S;Peters JW;Pelmenschikov V;Case DA;Cramer SP
通讯作者：
Cramer SP
影响因子：
4.8
作者：
Germer, Frauke;Zebger, Ingo;Appel, Jens
通讯作者：
Appel, Jens
影响因子：
2.9
作者：
TSUBAKI, M;SRIVASTAVA, RB;YU, NT
通讯作者：
YU, NT