Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases
Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases
复制标题
DOI：
10.1038/s41467-018-07140-x
复制
发表时间：
2018-11-09
影响因子：
16.6
通讯作者：
Winkler, Martin
中科院分区：
文献类型：
--
作者：
Duan, Jifu;Senger, Moritz;Winkler, Martin
[FeFe]-氢化酶无与伦比的催化周转率需要极其有效的质子转移 (PT) 途径，以将质子作为底物或产物在本体水和催化中心之间穿梭。对于梭菌 [FeFe]-氢化酶 CpI，已经提出并分析了这样的途径，但主要是基于理论基础。在这里，从动力学、光谱学和晶体学角度对两种不同 [FeFe]-氢化酶（CpI 和 HydA1）的 11 种酶变体（在假定的 PT 途径中进行了取代）进行了动力学、光谱学和晶体学检查，以为其在氢转换中的作用提供可靠的实验证据。通过定点诱变靶向 PT 途径的关键残基显着改变了这些变体的 pH 活性谱，并且在 H-2 存在的情况下，它们的辅因子被捕获在表明质子转移被阻止的中间状态。此外，晶体结构连贯地解释了残留活性的各个水平，证明了例如被困的 H2O 分子如何挽救中断的 PT 通路。这些特征提供了确凿的证据，证明目标位置对于催化质子转移确实至关重要。
The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H-2 their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H2O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.