Volbeda Anne, Martin Lydie, Barbier Elodie, Gutiérrez-Sanz Oscar, De Lacey Antonio L, Liebgott Pierre-Pol, Dementin Sébastien, Rousset Marc, Fontecilla-Camps Juan C
University Grenoble Alpes, IBS, 38044, Grenoble, France.
CEA, IBS, 38044, Grenoble, France.
J Biol Inorg Chem. 2015 Jan;20(1):11-22. doi: 10.1007/s00775-014-1203-9. Epub 2014 Oct 15.
Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-'Sox' species.
催化失活的对氧气敏感的[NiFe]氢化酶的特征是顺磁性Ni-A和Ni-B状态的混合物。暴露于氧气时，部分还原状态的酶优先形成未就绪的Ni-A状态。由于部分氧气还原应产生过氧化物中间体，该物种先前被认为是在已知含有Ni-A状态的[NiFe]氢化酶制剂中观察到的拉长的Ni-Fe桥连电子密度。然而，基于该状态对紫外线照射的稳定性以及在化学或电化学氧化条件下厌氧产生该状态的可能性，这一观点受到了挑战。因此，我们考虑了Ni-A物种的替代结构，包括硫醇盐配体氧化为亚磺酸盐或亚磺酸。在这里，我们报告了新的和修订的[NiFe]氢化酶结构，并结合傅里叶变换红外光谱（FTIR）的相应表征得出结论，即Ni-A物种包含氧化的半胱氨酸和桥连氢氧化物配体，而不是我们先前提出的过氧化物配体。由于典型地形成未就绪氧化态的混合物，并且这些混合物还可以被X射线诱导的光电子还原，我们的分析变得困难。由于使用了具有特殊性质的脱硫弧菌果糖氧化还原[NiFe]氢化酶突变体，本研究得以进行。除了Ni-A状态外，还报告了两种抗磁性未就绪状态的晶体学结果，从而提出了修订的氧化失活Ni-SU模型和一种以过硫化物离子为特征的新结构，该离子被指定为Ni-'Sox'物种。
