Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states
Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states
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DOI：
10.1007/s00775-014-1203-9
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发表时间：
2015-01-01
影响因子：
3
通讯作者：
Fontecilla-Camps, Juan C.
中科院分区：
文献类型：
--
作者：
Volbeda, Anne;Martin, Lydie;Fontecilla-Camps, Juan C.
无催化活性的氧化型O-2-敏感性[NiFe]-氢化酶的特征在于具有顺磁性Ni-A和Ni-B态的混合物。在O-2暴露时，处于部分还原状态的酶优先形成未准备好的Ni-A状态。由于部分O-2还原应产生过氧化物中间体，这种物质以前被分配到延长的Ni-Fe桥电子密度观察到的制备[NiFe]-氢化酶已知含有Ni-A状态。然而，基于这种状态对UV光暴露的稳定性以及在化学或电化学氧化条件下厌氧产生它的可能性，这一主张受到了挑战。因此，我们已经考虑了Ni-A物质的替代结构，包括硫醇盐配体氧化为次磺酸盐或次磺酸。在这里，我们报告新的和修订的[NiFe]-氢化酶的结构和结论，考虑到相应的表征傅里叶变换红外光谱（FTIR），Ni-A物种包含氧化的半胱氨酸和桥接氢氧化物配体，而不是我们之前提出的过氧化物配体。我们的分析呈现困难的典型形成的混合物的未准备好的氧化状态，此外，可以减少X射线诱导的光电子。本研究可以进行感谢使用脱硫弧菌fructosovorans [NiFe]-氢化酶突变体具有特殊性能。除了Ni-A状态，晶体学结果也报告了两个抗磁性的未准备状态，允许修改的氧化非活性Ni-SU模型的建议和一个新的结构，其特征在于由一个过硫化物离子，被分配到一个Ni-'S-ox'物种。
Catalytically inactive oxidized O-2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O-2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O-2 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-'S-ox' species.