Abstract：
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.
摘要：
催化惰性的氧化O2敏感的[NiFe]氢化酶的特征在于顺磁性Ni-A和Ni-B状态的混合物。在O2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。因为部分O2还原应该产生过氧化物中间体，该物种先前被分配给已知包含Ni-A状态的[NiFe]氢化酶制剂中观察到的细长Ni-Fe桥接电子密度。然而,基于该状态对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,该提议受到了挑战。因此,我们已经考虑了Ni-A物种的替代结构，包括将硫醇盐配体氧化为次磺酸或次磺酸。这里,我们报告了新的和修订的[NiFe]氢化酶结构，并得出结论，考虑到傅里叶变换红外光谱（FTIR）的相应表征，Ni-A物种包含氧化的半胱氨酸和桥接的氢氧化物配体，而不是我们之前提出的过氧化物配体。我们的分析是困难的典型的形成的混合物的未准备氧化状态，此外，可以被X射线诱导的光电子还原。由于使用了具有特殊特性的脱硫弧菌果聚糖[NiFe]氢化酶突变体，因此可以进行本研究。除了Ni-A状态,还报告了两种反磁性未准备状态的晶体学结果，允许提出修订的氧化非活性Ni-SU模型和以分配给Ni-'Sox'物种的过硫离子为特征的新结构。
