Resonance Raman Spectroscopic Analysis of the [NiFe] Active Site and the Proximal [4Fe-3S] Cluster of an O2-Tolerant Membrane-Bound Hydrogenase in the Crystalline State
Resonance Raman Spectroscopic Analysis of the [NiFe] Active Site and the Proximal [4Fe-3S] Cluster of an O2-Tolerant Membrane-Bound Hydrogenase in the Crystalline State
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DOI：
10.1021/acs.jpcb.5b04119
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发表时间：
2015-10-29
影响因子：
3.3
通讯作者：
Hildebrandt, Peter
中科院分区：
文献类型：
--
作者：
Siebert, Elisabeth;Rippers, Yvonne;Hildebrandt, Peter
我们应用共振拉曼（RR）光谱对耐氧2膜结合[NiFe]氢化酶（MBH）的单蛋白晶体进行了研究，该酶催化氢-2分裂为质子和电子。利用统计方法和全局拟合程序，对65 MBH样品在不同氧化还原状态下的RR光谱进行了分析，根据活性位点和前所未有的近端[4Fe-3S]簇的各自成分光谱进行了分析。将各辅因子的组成谱与量子力学/分子力学（QM/MM）方法计算得到的谱进行了比较。因此，最近发现的[4Fe-3S]簇中一个铁的羟基配位得到了证实。氧化MBH晶体的红外（IR）显微镜显示，[NiFe]活性位点位于Ni-r- b [Ni(III)]和Ni-r- s [Ni(II)]状态，而这些晶体的RR测量显示，Ni-a- s [Ni(II)]状态是这些晶体的主要光谱成分，表明它是通过假设的桥接羟基或水配体的光解作用在原位形成的。在QM/MM计算的基础上，单个频带频率可以与Ni-a-S态和Ni-L态的结构参数相关联。Ni-L态是由h -2还原活性位态的桥接氢化物光解作用形成的。
We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O-2-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H-2 into protons and electrons. RR spectra taken from 65 MBH samples in different redox states were analyzed in terms of the respective component spectra of the active site and the unprecedented proximal [4Fe-3S] cluster using a combination of statistical methods and global fitting procedures. These component spectra of the individual cofactors were compared with calculated spectra obtained by quantum mechanics/molecular mechanics (QM/MM) methods. Thus, the recently discovered hydroxyl-coordination of one iron in the [4Fe-3S] cluster was confirmed. Infrared (IR) microscopy of oxidized MBH crystals revealed the [NiFe] active site to be in the Ni-r-B [Ni(III)] and Ni-r-S [Ni(II)] states, whereas RR measurements of these crystals uncovered the Ni-a-S [Ni(II)] state as the main spectral component, suggesting its in situ formation via photodissociation of the assumed bridging hydroxyl or water ligand. On the basis of QM/MM calculations, individual band frequencies could be correlated with structural parameters for the Ni-a-S state as well as for the Ni-L state, which is formed upon photodissociation of the bridging hydride of H-2-reduced active site states.