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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• 作者：Elisabeth Siebert ; Yvonne Rippers ; Stefan Frielingsdorf ; Johannes Fritsch ; Andrea Schmidt ; Jacqueline Kalms ; Sagie Katz ; Oliver Lenz ; Patrick Scheerer ; Lars Paasche ; Vladimir Pelmenschikov ; Uwe Kuhlmann ; Maria Andrea Mroginski ; Ingo Zebger ; Peter Hildebrandt
• 刊名：Journal of Physical Chemistry B
• 出版年：2015
• 出版时间：October 29, 2015
• 年：2015
• 卷：119
• 期：43
• 页码：13785-13796
• 全文大小：580K
• ISSN：1520-5207

文摘

We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O₂-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H₂ 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 H₂-reduced active site states.