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[FeFe]-Hydrogenase Maturation: H-Cluster Assembly Intermediates Tracked by Electron Paramagnetic Resonance, Infrared, and X-Ray Absorption Spectroscopy

preprint
submitted on 21.04.2020 and posted on 22.04.2020 by Brigitta Németh, Moritz Senger, Holly J. Redman, Pierre Ceccaldi, Joan Broderick, Ann Magnuson, Sven T. Stripp, Michael Haumann, Gustav Berggren
[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe-4S] cubane cysteine-linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt = NH(CH2S)2). [FeFe]-hydrogenase apo-protein binding only the [4Fe-4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe]adt,). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined in situ electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe]adt. The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred´-CO like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster.

History

Email Address of Submitting Author

gustav.berggren@kemi.uu.se

Institution

Uppsala Universitet

Country

Sweden

ORCID For Submitting Author

0000-0002-6717-6612

Declaration of Conflict of Interest

No conflict of interest

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