[FeFe]-hydrogenase maturation: H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy.

ABSTRACT: [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 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.

SUBMITTER: Nemeth B 

PROVIDER: S-EPMC7399679 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature