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.Graphic abstract
Highlights
[FeFe]-hydrogenases utilize a unique cofactor denoted the H-cluster to catalyze the reversible interconversion of protons and electrons to molecular hydrogen with high turnover frequencies [1,2,3]
To gain further insight into the assembly process, we probed H-cluster intermediates using a combination of freeze-quench electron paramagnetic resonance (EPR) and X-ray absorption (XAS) spectroscopy, as well as in situ Fourier-transform infrared (FTIR) spectroscopy
The formation of the H-cluster starting from apo-HydA and [2Fe]adt was monitored on a seconds to minutes timescale using a combination of EPR, FTIR, and X-ray absorption spectroscopy
Summary
[FeFe]-hydrogenases utilize a unique cofactor denoted the H-cluster to catalyze the reversible interconversion of protons and electrons to molecular hydrogen with high turnover frequencies [1,2,3]. HydF seems to serve mainly as a scaffold protein, which in its holo-form carries a [2Fe]H subsite-like pre-catalyst that is spontaneously transferred to apo-HydA1 containing [4Fe–4S]H but lacking [2Fe]H [14, 25, 26]. Protein film electrochemistry studies of H-cluster assembly have suggested that the H-cluster formation is at least a three-step process, with initial rapid formation of a [4Fe–4S]H/[2Fe]adt adduct being followed by fusion of the two complexes and final CO release [33]. Fusion of the Fe(I)Fe(I) pre-catalyst and the [4Fe–4S]H2+ cluster on HydA1 results in a rapid electron transfer event to yield an Fe(I)Fe(II) state of the [2Fe]H site retaining the fourth CO ligand. FTIR spectroscopy further identified one-electron reduced states (Hred, Hred′) accumulating in parallel to Hox
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