The final steps of [FeFe]-hydrogenase maturation

Abstract

The active site (H-cluster) of [FeFe]-hydrogenases is a blueprint for the design of a biologically inspired H2-producing catalyst. The maturation process describes the preassembly and uptake of the unique [2FeH] cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the [FeFe]-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the [2FeH] cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged [2FeH] precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the [2FeH] cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor.

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

“…In vitro assembly of the H-cluster. Our present data (in combination also with earlier reports, refs [32,33]…”

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

“…The three methods consistently reveal that the first spectrally identified intermediate that accumulates to a significant extent already corresponds to the completely assembled H-cluster, which resides in the oxidized CO-inhibited state (Hox-CO). Accordingly, the fusion of the initial [4Fe-4S] 2+ and Fe(I)Fe(I) complexes has to be accompanied by the loss of one electron to result in the [4Fe-4S] 2+ -[Fe(I)Fe(II)] configuration of the H-cluster in Hox-CO [14,32,33]. A more reduced H-cluster species, Hred′-CO, could be observed only as a minor species in the early FTIR spectra, concomitantly with Hox-CO, and no other reduced species were detected at short mixing periods.…”

“…However, structural and spectroscopic information on the postulated intermediates is lacking. Thus, while the semi-synthetic approach has proven to be a powerful tool for elucidating [14]); b the complete H-cluster in the Hox state featuring a bridging CO ligand and an apical vacancy at the distal iron site of the diiron sub-complex (in a bacterial [FeFe]-hydrogenase, PDB-ID 4XDC, ref [15]); and c the synthetic diiron complex, [2Fe] adt , used for enzyme activation (color code: Fe, orange; O, red; N, blue; C, gray; H, white; protons were not resolved in protein structures; cys cysteine) the process of H-cluster formation [28,32,33], the details of the reaction between [4Fe-4S] H and the [2Fe] adt pre-catalyst still remain insufficiently understood.…”

“…Considering the final structure of the [2Fe] H subsite and the structure of the [2Fe] adt like pre-catalyst on HydF, the H-cluster assembly in vivo is a sophisticated multi-step process. Earlier H-cluster assembly studies have suggested that the reaction is initiated by the transfer of the pre-catalyst to the active site through a positively charged channel in the apo-protein [ 14 , 32 , 33 ]. In addition to the release of the pre-catalyst from HydF and its transfer to the binding site in HydA1, key steps must include the coordination of the bridging cysteine, release of a CO ligand, and diatomic ligand rearrangement, resulting in the formation of the so-called “inverted pyramid structure” of [2Fe] H with a bridging CO ligand [ 34 ].…”

“…However, structural and spectroscopic information on the postulated intermediates is lacking. Thus, while the semi-synthetic approach has proven to be a powerful tool for elucidating the process of H-cluster formation [ 28 , 32 , 33 ], the details of the reaction between [4Fe–4S] H and the [2Fe] adt pre-catalyst still remain insufficiently understood.…”