Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases

Abstract

The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H2 their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H2O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.

The final steps of [FeFe]-hydrogenase maturation

“…FTIR spectroscopy conveys deeper insights into the vibrational modes of the Fe-bound CO and CN − ligands (2,100 to 1,780 cm −1 ), which can be monitored without the interference of the strong vibrational signals from water or the polypeptide backbone (amide bands). Noncatalytic and catalytic states can be accumulated under certain conditions, reflected by the appearance of state-specific patterns of CO and CN − stretching frequency signals of the [2Fe H ] site (37)(38)(39)(40)(41). We investigated the level of cofactor occupancy for all holoproteins via ATR-FTIR spectroscopy (Fig.…”

Monophosphine‐substituted diiron azadithiolate complexes: New syntheses, characterization and electrochemical properties

“…Furthermore, treatment of Fe 2 (μ‐SH) 2 (CO) 6 with monophosphine ligands L gave the intermediates Fe 2 (μ‐SH) 2 (CO) 5 L ( A ), which were condensed with a premixed solution of paraformaldehyde and ammonium carbonate to produce the phosphine‐substituted diiron azadithiolate complexes 1a – 1f in satisfactory yields of 32–43%. It is worth noting that compared with the traditional two‐step way to prepare diiron azadithiolate complexes Fe 2 [(μ‐SCH 2 ) 2 NH](CO) 5 L containing monophosphine ligands, our novel one‐pot reaction is more simple and efficient …”

“…They proposed that proton‐coupled electron transfer facilitates reduction of the [4Fe4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. The shuttling of protons from the protein surface to the H‐cluster involves a relay of amino acids, and adt ligand can further facilitate the proton transfer . Although the mechanism of the hydrogen conversion of [FeFe]‐hydrogenases is still unclear, the [2Fe2S] cluster may play a key role in the process of catalytic hydrogen evolution, resulting in the synthesis of diiron dithiolatocarbonyl complexes as models for the active site of the [FeFe]‐hydrogenases .…”

New Approaches to an Old Problem: Original Techniques in [FeFe]-hydrogenase Research

“…[38][39][40] It starts at the H-cluster with a cysteine residue (C1) responsible for proton transfer to the H-cluster and continues with a serine (S1), glutamate (E1, E2), and an arginine residue (R). 41,42 A methionine 'above' the H-cluster (M2) was discussed as hydrogen-bonding partner to the adt ligand possibly providing an alternative proton transfer trajectory. 43 However, in Group C [FeFe]-hydrogenases neither cysteine C1 nor methionine M2 are conserved.…”