Mechanistic and spectroscopic investigations of the radical SAM maturases HydE and HydG for [FeFe]-hydrogenase

Abstract

While biochemical, spectroscopic, and analytical investigations helped classify multiple phylogenetically distinct hydrogenases it was not until 2004 that Peters et al. gave the world a look at the non-proteinaceous component of the active site of a hydrogenase enzyme. The active site (H-cluster) of [FeFe]-hydrogenase was found to possess a typical [4Fe-4S] cluster bridged by the sulfur of a cysteinyl group to an iron of a uniquely decorated 2Fe subcluster that serves as the site of molecular hydrogen synthesis and oxidation. The subcluster contains two irons bridged by a dithiomethylamine (DTMA) group and a carbon monoxide ligand. In addition each iron is coordinated by a carbon monoxide and cyanide ligand. Posewitz et al. in 2004 were the first to shed light on the syntheses of these non-proteinaceous ligands when through an insertional mutagenesis study of a hydrogen producing green alga they found two radical SAM enzymes, HydE and HydG, that were required for the maturation of [FeFe]- hydrogenase. HydG has been extensively studied and been shown to produce the diatomic ligands of the H-cluster from tyrosine. In this work the substrate specificity and active site of HydG was investigated. These investigations led to a refinement of the location and mechanism of H-atom abstraction of the substrate HydG and support the identity of the C-terminal FeS cluster as a [4Fe-4S] cluster that is responsible in the later steps of diatomic production. While several crystal structures of HydE have been published, the work reported herein is the first to propose a substrate and reaction mechanism for HydE. The results point to commonly biologically available low molecular weight thiols such as L-cysteine, L-homocysteine, and mercaptopyruvate as likely substrates. More recent work has implicated mercaptopyruvate as the substrate given glyoxylate was produced under turnover conditions. Our proposed mechanism involves formation of thioformaldehyde from mercaptopyruvate. Two thioformaldehyde units may be condensed with ammonia forming the DTMA precursor. While many details remain unsolved regarding the maturation of [FeFe]-hydrogenase, our findings regarding HydE and HydG are important steps forward in the understanding of biological catalysts of hydrogen production.