Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thermatoga maritima

• Published in: FEBS letters 2005-09, Vol.579 (22), p.5055-5060
• Main Authors: Rubach, Jon K., Brazzolotto, Xavier, Gaillard, Jacques, Fontecave, Marc
• Format: Article
• Language: English
• Subjects: 1,4-dithio-d,l-threitol; 5′-deoxyadenosine; AdoH; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; DTT; Escherichia coli Proteins - metabolism; Fe-only hydrogenase; HydE; HydG; Hydrogenase - biosynthesis; Hydrogenase - chemistry; Hydrogenase - genetics; IPTG; Iron - metabolism; Iron-Sulfur Proteins - biosynthesis; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - genetics; isopropyl-β-d-thiogalactopyranoside; Maturation; Molecular Sequence Data; Molecular Structure; Radical-SAM; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; S-adenosylmethionine; S-Adenosylmethionine - metabolism; SAM; Sequence Alignment; Sulfur - metabolism; Sulfur donor; Thermotoga maritima; Thermotoga maritima - enzymology; Trans-Activators - metabolism
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2005.07.092

Fe-only hydrogenases contain a di-iron active site complex, in which the two Fe atoms have carbon monoxide and cyanide ligands and are linked together by a putative di(thiomethyl)amine molecule. We have cloned, purified and characterized the HydE and HydG proteins, thought to be involved in the biosynthesis of this peculiar metal site, from the thermophilic organism Thermotoga maritima. The HydE protein anaerobically reconstituted with iron and sulfide binds two [4Fe–4S] clusters, as characterized by UV and EPR spectroscopy. The HydG protein binds one [4Fe–4S] cluster, and probably an additional one. Both enzymes are able to reductively cleave S-adenosylmethionine (SAM) when reduced by dithionite, confirming that they are Radical-SAM enzymes.