title: Identification of an Isothiocyanate on the HypEF Complex Suggests a Route for Efficient Cyanyl-Group Channeling during [NiFe]-Hydrogenase Cofactor Generation.
abstract: [NiFe]-hydrogenases catalyze uptake and evolution of H2 in a wide range of microorganisms. The enzyme is characterized by an inorganic nickel/ iron cofactor, the latter of which carries carbon monoxide and cyanide ligands. In vivo generation of these ligands requires a number of auxiliary proteins, the so-called Hyp family. Initially, HypF binds and activates the precursor metabolite carbamoyl phosphate. HypF catalyzes removal of phosphate and transfers the carbamate group to HypE. In an ATP-dependent condensation reaction, the C-terminal cysteinyl residue of HypE is modified to what has been interpreted as thiocyanate. This group is the direct precursor of the cyanide ligands of the [NiFe]-hydrogenase active site cofactor. We present a FT-IR analysis of HypE and HypF as isolated from E. coli. We follow the HypF-catalyzed cyanation of HypE in vitro and screen for the influence of carbamoyl phosphate and ATP. To elucidate on the differences between HypE and the HypEF complex, spectro-electrochemistry was used to map the vibrational Stark effect of naturally cyanated HypE. The IR signature of HypE could ultimately be assigned to isothiocyanate (-N=C=S) rather than thiocyanate (-S-C≡N). This has important implications for cyanyl-group channeling during [NiFe]-hydrogenase cofactor generation.
authors.affiliation: Department of Physics, Freie Universität Berlin, Berlin, Germany.
authors.forename: Sven T
authors.initials: ST
authors.lastname: Stripp
authors.affiliation: Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Lindenstrauss
authors.affiliation: Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany.
authors.forename: R Gary
authors.initials: RG
authors.lastname: Sawers
authors.affiliation: Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany.
authors.forename: Basem
authors.initials: B
authors.lastname: Soboh
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D017879:Isothiocyanates / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D043367:Ralstonia / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; C108878:HypF protein, Bacteria; D017879:Isothiocyanates; C085871:hypE protein, Bacteria; C037152:isothiocyanic acid