Ralstonia eutropha H16 Soluble Hydrogenase complex:

- NiFe active site deeply embedded within protein meaning that ligands must pass through protein channels

- Oxygen insensitivity perhaps due to the active site having cyanides bound to the Ni/Fe

- contained on PHG1 mega plasmid

- HoxHY dimer (HoxH and HoxY genes) = hydrogenase

- HoxFU (HoxF and HoxU genes) = NADH-dehydrogenase

- HoxE subunit?

- two additional subunits (HoxI gene)

- FMN (Flavin mononucleotide) cofactor also essential

- hypX necessary for assembly of active site

- HoxI perhaps has a role in the activation of the enzyme by binding NADPH

- (S/T)RRxFxK leader causes peptide to be translocated to periplasm through the Tat mechanism

Maturation of SH:

- HypABCDEF(and F1) req'd for maturation of all NiFe h2ases

- SH also requires HypX (involved in binding of CN- to active site)

Project:

- is it possible to express functional SH in E coli by transfecting with the individual genes? Would be good for future purposes plus for screening (high expression!)

- If not can PHG1 be expressed in E coli?

- If not, use Ralstonia eutropha as host