Hydrogenases catalyze the synthesis and decomposition of molecular hydrogen (2H+ + 2e– H2). It has been widely accepted that the active site of the hydrogenases are highly sensitive to O2 and some mechanism should be required for soluble [NiFe]-hydrogenase (SH) to protect from the O2-attack. In order to obtain the insights into the O2-protection mechanism and molecular evolutional relationship between SH and complex I, we have determined the X-ray crystal structures of SH.

X-ray absorption spectroscopy (XAS) is a powerful probe to elucidate the geometry and electronic structure of metalloprotein. The Cu K-edge and S K-edge XAS of pseudoazurin (PAz) and its Met16 variants were measured. XANES of Cu K-edge showed the effective nuclear charge (Zeff) of Cu ion in Met16 aromatic substituents (Met16Phe, Met16Tyr, Met16Trp) have lower values associated with the increasing of “axial” component in the Cu active site. The Cu-Ligand distances at the active site were estimated from EXAFS. This is the first EXAFS observation of the Cu-SMet bonding for all of blue copper proteins, and the Cu-SMet86 distance in the “rhombic” site of Met16Val was determined to be 2.48 Å.