Structural differences between the active sites of the Ni-A and Ni-B states of the [NiFe] hydrogenase: an approach by quantum chemistry and single crystal ENDOR spectroscopy

The two resting forms of the active site of [NiFe] hydrogenase, Ni-A and Ni-B, have significantly different activation kinetics, but reveal nearly identical spectroscopic features which suggest the two states exhibit subtle structural differences. Previous studies have indicated that the states differ by the identity of the bridging ligand between Ni and Fe; proposals include OH-, OOH-, H2O, O2-, accompanied by modified cysteine residues. In this study, we use single crystal ENDOR spectroscopy and quantum chemical calculations within the framework of density functional theory (DFT) to calculate vibrational frequencies, H-1 and O-17 hyperfine coupling constants and g values with the aim to compare these data to experimental results obtained by crystallography, FTIR and EPR/ENDOR spectroscopy. We find that the Ni-A and Ni-B states are constitutional isomers that differ in their fine structural details. Calculated vibrational frequencies for the cyano and carbonyl ligands and H-1 and O-17 hyperfine coupling constants indicate that the bridging ligand in both Ni-A and Ni-B is indeed an OH- ligand. The difference in the isotropic hyperfine coupling constants of the beta-CH2 protons of Cys-549 is sensitive to the orientation of Cys-549; a difference of 0.5 MHz is observed experimentally for Ni-A and 1.9 MHz for Ni-B, which results from a rotation of 7 degrees about the C alpha-C beta-S gamma-Ni dihedral angle. Likewise, the difference of the intermediate g value is correlated with a rotation of Cys-546 of about 10 degrees.