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

Abstract

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, O(2-), 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, (1)H and (17)O 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 (1)H and (17)O 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 β-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 Cα-Cβ-Sγ-Ni dihedral angle. Likewise, the difference of the intermediate g value is correlated with a rotation of Cys-546 of about 10 degrees.

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

“…The isotope labelling study instead proposed that the Ni-A state could contain a bridging hydroxide in a different orientation to that seen in Ni-B ( Figure 8 ii). This is supported by recent work by Barilone et al [ 55 ] who used single crystal ENDOR spectroscopy to confirm the Ni-A bridging ligand as a hydroxide, again suggesting that the difference in the structure of Ni-B and Ni-A is due to rotation at the nickel, specifically identifying a cysteine side chain as the mobile element. Altogether, the issue of the structural identity of Ni-A remains contentious, rendering the task of explaining why O 2 sensitive NiFe hydrogenases are slow to reactivate very difficult.…”

Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from E. coli

“…In the presence of O 2 , two different O 2 -inhibited, "super-oxidized" states may be enriched: Ni-A and Ni-B (Ni 3+ /Fe 2+ ). Ni-A represents an "unready" species that converts slowly into Ni-SI while Ni-B is readily activated under reducing conditions [42,43] [44][45][46]. On structural level the kinetic activation differences between Ni-A and Ni-B remain elusive.…”

A strenuous experimental journey searching for spectroscopic evidence of a bridging nickel–iron–hydride in [NiFe] hydrogenase

“…The Ni is coordinated to the protein matrix by four cysteinyl thiolates (S), two of which serve as bridging ligands to Fe. In addition to these two S ligands, there could be a third ligand to bridge the two metal atoms in several enzymatic states; for example, a hydroxide (Dole et al, 1997;Gu et al, 2003;Gastel et al, 2005;Volbeda et al, 2015;Barilone et al, 2015) and a possible hydride (Ni-H-Fe) in the active forms Ni-C and Ni-R (Dole et al, 1997;Amara et al, 1999;Brecht et al, 2003;Foerster et al, 2003) (Fig. 1a).…”