¹⁷O ENDOR detection of a solvent-derived Ni-(OH_x)-Fe bridge that is lost upon activation of the hydrogenase from Desulfovibrio gigas

Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = 1/2) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O^2- or OH^-) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW ¹⁷O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H₂¹⁷O, on the active Ni-C (S = 1/2) form prepared by H²-reduction of Ni-A in H₂¹⁷O, and also on Ni-A formed by reoxidation of Ni-C in H₂¹⁷O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H₂¹⁷O solvent. However, after a reduction/reoxidation cycle (Ni-A → Ni-C → Ni-A), an ¹⁷O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A(¹⁷O) shows that the incorporated ¹⁷O has a roughly axial hyperfine tensor, A(¹⁷O) ≈ [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a_iso(¹⁷O) ≈ 11 MHz) of the hyperfine interaction indicates that a solvent-derived ¹⁷O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O^2- or OH^-) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small ⁵⁷Fe hyperfine coupling seen previously for Ni-A (A(⁵⁷Fe) ∼ 0.9 MHz) is now shown to persist in Ni-C, A(⁵⁷Fe) ∼ 0.8 MHz. However, the ¹⁷O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme's activity.