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

Marta Carepo, David L. Tierney, Carlos D. Brondino, Tran Chin Yang, Ana Pamplona, Joshua Telser, Isabel Moura, José J.G. Moura*, Brian M Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

113 Scopus citations

Abstract

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 (O2- 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 (aiso(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 (O2- 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.

Original languageEnglish (US)
Pages (from-to)281-286
Number of pages6
JournalJournal of the American Chemical Society
Volume124
Issue number2
DOIs
StatePublished - Jan 16 2002

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Fingerprint Dive into the research topics of '<sup>17</sup>O ENDOR detection of a solvent-derived Ni-(OH<sub>x</sub>)-Fe bridge that is lost upon activation of the hydrogenase from Desulfovibrio gigas'. Together they form a unique fingerprint.

Cite this