Investigation of the coordination structures of the molybdenum(v) sites of sulfite oxidizing enzymes by pulsed EPRspectroscopy

Abstract

Sulfite oxidizing enzymes (SOEs) are physiologically vital and occur in all forms of life. During the catalytic cycle the five-coordinate square-pyramidal oxo-molybdenum active site passes through the Mo(V) state, and intimate details of the structure can be obtained from pulsed EPR spectroscopy through the hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of nearby magnetic nuclei (e.g., ¹H, ²H, ¹⁷O, ³¹P) of the ligands. By employing spectrometer operational frequencies ranging from ∼4 to ∼32 GHz, it is possible to make the nuclear Zeeman interaction significantly greater than the hfi and nqi, and thereby simplify the interpretations of the spectra. The SOEs exhibit three general types of Mo(V) structures which differ in the number of nearby exchangeable protons (one, two or zero). The observed structure depends upon the organism, pH, anions in the medium, and method of reduction. One type of structure has a single exchangeable Mo–OH proton approximately in the equatorial plane and a large isotropic hfi (e.g., low pH form of chicken SOE, low pH form of plant SOE reduced by Ti(III)); the second type has two exchangeable protons with distributed orientations out of the equatorial plane and very small (or zero) isotropic hfi (e.g., high pH form of chicken SOE, high pH form of plant SOE reduced by sulfite); the third type has no nearby exchangeable protons and a coordinated oxyanion (e.g., phosphate inhibited chicken SOE, low pH form of plant SOE reduced by sulfite). An additional structural conclusion is that the orientation angle of any exchangeable equatorial ligand (OH, OH₂, PO₄³⁻) is not uniquely fixed, but is distributed around its central value by up to ±20° (depending on pH, the type of the ligand and the type of enzyme). An unexpected finding was that the axial oxo group of SOEs exchanges with ¹⁷O in solutions enriched in H₂¹⁷O. The first determination of oxo ¹⁷O nqi parameters for a well-characterized model compound, [Mo¹⁷O(SPh)₄]⁻, clearly demonstrated that ¹⁷O nqi parameters can distinguish between oxo and OH₂ ligands.