Direct Detection and Characterization of Chloride in the Active Site of the Low-pH Form of Sulfite Oxidase Using Electron Spin Echo Envelope Modulation Spectroscopy, Isotopic Labeling, and Density Functional Theory Calculations

Abstract

Electron spin echo envelope modulation (ESEEM) investigations were carried out on samples of the low-pH (lpH) form of vertebrate sulfite oxidase (SO) prepared with 35 Cl-and 37 Cl-enriched buffers as well as with buffer containing the natural abundance of Cl isotopes. The isotope-related changes observed in the ESEEM spectra provide direct and unequivocal evidence that Cl − is located in close proximity to the Mo(V) center of lpH SO. The measured isotropic hyperfine interaction constant of about 4 MHz ( 35 Cl) suggests that the Cl − ion is either weakly coordinated to Mo(V) at its otherwise vacant axial position, trans to the oxo ligand, or is hydrogen-bonded to the equatorial exchangeable OH ligand. Scalar relativistic all-electron density functional theory (DFT) calculations of the hyperfine and nuclear quadrupole interaction parameters, along with steric and energetic arguments, strongly support the possibility that Cl − is hydrogen-bonded to the equatorial OH ligand rather than being directly coordinated to the Mo(V).

The Mononuclear Molybdenum Enzymes

“…303 Although analysis of 35 Cl versus 37 Cl coupling in the low pH EPR signal was initially interpreted as evidence for direct coordination of chloride to molybdenum at the vacant ligand position trans to the apical Mo=O group, 304 a subsequent examination of the quadrupole interaction parameters for 35 Cl in conjunction with density functional calculations has concluded that chloride is not directly coordinated to the molybdenum but instead merely bound near the molybdenum center. 305 This latter interpretation is in agreement with the subsequently determined X-ray crystal structure of the recombinant chicken enzyme, showing chloride in the substrate binding site, 269 and with XAS analysis of the analogous bromide and iodide complexes of sulfite oxidase, which indicate that the halide binding site is some 5 Å from the molybdenum. 306 The effect of chloride on the low-pH EPR signal of both human and A. thaliana sulfite oxidases (see below), as well as a closely related sulfite dehydrogenase from the bacterium Starkeya novella , has also shown that depletion of chloride results in accumulation of a “blocked” form of the low-pH signal that lacks strong proton hyperfine coupling and has sulfate trapped coordinated to the molybdenum in the ligand position otherwise occupied by the catalytically labile equatorial Mo=O.…”

Integrated refocused virtual ESEEM: Detection of nuclear transition spectra without dead time and blind spots

“…The lines of the OH ligand proton now are clearly seen. The high-frequency feature belongs to the OH ligand proton only, while the low-frequency one is also contributed to by the nearby chlorine nucleus [45]. This high-frequency part of the RV ESEEM spectrum is in good agreement with the Davies ENDOR spectrum recorded in similar conditions earlier [38] (Fig.…”

Identity of the Exchangeable Sulfur-Containing Ligand at the Mo(V) Center of R160Q Human Sulfite Oxidase

“…As we (and others) have previously demonstrated, 11, 18, 29, 30, 65 the structure of an unknown system can be inferred through the general agreement or disagreement of its spectroscopic parameters with those calculated from theory for a given model. The goal of the calculations is not to exactly replicate the experimental results by calculating the properties for a very large number of conceivable models.…”

“…11–18 The results of numerous EPR investigations, which involved high-resolution pulsed EPR techniques such as electron spin echo (ESE) envelope modulation (ESEEM) and pulsed electron-nuclear-double resonance (ENDOR) have been summarized in recent reviews. 11, 19 For wild type ( wt ) vertebrate SO in the Mo(V) state and in the absence of inhibiting anions ( e.g ., PO 4 3− , AsO 4 3− ) the exchangeable equatorial ligand is known to be hydroxide.…”