High-Resolution EPR Spectroscopy of Mo Enzymes. Sulfite Oxidases: Structural and Functional Implications

Abstract

Sulfite oxidases (SOs) are physiologically vital Mo-containing enzymes that occur in animals, plants, and bacteria and which catalyze the oxidation of sulfite to sulfate, the terminal reaction in the oxidative degradation of sulfur-containing compounds. X-ray structure determinations of SOs from several species show nearly identical coordination structures of the molybdenum active center, and a common catalytic mechanism has been proposed that involves the generation of a transient paramagnetic Mo(V) state through a series of coupled electron-proton transfer steps. This chapter describes the use of pulsed electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopic techniques to obtain information about the structure of this Mo(V) species from the hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of nearby magnetic nuclei. Variable frequency instrumentation is essential to optimize the experimental conditions for measuring the couplings of different types of nuclei (e.g., 1 H, 2 H, 31 P, and 17 O). The theoretical background necessary for understanding the ESEEM and ENDOR spectra of the Mo(V) centers of SOs is outlined, and examples of the use of advanced pulsed EPR methods (RP-ESEEM, HYSCORE, integrated four-pulse ESEEM) for structure determination are presented. The analysis of variable-frequency pulsed EPR data from SOs is aided by parallel studies of model compounds that contain key functional groups or that are isotopically labeled and thus provide benchmark data for enzymes. Enormous progress has been made on the use of high-resolution variable-frequency pulsed EPR methods to investigate the structures and mechanisms of SOs during the past ~15 years, and the future is bright for the continued development and application of this technology to SOs, other molybdenum enzymes, and other problems in metallobiochemistry.

Structural Studies of the Molybdenum Center of Mitochondrial Amidoxime Reducing Component (mARC) by Pulsed EPR Spectroscopy and 17O-Labeling

“…This, however, does not necessarily mean that Ser is the ligand. In fact, the DFT calculations for Met and Cys (see Supplementary Information) resulted in 1 H and 17 O hfi parameters similar to those obtained for Ser, and all of the experimental EPR data for mARC-2 are generally similar to those obtained earlier for SO (24, 27). Therefore, it is unlikely that DFT calculations can distinguish between various possible Mo(V) centers with the general structures (MPT)Mo V O(OH)-O-… or (MPT)Mo V O(OH)-S-…, especially taking into account the unknown orientation of the protein-derived ligand.…”

Elucidating the Catalytic Mechanism of Sulfite Oxidizing Enzymes Using Structural, Spectroscopic, and Kinetic Analyses

“…The coordination structures of the Mo(V) sites of SOEs obtained by pulsed EPR spectroscopy have been discussed previously (46), and the applications of pulsed EPR methods to SOEs have also been extensively reviewed (47). Therefore, only a few more recent EPR structural results for SOEs are presented here.…”

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

“…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. 20 The orientation of this − OH ligand depends on the buffer pH, however, and the corresponding structural forms, which result in very different EPR spectra, were historically labeled “low-pH” ( lpH , pH ≤ 7) and “high-pH” ( hpH , pH ≥ 9).…”