Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for “difficult” nuclei

Abstract

The catalytic mechanisms of sulfite oxidizing enzymes (SOEs) have been investigated by multi-frequency pulsed EPR measurements of “difficult” magnetic nuclei (35,37Cl, 33S, 17O) associated with the Mo(V) center. Extensive DFT calculations have been used to relate the experimental magnetic resonance parameters of these nuclei to specific active site structures. This combined spectroscopic and computational approach has provided new insights concerning the structure/function relationships of the active sites of SOEs, including: (i) the exchange of oxo ligands; (ii) the nature of the blocked forms; and (iii) the role of Cl− in low pH forms.

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

“…11, 27 The observation of characteristic 33 S ESEEM has established that Species 1 is indeed a blocked form, as was initially postulated based on the lack of an observable − OH ligand proton. 24 17 O ESEEM has revealed two types of oxygen nuclei: (1) a strongly coupled oxygen with a hyperfine interaction ( hfi ) constant ( A ) of ~18 MHz and (2) a weakly coupled oxygen with A ≅ 5 MHz.…”

“…11 In SDH and all non-plant forms of SO, integral heme centers accept the electrons from the reduced molybdenum center.…”

“…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).…”

Bioinspired functional analogs of the active site of molybdenum enzymes: Intermediates and mechanisms

“…Electron transfer from Mo IV to Fe III heme can in principle precede or follow SO 4 2− dissociation [13]. Indeed, so-called blocked forms of the enzyme are suggested to possess a SO 4 2− product bound to Mo V [13] although a SO 3 2− substrate bound to Mo V was also detected under high SO 3 2− concentrations by EPR spectroscopy [35][36][37]. Hence, both product and substrate can block the active site.…”

“…The phosphane dissociation allows for coordination of hydroxide at the resulting free coordination site giving the above mentioned hydroxido oxido complex Mo V (L) 2 O(OH) and hence closing the catalytic cycle [93,96]. Interestingly, in a mutant of human sulfite oxidase strong EPR evidence suggests that the substrate sulfite can indeed coordinate to the molybdenum(V) center forming an off-loop species [35][36][37]. Similarly, the substrate xanthine can bind to Mo V in XO yielding an off-loop species [39].…”