Applications of pulsed EPR spectroscopy to structural studies of sulfite oxidizing enzymes

Sulfite oxidizing enzymes (SOEs), including sulfite oxidase (SO) and bacterial sulfite dehydrogenase (SDH), catalyze the oxidation of sulfite (SO32−) to sulfate (SO42−). The active sites of SO and SDH are nearly identical, each having a 5-coordinate, pseudo-square-pyramidal Mo with an axial oxo ligand and three equatorial sulfur donor atoms. One sulfur is from a conserved Cys residue and two are from a pyranopterindithiolene (molybdopterin, MPT) cofactor. The identity of the remaining equatorial ligand, which is solvent-exposed, varies during the catalytic cycle. Numerous in vitro studies, particularly those involving electron paramagnetic resonance (EPR) spectroscopy of the Mo(V) states of SOEs, have shown that the identity and orientation of this exchangeable equatorial ligand depends on the buffer pH, the presence and concentration of certain anions in the buffer, as well as specific point mutations in the protein. Until very recently, however, EPR has not been a practical technique for directly probing specific structures in which the solvent-exposed, exchangeable ligand is an O, OH−, H2O, SO32−, or SO42− group, because the primary O and S isotopes (16O and 32S) are magnetically silent (I = 0). This review focuses on the recent advances in the use of isotopic labeling, variable-frequency high resolution pulsed EPR spectroscopy, synthetic model compounds, and DFT calculations to elucidate the roles of various anions, point mutations, and steric factors in the formation, stabilization, and transformation of SOE active site structures.

Figure optionsDownload high-quality image (295 K)Download as PowerPoint slideHighlights
► Isotopic labeling, variable frequency pulsed EPR and density functional calculations are combined to determine the Mo(V) structures of SOEs.
► The 17O and 1H hfi parameters of the hpH and lpH forms of SO constrain the possible orientations of the OH ligand.
► lpH SO has a single Cl− that is hydrogen-bonded to the OH ligand in the equatorial plane of the 5-coordinate Mo(V) center.
► The “blocked” form of SO has coordinated sulfite.