Pulsed EPR investigations of the Mo(V) centers of the R55Q and R55M variants of sulfite dehydrogenase from Starkeya novella

摘要

Continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy have been used to characterize two variants of bacterial sulfite dehydrogenase (SDH) from Starkeya novella in which the conserved active-site arginine residue (R55) is replaced by a neutral amino acid residue. Substitution by the hydrophobic methionine residue (SDHR55M) has essentially no effect on the pH dependence of the EPR properties of the Mo(V) center, even though the X-ray structure of this variant shows that the methionine residue is rotated away from the Mo center and a sulfate anion is present in the active-site pocket (Bailey et al. in J Biol Chem 284:2053–2063, 2009). For SDHR55M only the high-pH form is observed, and samples prepared in H₂ 17O-enriched buffer show essentially the same 17O hyperfine interaction and nuclear quadrupole interaction parameters as SDHWT enzyme. However, the pH dependence of the EPR spectra of SDHR55Q, in which the positively charged arginine is replaced by the neutral hydrophilic glutamine, differs significantly from that of SDHWT. For SDHR55Q the blocked form with bound sulfate is generated at low pH, as verified by 33S couplings observed upon reduction with 33S-labeled sulfite. This observation of bound sulfate for SDHR55Q supports our previous hypothesis that sulfite-oxidizing enzymes can exhibit multiple pathways for electron transfer and product release (Emesh et al. in Biochemistry 48:2156–2163, 2009). At pH ≥ 8 the high-pH form dominates for SDHR55Q.