title: Studies of the Mo(V) Center of the Y343F Mutant of Human Sulfite Oxidase by Variable Frequency Pulsed EPR Spectroscopy.
abstract: The Mo(V) forms of the Tyr343Phe (Y343F) mutant of human sulfite oxidase (SO) have been investigated by continuous wave (CW) and variable frequency pulsed EPR spectroscopies as a function of pH. The CW EPR spectrum recorded at low pH (∼6.9) has g-values similar to those known for the low-pH form of the native vertebrate SO (original lpH form); however, unlike the spectrum of original lpH SO, it does not show any hyperfine splittings from a nearby exchangeable proton. The detailed electron spin echo (ESE) envelope modulation (ESEEM) and pulsed electron-nuclear double resonance (ENDOR) experiments also did not reveal any nearby protons that could belong to an exchangeable ligand at the molybdenum center. These results suggest that under low-pH conditions the active site of Y343F SO is in the "blocked" form, with the Mo(V) center coordinated by sulfate. With increasing pH the EPR signal from the "blocked" form decreases, while a signal similar to that of the original lpH form appears and becomes the dominant signal at pH>9. In addition, both the CW EPR and ESE-detected field sweep spectra reveal a considerable contribution from a signal similar to that usually detected for the high-pH form of native vertebrate SO (original hpH form). The nearby exchangeable protons in both of the component forms observed at high pH were studied by the ESEEM spectroscopy. These results indicate that the Y343F mutation increases the apparent pK(a) of the transition from the lpH to hpH forms by ∼2 pH units.
authors.affiliation: Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA.
authors.forename: Arnold M
authors.initials: AM
authors.lastname: Raitsimring
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Astashkin
authors.forename: Changjian
authors.initials: C
authors.lastname: Feng
authors.forename: Heather L
authors.initials: HL
authors.lastname: Wilson
authors.forename: K V
authors.initials: KV
authors.lastname: Rajagopalan
authors.forename: John H
authors.initials: JH
authors.lastname: Enemark