Direct detection of the proton-containing group coordinated to Mo(V) in the high pH form of chicken liver sulfite oxidase by refocused primary ESEEM spectroscopy: Structural and mechanistic implications

A refocused primary electron spin-echo envelope modulation (RP ESEEM) techn ique and an adjustable frequency S/C-band pulsed EPR spectrometer have been used to produce ESEEM spectra with the lines due to nearby protons being g reatly enhanced relative to those due to distant matrix protons. Applicatio n of this technique to the high pH (hpH) form of the Mo(V) center of sulfit e oxidase has enabled nearby protons to be directly detected for the first time. Simulation of the RP ESEEM spectrum of the hpH form, suggests the pre sence of two nearby protons that have distributed hyperfine interactions (h fi); these protons are ascribed to a Mo-V-OH group with strong H-bonding in teractions to other nearby proton donors or to the presence of a coordinate d H2O ligand. The RP ESEEM technique promises to be widely applicable to th e investigation of mutant forms of SO with altered Mo centers and paramagne tic centers in other metalloproteins where a nearby proton of interest is o ften masked by much more numerous distant protons and where high spectral r esolution is not required. The distinctive differences in the CW and pulsed EPR spectra of the lpH and hpH forms are proposed to result from differenc es of the Mo-V-OH torsional angle and variations in the H-bonding interacti ons, which control the orientation of the Mo-V-OH proton(s) relative to the half-filled 4d(xy) orbital. The large isotropic hfi for the lpH form is su ggested to result from an intramolecular MoV-OH ... S-cys hydrogen bonding interaction that places the proton of the Mo-V-OH group in the equatorial p lane of the square pyramidal oxo-Mo(V) center of SO.