ESEEM investigations of the high pH and low pH forms of chicken liver sulfite oxidase

Two-pulse and four-pulse electron spin-echo envelope modulation spectroscopy (ESEEMS) at two operational frequencies and two-dimensional hyperfine sublevel correlation spectroscopy (HYSCORES), have been used to probe the Mo-V coordination environment of sulfite oxidase in H2O and D2O solutions, buffered at pH 9.5 and 7.0 with similar to 100 mM Tris-type buffers. At pH 9.5 the ESEEM and HYSCORE results definitively reveal the presence of one solvent-exchangeable D(H) near the Mo-V center, probably in the form of a Mo-OH(D) moiety. The orientation of this group is not fixed (although it is substantially restricted) and thus gives rise to a distribution of hyperfine interaction (hfi) parameters. The resulting loss of amplitude makes direct observation of a proton-related line using ESEEM impossible. However, such a line is observable in ESEEM spectra of the comparable deuterated enzyme because the narrower distribution of hfi parameters leads to less line broadening of the ESEEM spectra. ESEEM and HYSCORE spectra of sulfite oxidase obtained at pH 7.0 show no evidence of nearby H(D) groups, other than the single exchangeable proton/deuteron readily detected by CW EPR spectroscopy, which is also believed to be part of a Mo-OH(D) moiety. The differences observed in the Mo ... H(D) hfi parameters of SO as the pH is varied probably result from relatively small displacements of the H(D), which move the nucleon in and out of a node of the singly occupied Mo d orbital.