ESEEM INVESTIGATIONS OF THE HIGH PH AND LOW PH FORMS OF CHICKEN LIVERSULFITE OXIDASE

Two-pulse and four-pulse electron spin-echo envelope modulation spectr oscopy (ESEEMS) at two operational frequencies and two-dimensional hyp erfine 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 re veal the presence of one solvent-exchangeable D(H) near the Mo-V cente r, 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 observab le in ESEEM spectra of the comparable deuterated enzyme because the na rrower distribution of hfi parameters leads to less line broadening of the ESEEM spectra. ESEEM and HYSCORE spectra of sulfite oxidase obtai ned at pH 7.0 show no evidence of nearby H(D) groups, other than the s ingle exchangeable proton/deuteron readily detected by CW EPR spectros copy, which is also believed to be part of a Mo-OH(D) moiety. The diff erences 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 M o d orbital.