THE METAL-BINDING SITE OF IMIDAZOLE GLYCEROL PHOSPHATE DEHYDRATASE - EPR AND ENDOR STUDIES OF THE OXO-VANADYL ENZYME

The apo protein of imidazole glycerol phosphate dehydratase (IGPD) fro m Saccharomyces cerevisiae combines stoichiometrically with certain sp ecific divalent metal cations to assemble the catalytically active for m comprising 24 protein subunits and tightly bound metal, VO2+ ions re act similarly but, uniquely, result in a metallo-protein (VO-IGPD) wit h neither catalytic activity nor the ability to bind to the reaction i ntermediate analogue, 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphona te. Since VO2+ apparently assembles the quaternary structure correctly , it is used in the present study as a spin probe to investigate the m etal centre coordination environment by electron paramagnetic resonanc e (EPR) and electron nuclear double resonance (ENDOR) spectroscopy, At neutral pH, the EPR spectrum of VO-IGPD reveals at least three distin ct VO2+ sub-spectra with one predominant at low pH. The spin Hamiltoni an parameters for some of the subspectra are consistent with V-51 havi ng nitrogen in the inner-sphere equatorial coordination environment fr om, most probably, multiple coordinating histidines. Further evidence for inner-sphere nitrogen ligands is obtained from ENDOR spectroscopy. The spectra of the low of region show signals from interactions with N-14 which are consistent with couplings to the imino nitrogen of coor dinated histidine residues. In addition a number of proton ENDOR line pairs are resolved. Of the few that disappear upon exchange of the pro tein into D2O. one most likely originates from the exchangeable proton of the N-H group of a coordinated histidine imidazole. H-1-ENDOR line pairs from nonexchangeable protons with splittings of approximately 3 MHz can be attributed to imidazole carbon protons. Thus, most of the couplings observed by ENDOR are consistent with being from the imidazo le heterocycle of one or more histidine ligands.