ELECTRON-PARAMAGNETIC RESONANCE STUDIES ON THE HIGH-SALT FORM OF BOVINE SPLEEN PURPLE ACID-PHOSPHATASE

The EPR spectra of the high-salt form of reduced bovine spleen purple acid phosphatase (BSPAP(r)) and its complexes with inhibitory tetrahedral oxyanions, AMP, and fluorine have been examined in the 4-30 K temperature range. The EPR spectrum of the high-salt form of BAPAP(r) is identical to that previously reported for the low-salt form (Averill et al. (1987) J. Am. Chem. Soc. 109, 3760-3767), indicating that the substantial differences in conformation of the two forms result in undetectable alterations in the electronic structure of the binuclear iron center. Phosphate, AMP, and arsenate all result in broadened, highly anisotropic EPR spectra with decreased values of the antiferromagnetic coupling constant, -2J, while molybdate and tungstate produce a sharp axial or slightly rhombic spectrum, respectively, and fluoride produces an anomalous spectrum with an inverted g-tensor. These results are consistent with binding of the two classes of oxyanions (and AMP) to distinct sites at or near the binuclear iron center, while fluoride binds in yet a third mode. EPR spectra of the BSPAP(r) complex with molybdate show altered relaxation behavior in the presence of phosphate, consistent with a 50% decrease in the magnitude of -2J, suggesting that phosphate binds to the molybdate complex to produce a ternary complex analogous to that proposed for molybdate inhibition on the basis of kinetics studies.