Electron paramagnetic resonance studies of Mn(II)Mn(II) interaction in yeast inorganic pyrophosphatase

Yeast inorganic pyrophosphatase, EC3.6.1.1. (PPase), has two high affinity Mn(II) sites per PPase subunit in the absence of phosphoryl ligands and three high affinity sites in the presence of a phosphoryl ligand such as hydroxymethane bisphosphonate (PCHOHP) [1]. We here present strong evidence from Mn(II) EPR studies that in the presence of PCHOHP 1) the Mn(II) ions in one pair of the divalent metal ion sites are close enough to one another to show significant magnetic (dipolar or exchange) interaction and 2) the Mn(II) ions in another pair of the sites show weaker magnetic interaction. Further, the Mn(II) ions in these latter sites have EPR spectra that are indistinguishable from one another. EPR Spectra of PPase, Mn(II) and PCHOHP Q-band (35 GHz) spectra for solutions of PPase and different amounts of Mn(II) in the absence of phosphoryl ligand are shown in Fig. 1(a) and 1(b). Spectrum 1(b) is quite similar to that for Mn(H2O)6+2 with respect to both g value (1.999 vs. 2.001, respectively) and peak to peak line width (1.75 gauss vs. 15 gauss, respectively) and is indicative of isotropic and relatively isolated Mn(II) sites. it should be noted, however, that raising the Mn(II) stoichiometry from one to two per subunit actually results in a modest decrease in spectral intensity, probably indicative of a weak Mn(II): Mn(II) magnetic interaction. The concentration of free Mn(II) is negligible in these solutions, since the dissociation constant of Mn(II) from each of two sites on a PPase subunit is equal to 10−5M [1]. In Fig. 2, Mn(II) spectra are shown, at increasing Mn(II) to PPase molar ratio, in the presence of the strong competitive inhibitor of PPase, PCHOHP [2]. It is quite clear that addition of PCHOHP leads to much more complex spectra compared to those seen in Fig. 1. The multiplicity of lines clearly distinguishable in Fig. 2a and 2b is similar to that reported by Markham in studies of Mn2+ bound to S-adenosyl-methionine synthetase [3], and is suggestive of Mn(II)Mn(II) coupling, although it must be noted that EPR spectra of the same solutions at X-band (9 GHz) did not show the same multiple line EPR spectral patterns, perhaps due to broadening effects, that Markham was able to demonstrate (data not shown). Alternatively, the spectra seen in Fig. 2a and 2b could be accounted for either as a superposition of two spectra with slightly different zero field splittings or by anisotropy in the −12 to +12 transition. In any event, the broadness of all of the peaks in Fig. 2a, b compared to those seen in Fig. 1 (even the relatively narrow peak at 12,275 gauss has a peak to peak line width of 27 gauss) and the marked decrease in intensity in the low field line at 12,275 gauss as more than one equivalent of Mn(II) is added, provide strong evidence for significantly enhanced Mn(II)Mn(II) magnetic interaction in the presence of PCHOHP [4]. EPR Spectra of PPase, Mn(II), PCHOHP and Ca2+ Based on our previous binding studies [1] it is evident that in the presence of 1.5–2.0 equivalents of limit of 7–8 A can be set for the separation between the Mn(II) ions in these two sites; beyond this limit, the two ions would have only a small (< 10%) broadening effect on one another [4]. This result is consistent with preliminary work Dunaway-Mariano and Villafranca as quoted in Knight et al. [7]. The addition of Ca2+ displaces Mn(II) from the partially filled site C and the displaced Mn(II) is redistributed amongst the partially filled sites A and B. Since site C is filled with diamagnetic Ca2+, the Mn(II)Mn(II) magnetic interaction between sites B and C is suppressed and a narrow line spectrum, corresponding to Mn(II) population of sites A and B, is observed. The increased intensity of this spectrum as a function of added Ca2+ is reasonable since the population of Mn(II) at these sites is increased by displaced Mn(II) from site C. Finally, from the observed lack of zero field splitting, the results presented in Figs. 1(b) and 3(d) show that the Mn(II) ions in sites A and B have ligand environments with near octahedral symmetry which are indistinguishable from one another, at least as judged by their EPR spectra.