Structural and exchange properties of “Co(III)-phenanthroline-ATP”: a labeling reagent for the active site of ATPases

Abstract

This paper reports on ¹H and ³¹P NMR as well as EPR measurements of the labeling reagent of ATPase sites, “Co(III)-(phen)-ATP.” This complex is found to be paramagnetic, as deduced both from its EPR spectrum and from the significant broadening, though almost unshifted, proton and phosphorus resonances. This paramagnetism is a result of the incorporation of the superoxide free-radical anion in the coordination sphere of the trivalent cobalt ion. Evidence for the presence of superoxide in the complex is based on competition experiments with cyanide, which is able to displace the superoxide anion. The latter was identified by its inducing effect on the photoreactivity of luminol. The displacement of superoxide by cyanide was accompanied by the abolition of the paramagnetism of the complex. The relative distances between the protons and phosphorus atoms of ATP and the superoxide anion in the complex were calculated using the NMR line-broadening data. Structural models compatible with the experimental results are proposed. Under conditions of excess of adenine nucleotides or phenanthroline, the coordinated ATP molecule becomes exchangeable. This phenomenon is attributed to the labilization of the cobaltic ion ligands induced by the superoxide anion.