Effects of magnesium ions on the relative conformation of nucleotide binding sites of F1-ATPases as studied by electron spin resonance spectroscopy.

Abstract

Cations like Mg2+ play an important role in the catalytic mechanism of F1-ATPases. In this study we applied ESR spectroscopy and used the ATP analog 2-azido-2',3'-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester)ATP (2-N3-SL-ATP) to investigate the effects of Mg2+ ions on the structure of the nucleotide binding sites of F1-ATPases from beef heart mitochondria (MF1) and from the thermophilic bacterium PS3 (TF1). The results demonstrated that Mg2+ ions not only influenced the binding of the nucleotide analogs to F1 but also altered the structure and geometry of the nucleotide binding sites. We observed that the dipolar interactions that are indicative of the close proximity of enzyme-bound 2-N3-SL-ANP (Vogel, P.D., Nett, J.H., Sauer, H.E., Schmadel, K., Cross, R.L., and Trommer, W.E. (1992) J. Biol. Chem. 267, 11982-11986) were only detectable in MF1-ATPase when the enzyme was preincubated with Mg2+ ions. In the absence of Mg2+, the enzyme exhibited ESR spectra indicative of spin label bound in at least two different environments (binding sites) with no dipolar interactions visible. TF1-ATPase did not exhibit clear dipolar interactions in the presence or absence of Mg2+. The ESR spectra of TF1 in the absence of Mg2+ indicated two different environments of the spin labels. Subsequent addition of Mg2+, however, led to exactly the same spectra as if the enzyme was incubated with the ions, indicating a rearrangement of the nucleotide binding sites. In summary, clear differences in the structures of the nucleotide binding sites of MF1 and TF1 in the presence or absence of Mg2+ were observed. Conformational differences between F1-bound spin-labeled nucleotides were also observed between TF1- and MF1-ATPases.