Using site-directed tryptophan fluorescence we studied nucleotide occupancy of the catalytic sites of Escherichia coli F1-ATPase, under conditions used previously for crystallization and X-ray structure analysis of the bovine mitochondrial enzyme [Abrahams et al. (1994) Nature 370, 621-628]. We found that only two of the three catalytic sites mere filled in the E. coli enzyme under these conditions (250 μM MgAMPPNP plus 5 μM MgADP), consistent with what was reported in the bovine F1 X-ray structure, However, subsequent addition of a physiological concentration of MgATP readily filled the third catalytic site. Therefore the enzyme form seen in the X-ray structure results from the fact that it is obtained under sub-saturating nucleotide conditions. The data show that the X-ray structure is compatible with a catalytic mechanism in which all three F1-ATPase catalytic sites must fill with MgATP to initiate steady-state hydrolysis [e,g, Weber and Senior (1996) Biochim. Biophys. Acta 1275, 101-104]. The data further demonstrate that the site-directed tryptophan fluorescence technique can provide valuable support for F1 crystallography studies.
Löbau, S., Weber, J., & Senior, A. E. (1997). Nucleotide occupancy of F1-ATPase catalytic sites under crystallization conditions. FEBS Letters, 404(1), 15–18. https://doi.org/10.1016/S0014-5793(97)00077-X