Mg²⁺ coordination in catalytic sites of F₁-ATPase

Coordination of the Mg²⁺ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F₁-ATPase was investigated. From the X-ray structure of the mitochondrial enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], it may be inferred that the hydroxyl of βThr-156 is a direct ligand of Mg²⁺, whereas the carboxyls of βGlu-181, βGlu-185, and βAsp- 242 might contribute via intervening water molecules. Elimination of each respective functional group by site-directed mutagenesis, followed by determination of Mg-nucleotide and uncomplexed nucleotide binding affinities using a tryptophan probe, showed that βThr-156, βGlu-185, and βAsp-242 are all involved in Mg²⁺ coordination, whereas βGlu-181 is not. A derived structural model for the octahedral coordination around the Mg²⁺ ion is presented. The results indicate that the ADP-containing site in the X-ray structure is the catalytic site of highest affinity. Correct Mg²⁺ coordination is required for catalytic activity at physiological rates. Elimination of any one of the Mg²⁺-coordinating residues led to complete loss of Mg²⁺-dependent nucleotide binding cooperativity of the catalytic sites.