The influence of the binding of ADP and ATP on the high-affinity Mn(II) binding site of chloroplast coupling factor 1 (CF1) was studied by analysis of field-dependent solvent proton and deuteron spin-lattice relaxation data. In order to characterize metal-nucleotide complexes of CF1 under conditions similar to those of the NMR experiments, the enzyme was analyzed for bound nucleotides and Mn(II) after incubation with AdN and MnCl2 and removal of labile ligands by extensive gel filtration chromatography. As isolated, the enzyme contained 1.3-1.4 mol of adenine nucleotide (mostly ADP) and 1.3 mol of Mg(II) per mole of CF1.After incubation with added Mn(II) and nucleotide, a total of three binding sites with high affinity for divalent metal ions (Mg2+ + Mn2+) and/or nucleotide (ATP or ADP) were found. In the field-dependent NMR experiments, the Mn(II) binding site of CF1 was studied for three mole ratios of added Mn(II) to CF1, 0.5, 1.0, and 1.5, in the presence of an excess of either ADP or ATP. The results were extrapolated to zero Mn(II) concentration to characterize the environment of the first Mn(II) binding site of CF1.In the presence of both adenine nucleotides, pronounced changes in the Mn(II) environment relative to that in Mn(II)-CF1 were evident; the local relaxation rate maxima were more pronounced and shifted to higher field strengths, and the relaxation rate per bound Mn(II) increased at all field strengths. Analysis of the data revealed that the number of exchangeable water molecules liganded to bound Mn(II) increased from one in the binary Mn(II)-CF1 complex to three and two in the ternary Mn(II)-ADP-CF1 and Mn(II)-ATP-CF1 complexes, respectively; these results suggest that a water ligand to bound Mn(II) in the Mn(II)-ADP-CF1 complex is replaced by the 7-phosphate of ATP in the Mn(II)-ATP-CF1 complex. The residence time of water within the inner coordination sphere was found to be 430 ns in the presence of ADP and 260 ns in the presence of ATP. The low-field electron spin relaxation time decreased in the ternary Mn(II)-AdN-CF1 complexes relative to binary Mn(II)-CF1, reflecting a lower Mn(II) site symmetry in the latter complex. A binding model is presented to account for these observations.
ASJC Scopus subject areas