Extended x-ray absorption fine structure of Mn²⁺ and Mn²⁺·ATP complex bound to coupling factor 1 of the H⁺-ATPase from chloroplasts

The spinach chloroplast ATPase, coupling factor 1, contains three tight Mn²⁺-binding sites which interact cooperatively. The bound manganese coordinations were studied by x-ray absorption fine structure analysis. Mn²⁺ was found to be bound to the enzyme with an average Mn-O bond length of 2.05 ± 0.15 Å, significantly shorter than the 2.15 ± 0.15 Å of the Mn-O bond of the average first hydration shell for Mn²⁺ in aqueous solution. On adding ATP to the manganese-enzyme mixture, a tertiary complex of Mn²⁺·ATP·enzyme was formed as indicated by the appearance of a second shell. Mn-P bond distances were estimated at 4.95 ± 0.15 Å in the tertiary Mn²⁺·ATP·enzyme complex, which was considerably longer than the Mn-P bond distance of 3.36 ± 0.15 Å for the Mn²⁺·ATP complex in aqueous solution. The Mn-P bond distance in the tertiary Mn²⁺·ATP·enzyme complex decreased to 4.32 ± 0.15 Å when selenite, a potent effector of ATPase activity, was added. Based on these results, it is suggested that the tertiary complex is required for catalysis. The stimulation of ATP hydrolysis by anions such as selenite may be the result of shortening the distance between Mn²⁺ and the ATP phosphates in the enzyme active site.