Cooperative Binding of Manganese(II) to Chloroplast Coupling Factor 1 Detected by NMR Proton Relaxation Enhancement

The binding of divalent manganese to soluble latent spinach chloroplast coupling factor 1 (CF1) was examined by measurements of the water proton spin-lattice relaxation enhancement (PRE), revealing positive cooperativity between high-affinity sites. A method that used only a single enhancement parameter, εb, for the quantitation of cooperative PRE data was derived. Application to the high-affinity sites yielded a value of 9.01 ± 0.11 for εb. Two high-affinity sites participated in cooperative binding, although the possibility that a third site was present was not eliminated. The apparent binding constant to site ratio, K/n, was found to be 3.4-3.7 μM, giving a value for K of approximately 7 μM.