Structure function relationship of vanadate bound to a single site in chloroplast CF1-ATPase as determined by X-ray absorption

The structure of vanadate, a phosphate analogue which was suggested to func tion in the presence of tightly bound ADP and divalent cations as a transit ion state inhibitor of CF1-ATPase, was investigated by X-ray absorption spe ctroscopy. Analysis of the vanadium K-edge was used for determination of th e structure of vanadate bound to a single site in CF1-ATPase containing a s ingle tightly bound ADP. There was a decrease in the intensity of the 1s-3d pre-edge transition and a change in the shape of two other shoulders at th e edge region upon binding of vanadate to CF1 in the presence of Mg2+ ions. The changes are due to alteration in the structure of vanadium from tetrah edral to a five-coordinated trigonal bipyramidal geometry. Comparison of th e pre-edge peak intensity of ADP-vanadate complex, and model compound resol ved by crystallography support the proposed structure of CF1-bound vanadate . V-51 NMR measurements were used to verify the pentacoordinated structure of ADP-vanadate complex used as a model in the X-ray absorption studies. Th e inhibition of a single and multiple site activity by vanadate and by MgAD P was measured. Vanadate inhibition of CF1-ATPase activity decreased more t han 90 fold in the presence of MgADP. A differential specificity of the inh ibition in single and multiple mode of activity was observed. It is suggest ed that ADP-vanadate binds to the active sites of the enzyme as a pentacoor dinated vanadium having approximate trigonal bipyramidal geometry. This str ucture is analogous to the proposed transition state of the phosphate durin g the synthesis and the hydrolysis of ATP by CF1.