HISTIDINE-49 IS NECESSARY FOR THE PH-DEPENDENT TRANSITION BETWEEN ACTIVE AND INACTIVE STATES OF THE BOVINE F1-ATPASE INHIBITOR PROTEIN

The role of the histidyl residue at position 49 (H49) of the bovine mi tochondrial F-1-ATPase inhibitor protein (F1I) was examined by site-di rected mutagenesis. Six amino acids (Q, E, K, V, L, and I) were substi tuted for H49 and the activities of the resulting inhibitor proteins w ere characterized with respect to pH. Each of the six mutations abolis hed the pH sensitivity which is characteristic of wild-type F1I. At pH 8.0, each of the mutations caused an increase in apparent maximum inh ibition and a decrease in apparent K-i relative to wild type. At pH 6. 7 the hydrophilic substitutions had little effect on apparent K-i, whi le the hydrophobic substitutions caused increases of 3.5- to 8.5-fold relative to wild type. The ratios of apparent K-i at pH 8.0 to apparen t K-i at pH 6.7 were in the range of 0.5 to 1.6 for the mutants, where as the wild-type value is 15.0. The mutations appear to shift the equi librium between active and inactive conformations of F1I toward the ac tive state. We find that H49 is required by F1I for sensitivity to pH and that it may facilitate the transition between active and inactive states of F1I. A possible role for H49 in the stabilization of the ina ctive state through participation in a multivalent complex with Zn2+ i s also discussed.