PROTEIN INHIBITOR OF MITOCHONDRIAL ATP SYNTHASE - RELATIONSHIP OF INHIBITOR STRUCTURE TO PH-DEPENDENT REGULATION

In the absence of an electrochemical proton gradient, the F-1 moiety o f the mitochondrial ATP synthase catalyzes the hydrolysis of ATP. This reaction is inhibited by a natural protein inhibitor, in a process ch aracterized by an increase in ATPase inhibition as pH is decreased fro m 8.0 to 6.0, In order to gain greater insight into the molecular and chemical events underlying this regulatory process, the relationships among pH, helicity of the inhibitor protein, and its capacity to inhib it F-1-ATPase activity were examined. First, peptides corresponding to four regions of the 82-amino-acid inhibitor protein were chemically s ynthesized and assessed for both retention of secondary structure, and capacity to inhibit F-1-ATPase activity. These studies showed that a region of only 24-amino-acid residues, from Phe 22 through Leu 45, acc ounts for the inhibitory capacity of the inhibitor protein, and that r etention of native helical structure in this region is not essential f or inhibition. Second, three mutants (33P34, 39P40, and 43P44) of the intact inhibitor protein were prepared in which a proline residue was inserted within the inhibitory region to disrupt native helical struct ure. The secondary structures and inhibitory capacities of these mutan ts were analyzed as a function of pH. These studies revealed that, des pite the initial loss of helical structure within the inhibitory regio n due to proline insertion, a further loss of helical structure is req uired to modulate inhibitory activity, These results suggest that a lo ss of helical structure outside the inhibitory region correlates with an increase in inhibitory capacity. Finally, two separate mutants (H48 A and H55A) were prepared in which a conserved histidine residue in th e wild-type inhibitor protein was replaced with an alanine. The second ary structures and inhibitory capacities of these mutants were also in vestigated as a function of pH. Results indicated that, although histi dine residues do not directly affect the inhibitory capacity of the pr otein, they are important for maintaining the inhibitor protein in an inactive form at high pH. Furthermore, these results show that loss in helical structure, although correlated with an increase in inhibitory capacity, is not essential for this function. These novel experiments are consistent with a model in which the inhibitor protein is envisio ned as consisting of two regions, an inhibitory region and a regulator y region. It is suggested that reduction of pH allows for the protonat ion of a histidine residue blacking the interaction between the two re gions, thus activating the inhibitory response. The pH reduction also correlates with a partial unfolding of the protein that may either cau se or result from the loss of interaction between the two helices. Thi s unfolding may be necessary for further optimization of inhibitor fun ction. (C) 1996 Academic Press, Inc.