Mitochondrial ATP Synthase Residue βArginine-408, Which Interacts with the Inhibitory Site of Regulatory Protein IF1, Is Essential for the Function of the Enzyme

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Abstract
Mitochondrial ATP synthase (F1F0-ATPase) is regulated by an intrinsic ATPase inhibitor protein, IF1. We previously found that six residues of the yeast IF1 (Phe17, Arg20, Glu21, Arg22, Glu25, and Phe28) form an ATPase inhibitory site [Ichikawa, N. and Ogura, C. (2003) J. Bioenerg. Biomembr. 35, 399–407]. In the crystal structure of the F1/IF1 complex [Cabezón, E. et al. (2003) Nat. Struct. Biol. 10, 744–750], the core residues of the inhibitory site interact with Arg408, Arg412 and Glu454 of the β-subunit of F1. In the present study, we examined the roles of the three β residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The βArg412 and βGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the βArg408 mutants (R408I and R408T) could not. The ATPase activity of isolated mitochondria was decreased in R412I, R412T, E454Q, and E454V mutant cells, and undetectable in R408I and R408T cells. The subunits of F1 (α, β, and γ) were detected in mitochondria from each mutant on immunoblotting, and the F1F0 complex was isolated from them. These results indicate that βArg408 is essential not for assembly of the F1F0 complex but for the catalytic activity of the enzyme. In the crystal structure of F1, βArg408 binds to αGlu399 in the αDP/βDP pair and seems to be important for formation of the closed αDP/βDP conformation. IF1 seems to disrupt this αDPGlu399/βDPArg408 interaction by binding to βDPArg408, and to interfere with the change from the open αDP/βDP conformation to the closed conformation that is required for catalysis by F1F0-ATPase.