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Journal of Biochemistry 2005 138(2):201-207; doi:10.1093/jb/mvi116
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© 2005 The Japanese Biochemical Society

Regular Paper

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

Naoki Ichikawa*, Naomi Chisuwa, Mamiko Tanase and Mai Nakamura

Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585

1 To whom correspondence should be addressed. E-mail: ichikawa{at}life.osaka-cu.ac.jp

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 ß-subunit of F1. In the present study, we examined the roles of the three ß residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The ßArg412 and ßGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the ßArg408 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 ({alpha}, ß, and {gamma}) were detected in mitochondria from each mutant on immunoblotting, and the F1F0 complex was isolated from them. These results indicate that ßArg408 is essential not for assembly of the F1F0 complex but for the catalytic activity of the enzyme. In the crystal structure of F1, ßArg408 binds to {alpha}Glu399 in the {alpha}DP/ßDP pair and seems to be important for formation of the closed {alpha}DP/ßDP conformation. IF1 seems to disrupt this {alpha}DPGlu399/ßDPArg408 interaction by binding to ßDPArg408, and to interfere with the change from the open {alpha}DP/ßDP conformation to the closed conformation that is required for catalysis by F1F0-ATPase.


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