Abstract
ATP synthesis from ADP, P(i), and Mg2+ takes place in mitochondria on the catalytic F1 unit (alpha3beta3gammedeltaepsilon) of the ATP synthase complex (F0F1), a remarkable nanomachine that interconverts electrochemical and mechanical energy, producing the high energy terminal bond of ATP. In currently available structural models of F1, the P-loop (amino acid residues 156GGAGVGKT163) contributes to substrate binding at the subunit catalytic sites. Here, we report the first transition state-like structure of F1 (ADP.V(i).Mg.F1) from rat liver that was crystallized with the phosphate (P(i)) analog vanadate (VO(3-)4 or V(i)). Compared with earlier "ground state" structures, this new F1 structure reveals that the active site region has undergone significant remodeling. P-loop residue alanine 158 is located much closer to V(i) than it is to P(i) in a previous structural model. No significant movements of P-loop residues of the subunit were observed at its analogous but noncatalytic sites. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, thus facilitating the dehydration of ADP and P(i). This new crystallographic study provides strong support for the catalytic mechanism of ATP synthesis deduced from earlier biochemical studies of liver F1 conducted in the presence of V(i) (Ko, Y. H., Bianchet, M., Amzel, L. M., and Pedersen, P. L. (1997) J. Biol. Chem. 272, 18875-18881; Ko, Y. H., Hong, S., and Pedersen, P. L. (1999) J. Biol. Chem. 274, 28853-28856).
Highlights
The mammalian mitochondrial ATP synthase (F0F1) is a large protein complex (Fig. 1A) located in the inner membrane, where it catalyzes ATP synthesis from ADP, Pi, and Mg2ϩ at the expense of an electrochemical gradient of protons generated by the electron transport chain
During ATP synthesis in intact mitochondria, it is strongly believed, based on three-dimensional structures of F1 [9, 10] and single molecule technology applied to the simpler bacterial enzymes [11, 12], that the proton-driven motor contained within F0 drives the ATP hydrolysisdriven motor (i.e. F1) in the reverse direction to make ATP (Fig. 1A)
For the past 3 decades, a major objective related to work on the mitochondrial ATP synthase (F0F1) has been to understand the mechanism by which ATPMg is made from ADP, Pi, and Mg2ϩ, a process that takes place primarily on  subunits
Summary
No significant movements of P-loop residues of the ␣ subunit were observed at its analogous but noncatalytic sites. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, facilitating the dehydration of ADP and Pi. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, facilitating the dehydration of ADP and Pi This new crystallographic study provides strong support for the catalytic mechanism of ATP synthesis deduced from earlier biochemical studies of liver F1 conducted in the presence of Vi More recent studies show that in mitochondria, the entire “double motor” ATP synthase complex is itself in complex formation with the phosphate carrier and adenine nucleotide carrier, forming an ATP synthase-phosphate carrier-adenine nucleotide carrier supercomplex named the “ATP synthasome” [13, 14]
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