Mitochondrial ATP Synthase

Abstract

ATP synthesis from ADP, P i , and Mg 2؉ takes place in mitochondria on the catalytic F 1 unit (␣ 3 ␤ 3 ␥␦⑀) of the ATP synthase complex (F 0 F 1 ), a remarkable nanomachine that interconverts electrochemical and mechanical energy, producing the high energy terminal bond of ATP. In currently available structural models of F 1 , the P-loop (amino acid residues 156 GGAGVGKT 163 ) contributes to substrate binding at the ␤ subunit catalytic sites. Here, we report the first transition state-like structure of F 1 (ADP⅐V i ⅐Mg⅐F 1 ) from rat liver that was crystallized with the phosphate (P i ) analog vanadate (VO 4 3؊ or V i ). Compared with earlier "ground state" structures, this new F 1 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 The mammalian mitochondrial ATP synthase (F 0 F 1 ) is a large protein complex (Fig. 1A) located in the inner membrane, where it catalyzes ATP synthesis from ADP, P i , and Mg 2ϩ at the expense of an electrochemical gradient of protons generated by the electron transport chain (reviewed in Refs. 1 and 2). Excluding regulators, the mammalian ATP synthase consists of 15 subunit types (3, 4), of which five in the stoichiometric ratio ␣ 3 ␤ 3 ␥␦⑀ comprise the F 1 catalytic unit (5), an ATP hydrolysis-driven motor, whereas an additional 10 comprise F 0 . One part of F 0 , containing subunits a and c, is embedded in the inner membrane to form a proton-driven motor, whereas a second part composed of subunits b, oligomycin sensitivity conferring protein (OSCP), and F 6 forms a side stalk (4, 6, 7) or "stator" extending from the membrane-embedded segment to the top of F 1 (4,8).During ATP synthesis in intact mitochondria, it is strongly believed, based on three-dimensional structures of F 1 (9, 10) and single molecule technology applied to the simpler bacterial enzymes (11,12), that the proton-driven motor contained within F 0 drives the ATP hydrolysisdriven motor (i.e. F 1 ) in the reverse direction to make ATP (Fig. 1A). In addition, 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).To fully understand how ATP synthases work, we must elucidate three different levels of mechanism (Fig. 1A), these being (a) the mechanism (el...

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

“…In addition, there are x-ray crystallographic structures of 39 Vi⅐enzyme complexes in the Protein Data Base, of which 21 have been clearly identified as representing reaction intermediates (17). Recently, for example Chen et al have determined the crystal structure of the mitochondrial F 1 -ATPase in a vanadateinduced transition-like state (44). Moreover, the nucleotide moiety trapped in Vi-trapped structures is always the nucleoside diphosphate (Fig.…”

Vanadium and proteins: Uptake, transport, structure, activity and function

“…The vanadate moiety is located next to the P-loop ( 156 GGAGVGKT 163 ) and several residues, not only from the P-loop, are involved in its stabilization, namely Glu188 that acts as the catalytic base [385]. Interestingly, compared to the previously obtained F 1 ATP synthase in the ground state with phosphate (PDB: 1MAB) The other structure was deposited in 2011: a 2.85 Å XRD resolution structure of the catalytic A subunit of A 1 A o ATP synthase from Pyrococcus horikoshii (PDB: 3P20) [389].…”

“…The first structure to be released was a 3.0 Å resolution XRD structure of the subunit F 1 from the mitochondrial rat ATP synthase (PDB: 2F43) [385] representing a transition state containing ADP, vanadate(V) and Mg 2+ . The vanadate moiety is located next to the P-loop ( 156 GGAGVGKT 163 ) and several residues, not only from the P-loop, are involved in its stabilization, namely Glu188 that acts as the catalytic base [385].…”

The Transition-Like State and Pi Entrance into the Catalytic A Subunit of the Biological Engine A-ATP Synthase

“…10 F236 stabilizes this arched loop and is therefore one of the critical residues in the P-loop sequence (GPFGSGKT) of subunit A. F236 is the equivalent amino acid to the alanine in subunit β of F-ATP synthases (GGAGVGKT), which is a key residue in the catalytic process inside the β-subunit, moving towards the γ-phosphate of ATP during catalysis, as described for the rat liver F-ATP synthase. 11 Based on these, it was concluded that the differences in amino acid sequence and the different conformation of the P-loops result in altered nucleotide binding, nucleotide specificity, and nucleotide accessibility in A-and F-ATP synthases or V-ATPases, indicating that the A-ATP synthases might have a different catalytic mechanism. 10 Besides the formation of an electrochemical gradient by the membrane-integrated complexes, at least five steps inside the catalytic A-subunit are critical for catalysis and its regulation, namely, substrate entrance, phosphate and nucleotide binding, transition-state formation, ATP formation, and product release.…”