Abstract
In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF1. Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F1-ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.
Highlights
F1FO ATP synthases are energy-transducing enzymes that use the energy stored in electrochemical proton motive forces across the membranes of bacteria, chloroplasts or mitochondria to generate ATP from ADP and inorganic phosphate [1]
The biochemical work of Garcıa-Trejo and co-workers [29,30,36], together with the structure of P. denitrificans ATP synthase that revealed the z subunit bound in a manner analogous to the eukaryotic inhibitor protein IF1 [13], indicated that the z subunit is responsible for preventing ATP hydrolysis and enforcing unidirectional catalysis in the P. denitrificans enzyme
Deleting the z subunit caused only very moderate increases in ATP hydrolysis, and the rates remain very low in comparison with the rates observed from the mammalian and E. coli enzymes
Summary
F1FO ATP synthases are energy-transducing enzymes that use the energy stored in electrochemical proton (or sodium) motive forces across the membranes of bacteria, chloroplasts or mitochondria to generate ATP from ADP and inorganic phosphate [1]. They catalyse by a mechanical rotary mechanism [1,2,3]. Under conditions of low proton-motive force and high ATP/ADP ratio the thermodynamics of the system favour ATP hydrolysis over ATP synthesis, and rotation may reverse to dissipate the energy stored in the high ATP/ADP ratio and build the proton-motive force. Should the proton-motive force not be usefully employed and lost to proton leak, the hydrolysis reaction is wasteful, and it has been assumed that this explains why many organisms have developed strategies to regulate and prevent it occurring
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