Deleting the IF ₁ -like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis

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.

ATP synthesis at physiological nucleotide concentrations

“…Apparent K m values for ADP and phosphate were 11 µM and 104 µM, respectively. Under fully saturating conditions using 50 μM ADP and 10 mM phosphate, a maximal ATP synthesis rate of ~250 ± 50 nmol ATP min −1 mg −1 was obtained at 25 °C, which is in good accordance to a recent report for E. coli IMVs 7 . At maximal tested ADP (5 mM) and P i (100 mM) concentrations, the ATP synthesis rate declined to ~70%.…”

“…In order to minimize wasteful ATP hydrolysis, organisms have evolved different regulatory mechanisms, such as the inhibitory proteins IF1 in mitochondria 5,6 . A similar function has been attributed to the ζ subunit of the F 1 F 0 ATP synthase of Paracoccus denitrificans , but is currently debated 7 . In other bacteria such as B acillus PS3 and E. coli , the ε subunit has been proposed to ratchet the rotor in the synthesis direction 8 by extending its C-terminal domain along the γ subunit 9–12 , a conformational change that might be controlled by ATP binding in some bacteria 13 .…”

“…In their studies, they find that membrane vesicles of P. denitrificans are not capable of ATP hydrolysis, neither under high or low pmf conditions, and conclude that the kinetic control of ATP synthesis is an intrinsic property of the F 1 F 0 ATP synthase. There is an ongoing discussion to determine if the backwards reaction in P. denitrificans is blocked by the ζ subunit or not 6,7,48 , or if the inhibitory effect is caused by Mg-ADP inhibition, as proposed earlier 49 .…”

An update of the chemiosmotic theory as suggested by possible proton currents inside the coupling membrane

“…IF1 knock down in HeLa cells [80] equipped with a FRET sensor for measuring the intracellular ATP concentration, surprisingly, did not affect cell viability or mitochondrial morphology, even though the cellular ATP concentration decreased by about a third. Moreover, recently it was reported that deleting the IF1-like ζ subunit from Paracoccus denitrificans has little influence on ATP hydrolysis by ATP synthase [81], confirming that the inhibition of the hydrolysis of ATP by uncoupled ATP synthase does not depend only on IF1 and that the subject requires further investigations. Also, in bacteria, when the concentration of the ATP reaches the physiological value, the ɛ subunit inhibits ATP synthase by binding to the c ring, and it has been proposed as a target for the design of anti-tuberculosis drugs [82].…”

The regulatory subunit ε in Escherichia coli FOF1-ATP synthase

“…The inhibited state of Ct-F 1 in these structures is likely due to unusual occupancy of the ‘open’ β by ADP (no Mg2+) and Pi. This inhibited state may be distinct from the MgADP-inhibited state trapped by azide in several structures of Mi-F 1 [116], as the Ct-F 1 structures occupy a significantly different rotary position than azide-inhibited Mi-F 1 (11° difference, as aligned by the γ-core). Also, although not observed yet structurally, Ct-F 1 can enter a state inhibited by the εCTD, as demonstrated earlier [50].…”