Functional transitions of F₀F₁‐ATPase mediated by the inhibitory peptide IF1 in yeast coupled submitochondrial particles

Abstract

The mechanism of inhibition of yeast F 0 F 1 -ATPase by its naturally occurring protein inhibitor (IF1) was investigated in submitochondrial particles by studying the IF1-mediated ATPase inhibition in the presence and absence of a protonmotive force. In the presence of protonmotive force, IF1 added during net NTP hydrolysis almost completely inhibited NTPase activity. At moderate IF1 concentration, subsequent uncoupler addition unexpectedly caused a burst of NTP hydrolysis. We propose that the protonmotive force induces the conversion of IF1-inhibited F 0 F 1 -ATPase into a new form having a lower affinity for IF1. This form remains inactive for ATP hydrolysis after IF1 release. Uncoupling simultaneously releases ATP hydrolysis and converts the latent form of IF1-free F 0 F 1 -ATPase back to the active form. The relationship between the different steps of the catalytic cycle, the mechanism of inhibition by IF1 and the interconversion process is discussed.Keywords: ATP synthase; catalytic state; inhibitory peptide; latent ATPase; protonmotive force; yeast.Energy-driven ATP synthesis in energy-transducing membranes is carried out by membrane-bound F 0 F 1 -ATPase complex or ATP synthase [1]. The extrinsic F 1 subcomplex is composed of five types of subunits in the stoichiometry a 3 b 3 cde. Three fast-exchangeable nucleotide-binding sites, presumably catalytic, and three slow-exchangeable nucleotide-binding sites, certainly noncatalytic, reside in a/b interfaces [2,3] (an alternative point of view can be found in [4]). The membranous F 0 subcomplex promotes proton translocation, energetically coupled to ATP synthesis/ hydrolysis via a stalk connecting F 0 and F 1 . F 1 can be biochemically separated from F 0 and remains competent for uncoupled ATP hydrolysis. Numerous data, including direct observations of the rotation of the central axis of F 0 F 1 relative to a 3 b 3 crown during ATP hydrolysis [5,6], indicate that F 0 F 1 is a rotary motor, with an asymmetrical rotor composed, in mitochondria, of c, d and e subunits, anchored to a membranous decameric ring of c subunits thought to compose a proton-driven turbine [7].Mitochondrial F 0 F 1 is regulated by a naturally occurring inhibitor protein called IF1 [8]. IF1 is a small acid-and heat-stable protein, which stoichiometrically binds to the F 1 sector of ATP synthase and inhibits ATP hydrolysis [8][9][10][11][12][13][14][15][16][17][18][19]. Dl H þ is believed to favour the release of IF1 from ATP synthase [10][11][12][13][14][15][16][17] or, alternatively, to shift this regulatory peptide from an inhibitory to a silent position on the enzyme [10,18,19]. The IF1 binding site was proposed to be located close to the C-terminus proximal DELSEED loop of the b-subunit [20], close to the catalytic site [21], at the a/b interface [22], or at the a/c interface [23]. A recent tridimensional model of the bovine MF 1 -IF1 complex drawn from radiocristallographic data showed IF1 bound at the a/b interface, and interacting weakly with c [24].The F 0 F 1 -IF1 interaction is m...

The Binding Mechanism of the Yeast F1-ATPase Inhibitory Peptide

“…5b), yet much slower than at high MgATP concentration (see below). Assuming that k app is proportional to the IF1 concentration (37,41) Fig. 6 shows the relationship between MgATP concentrations and the rate of IF1 inhibition.…”