" Studies on the mechanism of oxidative phosphorylation : Effects of specific FO modifiers on ligand - induced conformation changes of F 1

Aurovertin is a fluorescent antibiotic that binds to the catalytic 13 subunits of the mitochondrial FjATPase and inhibits ATP synthesis and hydrolysis. ATP, ADP, and membrane energization in submitochondrial particles (SMP) alter the fluorescence of Fl-bound aurovertin. These fluorescence changes are considered to be in response to the conformation changes of Fl-ATPase. This paper shows that the ATP-induced fluorescence change of aurovertin bound to SMP or complex V (purified ATP synthase complex F-jF1) is inhibited when these preparations are pretreated with oligomycin or N,N'-d yclohexylcarbodlimide (DCCD). This inhibition is not seen with isolated Fl-ATPase. These and other results have suggested that modifications of the DCCD-binding protein in the membrane sector (FO) of the ATP synthase complex are communicated to F1, thereby altering the binding characteristics of ATP to the 13 subunits. By analogy, it is proposed that modifications (e.g., protonation/deprotonation) of the DCCD-binding protein effected by protonic energy alter the conformation of F1 and bring about the substrate/product binding changes that appear to be essential features of the mechanism and regulation of oxidative phosphorylation. The following recept findingshave suggested that the mitochondrial Fl-ATPase undergoes catalytically significant conformation changes during oxidative phosphorylation. (i) In agreement with an early prediction ofBoyer et al. (1, 2), it has been shown that F1-bound AIP and Pi can form F1-bound ATP in an isoenergetic process involving negligible free energy change (3-6). The energy-requiring steps in oxidative phosphorylation are considered to be substrate (ADP and Po)-binding (1, 7) and product (ATP)-releasing (1, 4, 8). (ii) The three catalytic sites on isolated Fl-ATPase interact, resulting in negative cooperativity with respect to substrate (MgATP) concentration and positive catalytic cooperativity in the sense that substrate binding to the second and third sites greatly enhances turnover by increasing the rate of product (ADP) removal from the first site (4, 8-11). Some of these cooperative effects have also been observed in the direction ofATP synthesis (ref. 12; unpublished data). (iii) In oxidative phosphorylation catalyzed by phosphorylating submitochondrial particles (SMP), partial uncoupling or attenuation of the rate of respiration not only diminishes the apparent Vmax (VmPx) for ATP synthesis but also alters the apparent Km (KamPP) for ADP and Pi. Partial uncoupling increases the KmPP for ADP and Pi, with ln(Vmjx/Km) being a linear function of the uncoupler concentration (i.e., the free energy of the system) (ref. 7; see also ref. 13). Attenuation of the rate of respiration up to -60% by an electron-transfer inhibitor decreases these KamPP values; double-reciprocal plots of VmPFPx for ATP synthesis versus ADP or Pi concentrations at several fixed concentrations of the electron-transfer inhibitor is a set of parallel lines for either ADP or Pi as the variable substrate (14). Under these conditions, there is no change in Vrnax/Km and little or no change in the membrane potential Adi (14). (iv) In SMP, the energy-induced fluorescence response of bound aurovertin, a reporter of F1 conformation change (15, 16), decreases in parallel with decreases in Ale and VAmPaP for ATP synthesis, as the respective assay systems are treated with incremental amounts of an uncoupler (14). Thus, it appears that energy-induced affinity changes of F1 for ATP, ADP, and Pi and the changes in K"mPP for ADP and Pi in response to alterations of the transmembrane electrochemical potential of protons, AiAiH+, and respiration rate are essential features of the mechanism and regulation of oxidative phosphorylation. This paper is concerned with the role played by F0 in the conformation changes of F1. The results suggest that modifications at the N,N'-dicyclohexylcarbodiimide (DCCD)binding protein influence the ligand-induced conformation changes of the catalytic p subunits of Fl. MATERIALS AND METHODS SMP (17), complex V (ATP synthase complex F -F1) (18), and Fl-ATPase (19) were prepared from bovine heart mnitochondria by published procedures. SMP were washed once with a buffer containing 0.25 M sucrose and 10 mM Tris acetate (pH 7.5) and was suspended in the same buffer at 40-60 mg of protein per ml. The heat-activation step in the F1-ATPase preparation was omitted. Protein concentration was determined by the biuret method (20) in the presence of 0.1% deoxycholate or by the method of Lowry et al. (21). Inhibitors were added in ethanolic solution at the concentrations indicated to SMP (10 mg of protein per ml), complex V (5 mg of protein per ml), or Fl-ATPase (1 mg of protein per ml) in a buffer containing 0.25 M sucrose and 50 mM Tris acetate (pH 7.5). The mixture was then incubated at 00C (SMP and complex V) or at room temperature (Fl-ATPase) and sampled for measurements of aurovertin fluorescence and ATPase activity. Aurovertin fluorescence was monitored by an SLM photon-counting spectrofluorometer at 30'C in the above buffer. When SMP were used, the assay mixtures contained, in addition, 0.5 mM EDTA, 3 mM MgCl2, and 20mM potassium phosphate (pH 7.5) (15). The amounts of enzyme added per ml to the assay medium were: 0.2 mg of SMP, 0.094 mg of complex V, and 0.025 mg of Fl-ATPase. Aurovertin was added at 0.63 ,uM from a stock solution in absolute ethanol whose concentration had been determined by using an extinction coefficient of 42,700 M-l cm-1 (22). Additions of ATP, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), succinate, etc., were made as indicated in the figures. The wavelengths of excitation and emission were 366 Abbreviations: SMP, phosphorylating submitochondrial particles; F1, F1-ATPase; FO, membrane sector of the ATP synthase complex; TNP-ATP, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; DCCD, N,N'-dicyclohexylcarbodiimide; Ph3SnCl, triphenyltin chloride; Bu3SnCl, tributyltin chloride; VamP. and K.IPP, apparent Vma and Km. *To whom reprint requests should be addressed. 7550 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 82 (1985) 7551 and 470 nm, respectively. TNP-ATP showed no fluorescence with this setting of wavelengths within the concentration range used and had only a small filter effect on the aurovertin fluorescence (<2% decrease by 0.4 ,uM TNP-ATP). ATPase activity was measured at 30'C by the ATP regenerating system as described (10) in the presence of 50 pug of SMP, 25 gg of complex V, or 1 gg of Fl-ATPase per ml. In the assay of SMP for ATPase activity, 13 gM rotenone and 5 ,uM carbonylcyanide m-chlorophenylhydrazone were also present (23). Oligomycin sensitivity of ATPase activity was estimated by addition of 5 pug of oligomycin per ml to the assay mixtures. The assay system was not affected by the concentrations of inhibitors carried with the samples. The sources of chemicals used were as follows: ATP and oligomycin were from Boehringer Mannheim; pyruvate kinase, lactic dehydrogenase, phosphoenolpyruvate and p(chloromercuri)benzoic acid, ClHgC6H4COOH, from Sigma; DCCD and tributyltin chloride (Bu3SnCl) from Aldrich; triphenyltin chloride (Ph3SnCl), from ICN; and TNP-ATP, from Molecular Probes, Junction City, OR. Other chemicals were reagent grade. Aurovertin and venturicidin were gifts, respectively, from R. B. Beechey (University College of Wales) and D. E. Griffiths (University of Warwick).