Oligomycin binding

B. The ATP synthase inhibitor oligomycin binds directly to the enzyme complex and plugs up the H channel, which blocks ATP formation. 

Ej form can be stabilized by means other than phosphorylation, for instance by binding the non-phosphorylating substrate analog CrATP (Vilsen et al., 1987 Vilsen and Andersen, 1992b), or by oligomycin binding in the case of Na+-K+-ATPase (Esmann and Skou, 1985). 

The spheres removed from SMPs do not support ATP synthesis but do hydrolyze ATP to ADP and phosphate. Thus, ATP synthesis is carried out by Fq/Fi—ATPase (ATP synthase). The subscript o in Fq indicates that it contains the site at which a potent antibiotic inhibitor, oligomycin, binds and inhibits oxidative phosphorylation. Oligomycin does not bind Fi-ATPase and does not inhibit ATP hydrolysis to ADP and phosphate. 

The antibiotic oligomycin binds to a specific protein of the FO complex, blocks the flow of protons through the FO channel, and inhibits oxidative phosphorylation directly. 

The Fo domain is also a site of action for inhibitors, the best known of which is oligomycin (Table 13.1.4), which acts to inhibit proton conduction [128]. The oligomycin binding site has been localized to the a and c subunits of the Fq domain... 

FnFi-ATPase CONTENT IN CHROMATOPHORES OF RHODOSPIRILLUM RUBRUM AND STOICHIOMETRY OF OLIGOMYCIN BINDING... 

FqFi-ATPase Content in Chromatophores of Rhodospirillum Rubrum and Stoichiometry of Oligomycin Binding 173... 

ATP synthase Oligomycin Binds to OSCP in stalk and blocks H pore... 

One striking characteristic of the coupling ATPase of energy-transducing membranes, apart from the extraordinarily large number of different polypeptide subunits, is the existence of two different polypeptides involved in the response of the enzyme to the inhibitor oligomycin. One binds the inhibitor, the other, separated in space from the former by possibly as much as 10 to 15 A, confers oligomycin sensitivity to the entire enzyme complex. How could the transfer of information between these two polypeptide subunits and their concerted interaction with the ATPase proper be visualized ... 

Synthesis of ATP by mitochondria is inhibited by oligomycin, which binds to the OSCP subunit of ATP synthase. On the other hand, there are processes that require energy from electron transport and that are not inhibited by oligomycin. These energy-linked processes include the transport of many ions across the mitochondrial membrane (Section E) and reverse electron flow from succinate to NAD+ (Section C,2). Dinitrophenol and many other uncouplers block the reactions, but oligomycin has no effect. This fact can be rationalized by the Mitchell hypothesis if we assume that Ap can drive these processes. 

As noted earlier, studies with inhibitors have been of great value. One mole of ouabain binds per enzyme complex and inhibits all enzyme functions. It provides a convenient marker for the extracellular surface of the enzyme. Oligomycin inhibits the (Na+, K+)-ATPase but not the K+-phosphatase reaction. It stimulates the ADP/ATP exchange reaction and this led to the postulate for two phosphoenzymes in the reaction scheme. Anomalous kinetic behaviour for (Na+, K+)-ATPase, over some years, was eventually recognized57 to be due to a vanadate impurity in ATP, which binds with high affinity to the low affinity ATP site and with low affinity to the high affinity ATP site. In accord with this, vanadate effectively inhibits the K+-phosphatase... 

ATP synthase activity can be restored by adding back the F] complex to the depleted membranes. The F[ complexes bind to membrane channels known as the F complex, which are also composed of multiple subunits. The polypeptides of the F0 component are very hydrophobic and form a proton transport channel through the membrane, which links the proton gradient to ATP synthesis. This channel appears to be lined with hydrophilic residues such as seryl, threonyl and carboxyl groups. The stalk that connects the F, to the F complex comprises one copy each of the polypeptide known as the oligomycin-sensitivity-conferring protein (OSCP) and another protein known as F6. 

Inhibition of ATP synthase (energy transfer) reduces proton flow from the inter-membrane space to the matrix, which inhibits electron flow in the respiratory chain. Oligomycin, a macrolide antibiotic, prevents phosphoryl group transfer of ATP synthase. Dicyclohexylcarbodimide (DCCD) binds to and inhibits ATP synthase. Similar to the inhibitors of Complexes I, III, and IV, energy transfer inhibitors cause accumulation of reactive electrons and generate ROS. 

The antibiotics oligomycin and aurovertin and the reagents N,N -dicyclohexylcarbodiimide (DCCD) and 4-chloro-7-nitrobenzofurazan (Nbf-Cl) inhibit ATP synthesis by binding to different sites of ATP synthase. Oligomycin and DCCD bind to proteolipid (Fq) components and block translocation of protons, aurovertin binds to a specific )8-subunit of Fi, and Nbf-Cl binds with a specific tyrosine residue of the y5-subunit of Fi. 

ATP synthase from DDT [l,l-bis-(p-chlorophenyl)-2,2,2-tridilorethane] susceptible insects, but not from DDT resistant strains, is inhibited by DDT, although at relatively high concentration. Recently, it has been suggested that this inhibition is associated with the presence of a specific protein in the Fo component at a site different from the binding sites of oligomycin and DCCD. Since this specific protein is only present in insect strains that are susceptible to DDT the authors conclude that this protein is the target for DDT and that inhibition of ATP synthase is its primary mode of action [14]. 

It has been shown for bovine heart FqFi that binding of 1 mol oligomycin per mol of enzyme is enough to inhibit catalysis completely (6). For the yeast enzyme, however a large excess of oligomycin has to be added for maximal inhibition (7). 

Fig. 3. Scatchard plots for the binding of oligomycin to chromatophores from R. rubrum at different concentrations of BChl. The results shown in Fig. 2 were used to construct the Scatchard plots, r denotes the fraction of inhibited FqFj and [u] denotes the free inhibitor concentration. The same symbols were used as in Fig. 2 for the different concentrations of BChl during incubation with oligomycin.

Four additional polypeptides have been identified in the membrane sector. One of these, a strongly hydrophobic protein, seems to contain the binding site of dicyclohexyl-carbodiimide, an inhibitor of oxidative phosphorylation, and probably also of oligomycin. These four polypeptides are coded for by the mitochondrial genome, the others by the nuclear genome. 

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