Regulatory Interplay between Proton Motive Force, ADP, Phosphate, and Subunit ϵ in Bacterial ATP Synthase

Abstract

ATP synthase couples transmembrane proton transport, driven by the proton motive force (pmf), to the synthesis of ATP from ADP and inorganic phosphate (P i ). In certain bacteria, the reaction is reversed and the enzyme generates pmf, working as a proton-pumping ATPase. The ATPase activity of bacterial enzymes is prone to inhibition by both ADP and the C-terminal domain of subunit ⑀. We studied the effects of ADP, P i , pmf, and the C-terminal domain of subunit ⑀ on the ATPase activity of thermophilic Bacillus PS3 and Escherichia coli ATP synthases. We found that pmf relieved ADP inhibition during steady-state ATP hydrolysis, but only in the presence of P i . The C-terminal domain of subunit ⑀ in the Bacillus PS3 enzyme enhanced ADP inhibition by counteracting the effects of pmf. It appears that these features allow the enzyme to promptly respond to changes in the ATP:ADP ratio and in pmf levels in order to avoid potentially wasteful ATP hydrolysis in vivo.ATP synthase (F O F 1 ) is a ubiquitous enzyme present in the plasma membrane of bacteria, the thylakoid membrane of chloroplasts, and the inner mitochondrial membrane. The enzyme catalyzes ATP synthesis coupled to transmembrane proton translocation 2 driven by the proton motive force (pmf).3 At low pmf the activity is reversed and the enzyme functions as a proton-pumping ATPase. ATP synthase consists of two distinct regions: the hydrophobic F O , which is embedded in the membrane, and the hydrophilic F 1 that protrudes ϳ100 Å from the membrane bilayer. F 1 contains three catalytic and three non-catalytic nucleotide-binding sites and is responsible for both ATP synthesis and hydrolysis reactions. F O performs transmembrane proton transport. The simplest subunit composition is found in bacteria (e.g. Escherichia coli or thermophilic Bacillus PS3; see Refs. 1 and 2 for reviews), where F 1 is a complex of five types of subunits at a stoichiometry of ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 , and F O is a complex of three types of subunits at a stoichiometry of a 1 b 2 c 10 .It is now widely accepted that the enzyme operates according to the "binding change mechanism" (see Ref. 3 and the references therein). Briefly, proton translocation is coupled to the rotation of the c-ring oligomer (4) together with the ␥⑀ complex relative to the rest of the enzyme (see Refs. 5-7 and references therein for details). Rotation of the ␥ subunit inside the ␣ 3 ␤ 3 hexamer causes sequential conformational changes to the catalytic sites. These sequential conformational changes result in substrate binding, the chemical step, and product release (8 -10). This "rotary binding change mechanism" is usually considered reversible, and numerous demonstrations of ATPdriven proton pumping support this assumption. However, several observations indicate that this is not the case. Many factors (e.g. products/substrates of catalysis, inhibitors, inorganic anions, etc.) affect ATP synthesis and the hydrolysis activities of the enzyme (see Ref. 11 for a detailed discussion). One of the most well known anisotropic reg...

Simple mechanism whereby the F 1 -ATPase motor rotates with near-perfect chemomechanical energy conversion

“…At around 1,800-1,900°, the magnetic bead resisted forced rotation; (θ B − θ TC ) dropped sharply. This occurred because of ADP inhibition, an inhibitory state common to F 1 s of many species (16)(17)(18)(19). As the forced rotation continued further, F 1 escaped from the inhibited state by the mechanical reactivation as reported (20).…”

miR-141 as a regulator of the mitochondrial phosphate carrier (Slc25a3) in the type 1 diabetic heart

“…Mitochondrial ATP synthase activity. ATP synthase activity was measured in mitochondrial subpopulations and cellular lysate as oligomycin-sensitive ATPase activity using an assay coupled with pyruvate kinase, which converts the ADP to ATP and produces pyruvate from phosphoenolpyruvate as previously described (10,14,33,36). Protein content was assessed as described above (5) with final values expressed as nanomoles consumed per minute per milligram of protein, which was equal to the nanomoles of NADH oxidized per minute per milligram of protein.…”

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

“…So in FATPase a synthesis of one ATP also drives 120 degrees rotation, which, however, requires a movement of 4 subunits c, hence 4 protons (Van Walraven et al 1996;Panke and Rumberg 1997;Stock et al 1999;Ferguson 2000;Seelert et al 2000;Stahlberg et al 2001). In the case of F-ATPase rotation and ATP synthesis is driven by trans-membrane delta pH, but it should be noted that both enzymes can work in both directions depending on the conditions Itoh et al 2004;Rondelez et al 2005;Feniouk et al 2007;Nakano et al 2008). Another difference between these related rotary engines is that whereas the c ring of the F-ATPase is built from identical c subunits, each having two trans-membrane alpha helices (Dmitriev et al 1999;Fillingame et al 2000), the c ring in V-ATPase consist of 4 c subunits and one copies of c' and c" subunits each (Hirata et al 1997;Powell et al 2000).…”