Proton transport coupled ATP synthesis by the purified yeast H+-ATP synthase in proteoliposomes

Abstract

The H(+)/ATP synthase from yeast mitochondria, MF₀F₁, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF₀F₁ liposomes were energized by acid-base transitions (DeltapH) and a K(+)/valinomycin diffusion potential (Deltaphi). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120s⁻¹ at a transmembrane pH difference of 3.2 units (at pH(in)=4.8 and pH(out)=8.0) and a Deltaphi of 133mV (Nernst potential). Functional studies showed that the monomeric MF₀F₁, was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of K(M) on pH(out) indicated that the substrate for ATP synthesis is the monoanionic phosphate species H₂PO⁻₄.

Macromolecular organization of ATP synthase and complex I in whole mitochondria

“…Given that the matrix pH is 7.9 (33), the cytosolic pH is 7.35 (34), and the outer membrane is freely permeable to ions, the nominal ΔpH across the mitochondrial inner membrane is 0.55 units, equivalent to 32 mV. However, in vitro studies indicate that the mitochondrial, chloroplast and bacterial ATP synthases all need a ΔpH close to 2 units, equivalent to approximately 120 mV, and a p-side pH close to 6, to produce enough ATP to sustain life (35)(36)(37)(38). We propose that the apparent paradox of a ΔpH that is seemingly insufficient and a p-side pH that is too high for efficient ATP production is resolved by the special geometry of the mitochondrial cristae.…”

Comparison of the H + /ATP ratios of the H + -ATP synthases from yeast and from chloroplast

“…Liposomes were prepared as follows: a dry lipid film was prepared by rotary evaporation of 10 mL chloroform containing 250 mg phosphatidylcholine and 12.5 mg phosphatidic acid, resuspended in 8 mL 10 mM Hepes (pH 7.6), 250 mM sucrose, 2 mM MgCl 2 , and 1 mM EDTA and sonicated in 2-mL portions with a 3-mmdiameter tip for a total of 80 s (Branson Sonifier 250, step 2, 60% output), resulting in a lipid concentration of 32.8 mg/mL. MF 0 F 1 or CF 0 F 1 were reconstituted into the liposome membrane, similar to the method described earlier (17). To 150 μL reconstitution buffer (80 mM Mops, 80 mM Mes, 80 mM Hepes, 48 mM KCl, 40 mM NaH 2 PO 4 , and 70-240 mM NaOH), 150 μL liposomes, 40 μL protein solution (2 μM), 2.4 μL MgCl 2 (1 M), 52 μL Triton X-100 [10% (wt/vol)], and 206 μL H 2 O were added to a final volume of 600 μL.…”

“…MF 0 F 1 from Saccharomyces cerevisiae cells of the strain YRD15 was isolated as previously described (17). The MF 0 F 1 complex was obtained in a buffer containing 20 mM Hepes/NaOH (pH 7.65), 250 mM sucrose, 1 mM EDTA, 4 mM MgCl 2 , 5 mM 6-aminohexanoic acid, 1 mM DTT, 100 mM NaCl, and 1 mM dodecyl maltoside (Glycon), with a protein concentration of 5-10 μM, rapidly frozen, and stored in liquid nitrogen.…”

“…Hence, their respective H + /ATP ratios, based on the assumptions mentioned above, should be 3.3 and 4.7. We have isolated the two complexes and reconstituted them into liposomes, which, if subjected to acid-base transitions to generate a high protonmotive force, were able to synthesize ATP at physiological rates (17,18). The technique of acid-base transitions offers the great advantages of (i) measuring the imposed transmembrane ΔpH with the accuracy of the pH electrode, thus making high-precision quantitative studies possible (18)(19)(20), and (ii) allowing the testing of ATP synthases from different species with the same method and under identical experimental conditions.…”

Nano breathers and molecular dynamics simulations in hydrogen-bonded chains

“…The proton dynamics in HB chains is often modeled by a characteristic non-linear substrate potential with two degenerate equilibrium positions. The solitons ensure the transport of energy and charges in bioenergetics, e.g., mitochondrial adenosine triphosphate formation [18,19], photophosphorylation in chloroplasts [20], anaerobic metabolism in Halobacterium [21], and proton migration in ice crystals [22], and explain some aspects of biological processes such as the duplication of deoxyribonucleic acid (DNA) and the transcription of messenger ribonucleic acid (mRNA) [23], the denaturation of DNA [24], and the molecular mechanism of muscle contraction [25,26]. As a consequence, a detailed understanding of the charge transfer mechanism of HB chains will have great impact on our picture of the localization of bioenergy in HB chains.…”