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

Abstract

F 0 F 1 -ATP synthases use the free energy derived from a transmembrane proton transport to synthesize ATP from ADP and inorganic phosphate. The number of protons translocated per ATP (H + /ATP ratio) is an important parameter for the mechanism of the enzyme and for energy transduction in cells. Current models of rotational catalysis predict that the H + /ATP ratio is identical to the stoichiometric ratio of c-subunits to β-subunits. We measured in parallel the H + /ATP ratios at equilibrium of purified F 0 F 1 s from yeast mitochondria (c/β = 3.3) and from spinach chloroplasts (c/β = 4.7). The isolated enzymes were reconstituted into liposomes and, after energization of the proteoliposomes with acid-base transitions, the initial rates of ATP synthesis and hydrolysis were measured as a function of ΔpH. The equilibrium ΔpH was obtained by interpolation, and from its dependency on the stoichiometric ratio,, finally the thermodynamic H + /ATP ratios were obtained: 2.9 ± 0.2 for the mitochondrial enzyme and 3.9 ± 0.3 for the chloroplast enzyme. The data show that the thermodynamic H + /ATP ratio depends on the stoichiometry of the c-subunit, although it is not identical to the c/β ratio. chemiosmotic theory | protonmotive force | bioenergetics | nanomachine C ells of all life kingdoms use H + -ATP synthases to produce the cellular energy carrier ATP from the energy of a transmembrane electrochemical potential difference of protons built up and maintained by proton transport mechanisms, such as the oxidative electron transport in mitochondria or the photoinduced electron transport in chloroplasts (1). The number of protons translocated for each synthesized ATP molecule (H + / ATP ratio) determines how large this difference of proton potential needs to be to maintain the high ATP/ADP ratio required by cell life. It is a key parameter in determining the flow of energy conversions in all living organisms. Ever since compelling experimental evidence in favor of a rotational mechanism for ATP synthases appeared (2-10), picturing them as molecular nanomachines in which two differently stepped motors (the membrane-embedded c-oligomer and the tripartite catalytic head) are connected by a central rotating shaft (γ/ε subunits), the general assumption has been that the H + /ATP ratio should coincide with the ratio of the number of proton-binding c-subunits to the three catalytic nucleotide-binding β-subunits. Structural data have shown that the number of c-subunit monomers varies according to species (ref. 11 and references therein), with numbers that are mostly not multiples of three. Consequences of the above assumption are (i) the H + /ATP ratio can vary among different species, and (ii) the H + /ATP ratio can be a noninteger number. The number of β-subunits is three in all F 0 F 1 analyzed so far, and the number of c-subunits varies between 8 and 15, resulting in predicted H + /ATP ratios between 2.7 and 5.0. To our knowledge, neither of the two assumptions above has yet been experimentally challenged with the required accu...

The Response of Cyclic Electron Flow around Photosystem I to Changes in Photorespiration and Nitrate Assimilation

“…The ratio of H + /ATP is still under debate, with mechanistic models predicting a requirement of 4.7 H + /ATP but experimental measurements indicating H + /ATP closer to 4 (Petersen et al, 2012). In this study, the rates of CEF needed to balance ATP/NADPH demand under all conditions were larger assuming a greater proton requirement for ATP synthase (4.7 as compared with 4 H + /ATP).…”

Twisting and subunit rotation in single FOF1-ATP synthase

“…New non-optical particle trapping devices such as the anti-Brownian electrokinetic trap [100][101][102][103][104] (ABEL trap) invented by Cohen and Moerner can be used to catch and actively push a single F O F 1 -ATP synthase to a target position for extended confocal FRET measurements [105]. Then, the different numbers of transported protons required to make one ATP molecule in the F O F 1 -ATP synthases of chloroplasts [3,106], bacteria [3] or mitochondria [107] (as calculated from ensemble measurements) can be compared to the step sizes of the rotary c-rings of the respective single F O motors.…”

Dynamic photosynthesis under a fluctuating environment: a modelling-based analysis

“…A similar mismatch between the predictions based on structural and biochemical methods was observed for Escherichia coli (Steigmiller et al, 2008) and Saccharomyces cerevisiae (Petersen et al, 2012). The H ) /ATP ratio for ATPase of S. oleracea was also calculated using a biochemical kinetic approach by Berry and Rumberg (1996) also resulting in a value of 4.…”

“…However, Turina et al (2003), Petersen et al (2012) and Steigmiller et al (2008) determined the H ) /ATP ratio for ATPase of S. oleraceae using biochemical equilibrium approaches and obtained a value of 4 in all experiments, suggesting a mismatch between the structure-based and biochemistry-based ratios. A similar mismatch between the predictions based on structural and biochemical methods was observed for Escherichia coli (Steigmiller et al, 2008) and Saccharomyces cerevisiae (Petersen et al, 2012).…”

“…The former value is obtained from biochemical assays, whereas the latter is derived from the structure of ATPase in Spinacia oleracea using different techniques (see Section 5.2.5 for details). Estimations from biochemical assays may be affected by some methodological artefact and discrepancies between these two methods have also been reported for the mitochondrial ATPase of other organisms (Steigmiller et al, 2008;Petersen et al, 2012). For this reason, I chose to perform the simulations in Chapter 5 with an HPR of 14.…”