Bernd Rumberg

The H+/ATP coupling ratio of the ATP synthase from thiol‐modulated chloroplasts and two cyanobacterial strains is four

In this paper the authors emphasise that the proton translocating ATP synthase from thiol‐modulated chloroplasts and two cyanobacterial strains has a coupling ratio of 4 protons per ATP synthesised or hydrolysed. This ratio is determined by several thermodynamic studies at equilibrium between phosphate potential (ΔG p) and proton gradient ( ), and is confirmed by measurement of proton flux during ATP hydrolysis. Ratios lower than 4 H+/ATP that have been published in the past have predominantly been determined with the oxidised chloroplast enzyme. Errors in these measurements will be discussed.

Evidence for electronic and ionic interaction between electron transport chains in chloroplasts

Abstract Electron transport, phosphorylation and the absorption changes of plastoquinone and chlorophyll-a1 have been measured as a function of 3(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) concentration. The differential effect of DCMU observed is not consistent with the concept of isolated chains of electron carriers. Two types of cooperation are realized. 1. 1. Electron exchange is possible between at least 10 chains within a common plastoquinone strand. This follows from the different sensitivity of the electron transfer reactions against DCMU with regard to short and long time excitation under uncoupled conditions. 2. 2. All the electron transport chains of one thylakoid seem to interact by means of the internal protons which are pumped into the common inner phase by the chains and react on them by means of pH-sensitive electron transfer reactions. This is the interpretation of the different DCMU dependence of basal, coupled and uncoupled electron transport.

Kinetic modeling of rotary CF0F1-ATP synthase: storage of elastic energy during energy transduction

F0F1-ATP synthase uses proton-motive force to produce ATP from ADP and Pi. With regard to its rotary mechanics, this energy transducing molecular machine assumes a unique position among all enzymes. In the work presented here we put forward a detailed functional model which is based on experimental results obtained with ATP synthase from spinach chloroplasts. We focus on the role of the elastic element, realized by the stalk-like subunit gamma, whose function is energy transduction between F0 and F1 taking into account the H+/ATP coupling ratio of four. Fitting parameters are the rate constants and the torsional rigidity of gamma, which have been adjusted according to the experimental results where the influence of transmembrane DeltapH on the rates of ATP synthesis/hydrolysis is put to the test. We show that the input and output of torsional energy are regulated by purely statistical principles, giving rise to the amount of transiently stored energy to be sliding, depending on DeltapH. During conditions of maximal turnover gamma turns out to be wound up towards 102 degrees which corresponds to a torque of 5.3. 10-20 N.m.

H+/ATP coupling ratio at the unmodulated CF0CF1-ATP synthase determined by proton flux measurements

Abstract The H + /ATP coupling ratio is determined kinetically by comparing ATP synthesis and proton flow across the ATP synthase in spinach thylakoids under conditions of continuous illumination. Net proton flow at steady-state is zero, therefore the initial efflux in the dark is taken. A new method of kinetic analysis of the relaxation of the transmembrane proton gradient is presented, allowing direct determination of phosphorylating and basal proton fluxes at the same time. Special care is taken for the influence of dark phosphorylation and the H + diffusion potential. The investigations give evidence of a H + /ATP coupling ratio of four for the oxidized or unmodulated state of the ATP synthase being at par with the coupling ratio for the reduced or modulated state of the enzyme.

Proton to electron stoichiometry in electron transport of spinach thylakoids

According to the concept of the Q-cycle, the H+/e- ratio of the electron transport chain of thylakoids can be raised from 2 to 3 by means of the rereduction of plastoquinone across the cytochrome b6f complex. In order to investigate the H+/e- ratio we compared stationary rates of electron transport and proton translocation in spinach thylakoids both in the presence of the artificial electron acceptor ferricyanide and in the presence of the natural acceptor system ferredoxin+NADP. The results may be summarised as follows: (1) a variability of the H+/e- ratio occurs with either acceptor. H+/e- ratios of 3 (or even higher in the case of the natural acceptor system, see below) are decreased towards 2 if strong light intensity and low membrane permeability are employed. Mechanistically this could be explained by proton channels connecting the plastoquinol binding site alternatively to the lumenal or stromal side of the cytochrome b6f complex, giving rise to a proton slip reaction at high transmembrane DeltapH. In this slip reaction protons are deposited on the stromal instead of the lumenal side. In addition to the pH effect there seems to be a contribution of the redox state of the plastoquinone pool to the control of proton translocation; switching over to stromal proton deposition is favoured when the reduced state of plastoquinone becomes dominant. (2) In the presence of NADP a competition of both NADP and oxygen for the electrons supplied by photosystem I takes place, inducing a general increase of the H+/e- ratios above the values obtained with ferricyanide. The implications with respect to the adjustment of a proper ATP/NADPH ratio for CO2 reduction are discussed.

The H+/ATP Coupling Ratio at the H+-ATP-Synthase of Spinach Chloroplasts is Four

The importance of the H+/ATP ratio, i.e. the number of H+ which have to be translocated across the ATP-synthase for each ATP molecule being synthesized or hydrolysed, has its origin in the treefold aspect of (1) mechanistic consequences with respect to the coupling process, (2) energetic consequences with respect to the attainable excess of ATP above ADP, (3) stoichiometric consequences with respect to the overall process of CO2 reduction.

Kinetic modelling of the proton translocating CF0CF1-ATP synthase from spinach

The rate of both ATP synthesis and hydrolysis catalysed by the thiol‐modulated and activated ATP synthase from spinach is measured as a function of all substrates including the protons inside the thylakoid lumen. The most important findings are: (1) sigmoid kinetics with respect to Hin +, (2) hyperbolic kinetics with respect to ADP, ATP and phosphate, with K m for phosphate and ADP decreasing upon increasing Hin +, (3) binding of ADP and phosphate in random order and competitive to ATP. Simulation of the complete set of experimental data is obtained by a kinetic model featuring Boyers binding‐change mechanism.

Kinetic modeling of the photosynthetic electron transport chain.

A simulation model of the photosynthetic electron transport chain operating under steady state conditions is presented. The model enables the calculation of (1) the rates of electron transport and transmembrane proton translocation, (2) the proton/electron stoichiometry, (3) the number of electrons stored in the different redox centers and (4) the stationary transmembrane pH difference. Light intensity and proton permeability of the thylakoid membrane are varied in order to compare the predictions of the model with experimental data. The routes of electron transport and proton translocation are simulated by two coupled arithmetic loops. The first one represents the sequence of reaction steps making up the linear electron transport chain and the Q-cycle. This loop yields the electron flow rate and the proton/electron ratio. The second loop balances the H+ fluxes and yields the internal H+ concentration. The bifurcation of the electron transport pathways at the stage of plastoquinol oxidation is obligatory. The first electron enters always the linear branch and is transferred to photosystem I. The electron of the remaining semiquinone can enter the Q-cycle or, alternatively, the semiquinone can be lost from the cytochrome b6f complex. The competition between these two reactions explains the experimentally observed variability of the proton/electron ratio. We also investigated additional model variants, where the variation of the proton/electron stoichiometry is attributed to other loss reactions within the cytochrome b6f complex. However, the semiquinone detachment seems to be the best candidate for a satisfactory description of the experimental data. Additional calculations were done in order to assess the effects of the movement of the Rieske protein on linear electron transport; it was found that this conformational change does not limit the electron transport rate, if it occurs with a time constant of at least 1000 s(-1).

Evidence for the participation of cytochrome b in the electron-transport system of photosynthesis

Abstract Rapid light-induced changes in the absorbancy of photosynthesizing broken chloroplasts (spinach) were measured in the blue region. New information was obtained by alternative illumination with red and far-red actinic light. Evidence is given that the complex overall difference spectrum is composed of three single difference spectra which are caused by the oxidation of chlorophyll a1−430−703 and cytochrome f-407−424−555 and the reduction of oxidized cytochrome b-412−435−(563). By analysis of the changes of cytochrome b it was possible to get the first reliable information about the participation of this substance in photosynthesis. The experimental results favour the assumption that cytochrome b is involved as electron acceptor of light reaction II in photosynthesis and that the electron transfer from cytochrome b to oxidized cytochrome f is the rate-limiting step in the electron transport from light reaction II to light reaction I.

Kinetics and energetics of redox regulation of ATP synthase from chloroplasts

The rate of ATP hydrolysis catalyzed by the membrane‐bound CF0F1 ATP synthase from chloroplasts served as a probe for the determination of the reduction grade of the enzyme treated with dithiothreitol (DTT) or thioredoxin. Rate constants for reduction were obtained. It turns out that reduction by thioredoxin is about a factor of 6,000 more effective than DTT reduction. The activation profiles with respect to ΔpH were obtained for reduced and oxidized ATPases. The activation curve of reduced enzyme turns out to have its half‐maximum degree of activation at ΔPH = 1.65, which is considerably lower than reported hitherto. The corresponding value of the oxidized enzyme has been obtained from the rate of ATP hydrolysis in the case of incomplete reduced ATPases, taking into account the aforementioned rate constants, and comes to ΔpH = 3.35.