Sodium,protons, and energy coupling in the methanogenic bacteria

In this review, I focus on the bioenergetics of the methanogenic bacteria, with particular attention directed to the roles of transmembrane electrochemical gradients of sodium and proton. In addition, the mechanism of coupling ATP synthesis to methanogenic electron transfer is addressed. Evidence is reviewed which suggests that the methanogens possess great diversity in their bioenergetic machinery. In particular, in some methanogens the primary ion which is translocated coupled to metabolic energy is the proton, while others appear to utilize sodium. In addition, ATP synthesis driven by methanogenic electron transfer is accomplished in some organisms by a chemiosmotic mechanism and is coupled by a more direct mechanism in others. A possible explanation for this diversity (which is consistent with the relatedness of these organisms to each other and to other members of the Archaebacteria as determined by molecular biological techniques) is discussed.     

ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. III. Characterization of the ATP formation onset lag and post-illumination phosphorylation for thylakoids exhibiting localized or bulk-phase delocalized energy coupling

When 100 mM KCl replaced sucrose in a chloroplast thylakoid stock suspension buffer, the membranes were converted from a localized proton gradient to a delocalized proton gradient energy coupling mode. The KCl-suspended but not the sucrose-suspended thylakoids showed pyridine-dependent extensions of the ATP onset lag and pyridine effects on post-illumination phosphorylation. The ATP formation assays were performed in a medium of identical composition, using about a 200-fold dilution of the stock thylakoid suspension; hence the different responses were due to the pretreatment, and not the conditions present in the phosphorylation assay. Such permeable buffer effects on ATP formation provide a clear indicator of delocalized proton gradients as the driving force for phosphorylation. The pyridine-dependent increases in the onset lags (and effects on post-illumination phosphorylation) were not due to different ionic conductivities of the membranes (measured by the 515 nm electrochromic absorption change), H⁺/e ^– ratios, or electron transport capacities for the two thylakoid preparations. Thylakoid volumes and [ 14C]pyridine equilibration were similar with both preparations. The KCl-induced shift toward a bulk-phase delocalized energy coupling mode was reversed when the thylakoids were placed back in a low-salt medium.Proton uptake, at the ATP-formation energization threshold flash number, was much larger in the KCl-treated thylakoids and they also had a longer ATP formation onset lag, when no pyridine was present. These results are consistent with the salt treatment exposing additional endogenous buffering groups for interaction with the proton gradient. The concomitant appearance of the pyridine buffer effects implies that the additional endogenous buffering groups must be located on proteins directly exposed in the aqueous lumen phase.Kinetic analysis of the decay of the post-illumination phosphorylation in the two thylakoid preparations showed different apparent first-order rate constants, consistent with there being two different compartments contributing to the proton reservoirs that energize ATP formation. We suggest that the two compartments are a membrane-phase localized compartment operative in the sucrose-treated thylakoids and the bulk lumen phase into which protons readily equilibrate in the KCl-treated thylakoids.     

Evidence that the intrinsic membrance protein LHCII in thylakoids is necessary for maintaining localized\Delta \tilde \mu _{H^ + } energy coupling

This work tested the hypothesis that thylakoid localized proton-binding domains, suggested to be involved in localized -driven ATP formation, are maintained with the involvement of several membrane proteins, including the LHCII (Laszlo, J. A., Baker, G. M., and Dilley, R. A. (1984) Biochim. Biophys. Acta 764, 160–169), which comprises about 50% of the total thylakoid protein. The concept we have in mind is that several membrane proteins cooperate to shield a localized proton diffusion pathway from direct contact with the lumen, thus providing a physical barrier to H⁺ equilibration between the sequestered domains and the lumen. A barely mutant,chlorina f ₂, that lacks Chl b and does not accumulate some of the LHCII proteins, was tested for its capacity to carry out localized-proton gradient-dependent ATP formation. Two previously developed assays permit clear discrimination between localized and delocalized gradient-driven ATP formation. Those assays include the effect of a permeable buffer, pyridine, on the number of single-turnover flashes needed to reach the energetic threshold for ATP formation and the more recently developed assay for lumen pH using 8-hydroxy-1,3,6-pyrene trisulfonic acid as a lumenally loaded pH-sensitive fluorescent probe. By those two criteria, the wild-type barley thylakoids revealed either a localized or a delocalized energy coupling mode under low- or high-salt storage conditions, respectively. Addition of Ca⁺⁺ to the high-salt storage medium caused those thylakoids to maintain a localized energy-coupling response, as previously observed for pea thylakoids. In contrast, thechlorina f ₂ mutant thylakoids had an active delocalized energy coupling activity but did not show localized energy coupling under any conditions, and added Ca⁺⁺ to the thylakoid storage medium did not alter the delocalized energy coupling mode. One interpretation of the results is that the absence of the LHCII polypeptides produces a leaky pathway for protons which allows the gradient to equilibrate with the lumen under all conditions. Another interpretation is possible but seems less likely, that being that the absence of the LHCII polypeptides in some way causes the proposed Ca⁺⁺ -gated H⁺ flux site on the membrane sector (CF₀) of the energy coupling complex to lose its gating function.     

pH effects on light dependence of the stoichiometry of proton influx and efflux to electron transport in spinach chloroplasts

Initial and steady state rates of proton transport at low light intensity have been measured and compared with steady state rates of electron transport in the pH range of 6.0–7.6 in envelope-free spinach chloroplasts. At pH 6–7, the H⁺/e^- values computed using the initial rate of proton transport varied with light intensity, from a value of 2 at low light to almost 5 at high light. In contrast, the H⁺/e^- values computed using the steady state rate of proton transport did not show a dependence on light intensity, having a constant value of 1.7±0.2. Likewise, at pH 7.6, the H⁺/e^- ratio, computed using either the initial or steady state rates of proton transport did not vary with light intensity but was constant at H⁺/e^-=1.7±0.1. Analysis of the light dependence of electron and proton transport allowed determination of (a) the quantam requirements of transport, (b) the rates of transport at light saturation, and (c) H⁺/e^- ratios for initial and steady state proton transport. Extrapolating the initial proton transport to zero light, we found that both H⁺/photon and H⁺/e^- values were not strongly dependent on pH, approaching a near constant value of 2.0. Using the initial rate of proton transport extrapolated to saturating light intensity we found the H⁺/e^- ratio to be strongly pH-dependent. We suggest that internal pH controls electron transport at high light intensities. The true stoichiometry is reflected only in measurements taken at low light using the initial proton transport data. Our findings and interpretation reconcile some conflicting data in the literature regarding the pH-dependence of the H⁺/e^- ratio and support the concept that internal pH controls noncyclic electron transport.Abbreviations Bicine N, N-bis [2-hydroxyethyl] glycine - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethansulfonic acid - MES 2-(N-morpholino) ethanesulfonic acid     

Proton transfer dynamics at membrane/water interface and mechanism of biological energy conversion

Proton transfer between water and the interior of membrane proteins plays a key role in bioenergetics. Here we survey the mechanism of this transfer as inferred from experiments with flash-triggered enzymes capturing or ejecting protons at the membrane surface. These experiments have revealed that proton exchange between the membrane surface and the bulk water phase proceeds at 1 msec because of a kinetic barrier for electrically charged species. From the data analysis, the barrier height for protons could be estimated as about 0.12 eV, i.e., high enough to account for the observed retardation in proton exchange. Due to this retardation, the proton activity at the membrane surface might deviate, under steady turnover of proton pumps, from that measured in the adjoining water phase, so that the driving force for ATP synthesis might be higher than inferred from the bulk-to-bulk measurements. This is particularly relevant for alkaliphilic bacteria. The proton diffusion along the membrane surface, on the other hand, is unconstrained and fast, occurring between the neighboring enzymes at less than 1 µsec. The anisotropy of proton dynamics at the membrane surface helps prokaryotes diminish the futile escape of pumped protons into the external volume. In some bacteria, the inner membrane is invaginated, so that the ejected pro tons get trapped in the closed space of such intracellular membrane sacks which can be round or flat. The chloroplast thylakoids and the mitochondrial cristae have their origin in these intracellular structures.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 308–314.Original Russian Text Copyright © 2005 by Mulkidjanian, Cherepanov, Heberle, Junge.This revised version was published online in April 2005 with corrections to the post codes.     

Models of cardiac excitation-contraction coupling in ventricular myocytes

Mathematical and computational modeling of cardiac excitation-contraction coupling has produced considerable insights into how the heart muscle contracts. With the increase in biophysical and physiological data available, the modeling has become more sophisticated with investigations spanning in scale from molecular components to whole cells. These modeling efforts have provided insight into cardiac excitation-contraction coupling that advanced and complemented experimental studies. One goal is to extend these detailed cellular models to model the whole heart. While this has been done with mechanical and electophysiological models, the complexity and fast time course of calcium dynamics have made inclusion of detailed calcium dynamics in whole heart models impractical. Novel methods such as the probability density approach and moment closure technique which increase computational efficiency might make this tractable.     

Absence of kinetic barrier for transfer of protons from aqueous phase to membrane-water interface

The kinetics of interfacial proton transfer reaction is an important factor in proton transport across membranes. The following experimental system was designed in order to measure this kinetics. Sonicated liposomes having the protonophore SF6847 was suspended in Tris buffer. Application of a temperature jump (in ∼ 3 μs) caused a drop in the aqueous phase pH which was subsequently sensed by the membrane-bound SF6847. The kinetics of this interfacial proton transfer reaction was monitored on μs timescales. The estimated bimolecular rate constant of 2×10¹¹ M⁻¹ s_#x2212;1 for this process show that there is no kinetic barrier for the transfer of protons from the aqueous phase to the membrane-water interface.     

How acidic is the lumen?

Proton motive force (pmf), established across the thylakoid membrane by photosynthetic electron transfer, functions both to drive the synthesis of ATP and initiate processes that down-regulate photosynthesis. At the same time, excessively low lumen pH can lead to the destruction of some lumenal components and sensitization of the photosynthetic apparatus to photoinhibition. Therefore, in order to understand the energy budget of photosynthesis, its regulation and responses to environmental stresses, it is essential to know the magnitude of pmf, its distribution between pH and the electric field () as well as the relationships between these parameters and G_ATP, and down-regulatory and inhibitory processes. We review past estimates of lumen pH and propose a model that can explain much of the divergent data in the literature. In this model, in intact plants under permissive conditions, photosynthesis is regulated so that lumen pH remains mod erate (between 5.8 and 6.5), where it modulates the activity of the violaxanthin deepoxidase, does not significantly restrict the turnover of the cytochrome b₆f complex, and does not destabilize the oxygen evolving complex. Only under stressed conditions, where light input exceeds the capacity of both photosynthesis and down-regulatory processes, does lumen pH decrease below 5, possibly contributing to photoinhibition. A value of n = 4 for the stoichiometry of protons pumped through the ATP synthase per ATP synthesized, and a minor contribution of to pmf, will allow moderate lumen pH to sustain the observed levels of G_ATP.     

Correlation between membrane-localized protons and flash-driven ATP formation in chloroplast thylakoids

Flash-driven ATP formation by spinach chloroplast thylakoids, using the luciferin luminescence assay to detect ATP formed in single turnover flashes, was studied under conditions where a membrane protein amine buffering pool was either protonated or deprotonated before the beginning of the flash trains. The flash number for the onset of ATP formation was delayed by about 10 flashes (from 15 to about 25) when the amine pool was deprotonated as compared to the protonated state. The delay was substantially reversed again by reprotonating the pool upon application of 20–30 single-turnover flashes and 8 min of dark before addition of ADP, P_i, and the luciferin system. In the case of deprotonation by desaspidin, the uncoupler was removed by binding to BSA before the reprotonating flashes were given. Reprotonation was carried out before addition of ADP and P_i, to avoid a possible interference by the ATP-ase, which can energize the system by pumping protons. The reprotonated state, as indicated by an onset lag of about 15 flashes rather than 25 for the deprotonated state, was stable in the dark over extended dark times. The number of protons released by 10 flashes is approximately 30 nmol H⁺ (mg chl)^–1, an amount similar to the size of the reversibly protonated amine group buffering pool. The data are consistent with the hypothesis that the amine buffering groups must be in the protonated state before any protons proceed to the coupling complex and energize ATP formation. Other work has suggested that the amine buffering pool is sequestered within membrane proteins rather than being exposed directly to the inner aqueous bulk phase. Therefore, it is possible that the sequested amine group array may provide localized association-dissociation sites for proton movement to the coupling complex.     

The kinetic mechanism by which CCCP (carbonyl cyanidem-Chlorophenylhydrazone) transports protons across membranes

Summary We demonstrate that a simple kinetic model describes the transport of protons across lipid bilayer membranes by the weak acid CCCP (carbonyl cyanidem-chlorophenylhydrazone). Four parameters characterize this model: the adsorption coefficients of the anionic and neutral forms of the weak acid onto the interface ( _A and _HA) and the rate constants for the movement of A^– and HA across the membrane (k _A andk _HA). These parameters were determined by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. From these equilibrium and steady state measurements on diphytanoyl phosphatidylcholine/chlorodecane membranes we found that _A= _HA=1.4 10^-3cm,k _A=175 s^–1 andk _HA=12,000 sec^–1. These parameters and our model describe our kinetic experiments if we assume that the protonation reactions, which occur at the interfaces, remain at equilibrium. The model predicts a single exponential decay of the current in a voltage-clamp experimetn. The model also predicts that the decay in the voltage across the membrane following an intense current pulse of short duration (50 nsec) can be described by the sum of two exponentials. The magnitudes and time constants of the relaxations that we observed in both voltage-clamp and charge-pulse experiments agree well with the predictions of the model for all values of pH, voltage and [CCCP].     

Calculated electrostatic gradients in recombinant human H-chain ferritin.

Calculations to determine the electrostatic potential of the iron storage protein ferritin, using the human H-chain homopolymer (HuHF), reveal novel aspects of the protein. Some of the charge density correlates well with regions previously identified as active sites in the protein. The three-fold channels, the putative ferroxidase sites, and the nucleation sites all show expectedly negative values of the electrostatic potential. However, the outer entrance to the three-fold channels are surrounded by regions of positive potential, creating an electrostatic field directed toward the interior cavity. This electrostatic gradient provides a guidance mechanism for cations entering the protein cavity, indicating the three-fold channel as the major entrance to the protein. Pathways from the three-fold channels, indicated by electrostatic gradients on the inner surface, lead to the ferroxidase center, the nucleation center and to the interior entrance to the four-fold channel. Six glutamic acid residues at the nucleation site give rise to a region of very negative potential, surrounding a small positively charged center due to the presence of two conserved arginine residues, R63, in close proximity (4.9 A), suggesting that electrostatic fields could also play a role in the nucleation process. A large gradient in the electrostatic potential at the 4-fold channel gives rise to a field directed outward from the internal cavity, indicating the possibility that this channel functions to expel cations from inside the protein. The 4-fold channel could therefore provide an exit pathway for protons during mineralization, or iron leaving the protein cavity during de-mineralization.     

Proton-linked sugar transport systems in bacteria

The cell membranes of various bacteria contain proton-linked transport systems ford-xylose,l-arabinose,d-galactose,d-glucose,l-rhamnose,l-fucose, lactose, and melibiose. The melibiose transporter ofE. coli is linked to both Na⁺ and H⁺ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H⁺ transporters inE. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H⁺, arabinose/H⁺, and galactose/H⁺ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning -helices, possibly in two groups of six, there is a central hydrophilic region, probably comprised largely of -helix; the highly conserved amino acid residues (40–50 out of 472–522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na⁺ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na⁺ transporter ofE. coli, and there is evidence for its structural similarity to glucose/H⁺ transporters in Plants.In vivo andin vitro mutagenesis of the lactose/H⁺ and melibiose/Na⁺ (H⁺) transporters ofE. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H⁺ transporter has been purified. The directions of future investigations are discussed.     

A shift in chloroplast energy coupling by KCl from localized to bulk phase delocalized proton gradients

Spinach thylakoids were resuspended in a buffer including either 200 mM sucrose or 100 mM KCl. singleturnover flash excitation and the luciferin-luciferase system were used to energize and follow ATP formation. The effect of the permeable buffer pyridine was measured on the ATP formation onset lag and on the post-illumination ATP yield. Consistent with a bulk phase delocalized proton gradient coupling model, thylakoids stored in 100 mM KCl exhibited an increase in these two parameters in the presence of pyridine. Thylakoids isolated in the absence of KCl showed no effect of pyridine on the two parameters indicating a localized energy coupling mode.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Delayed light studies on photosynthetic energy conversion. VIII. Evidence from millisecond emission of chloroplasts for two adenylate binding sites on membrane-bound coupling factor,CF1

Effects of adenylates on chloroplast delayed light emission, at millisecond dark times, are inverse to the previously characterized effects of adenylates on electron transport rates. Either ADP alone or ATP alone increase intensity of delayed light, while ADP plus P_i decrease it. ADP alone requires the presence of an electron acceptor to have this effect on delayed light, but ATP does not.All three adenylate effects are abolished by uncoupling with gramicidin, by partial removal of photophosphorylation coupling factor (CF₁) with EDTA, and by antibody to CF₁. Readdition of CF₁ re-established the adenylate effects in EDTA-stripped membranes. The three adenylate effects are differentially sensitive to pH, and pH differentially affected their abolition by antibody to CF₁. The two adenylate effects shown in the absence of P_i are exhibited at lower adenylate concentrations than the ADP plus P_i effect, and are also less sensitive to phloridzin.These results are discussed in terms of probable adenylate effects on membrane-bound chloroplast coupling factor, CF₁. At least two ADP binding sites would differ with respect to adenylate concentration for half maximal binding; pH of optimal binding capacity; phloridzin sensitivity; and functional regulation of electron transport, proton uptake, and energy storage within the membrane as measured by delayed light emission. It remains unclear whether the high affinity ADP binding site is identical to a high affinity ATP binding site on CF₁.     

Energy coupling and respiration inNitrosomonas europaea

Intact cells ofNitrosomonas europaea grown in an ammonium salts medium will oxidise ammonium ions, hydroxylamine and ascorbate-TMPD; there is no oxidation of carbon monoxide, methane or methanol. TheK _m value for ammonia oxidation is highly pH dependent with a minimum value of 0.5 mM above pH 8.0. This suggests that free ammonia is the species crossing the cytoplasmic membrane(s). The measurement of respiration driven proton translocation indicates that there is probably only one proton translocating loop (loop 3) association with hydroxylamine oxidation. The oxidation of endogenous substrates is sometimes associated with more than one proton-translocating loop. These results indicate that during growth hydroxylamine oxidation is probably associated with a maximum P/O ratio of 1.Abbreviations H⁺/O ratio g equiv. H⁺ translocated/g atom O consumed     

Energy coupling of membrane transport and efficiency of sucrose dissimilation in yeast

Proton coupled transport of α-glucosides via Mal11 into Saccharomyces cerevisiae costs one ATP per imported molecule. Targeted mutation of all three acidic residues in the active site resulted in sugar uniport, but expression of these mutant transporters in yeast did not enable growth on sucrose. We then isolated six unique transporter variants of these mutants by directed evolution of yeast for growth on sucrose. In three variants, new acidic residues emerged near the active site that restored proton-coupled sucrose transport, whereas the other evolved transporters still catalysed sucrose uniport. The localization of mutations and transport properties of the mutants enabled us to propose a mechanistic model of proton-coupled sugar transport by Mal11. Cultivation of yeast strains expressing one of the sucrose uniporters in anaerobic, sucrose-limited chemostat cultures indicated an increase in the efficiency of sucrose dissimilation by 21% when additional changes in strain physiology were taken into account. We thus show that a combination of directed and evolutionary engineering results in more energy efficient sucrose transport, as a starting point to engineer yeast strains with increased yields for industrially relevant products.     

An Issue of Originality and Priority: The Correspondence and Theories of Oxidative Phosphorylation of Peter Mitchell and Robert J.P. Williams, 1961–1980

In the same year, 1961, Peter D. Mitchell and Robert R.J.P. Williams both put forward hypotheses for the mechanism of oxidative phosphorylation in mitochondria and photophosphorylation in chloroplasts. Mitchell’s proposal was ultimately adopted and became known as the chemiosmotic theory. Both hypotheses were based on protons and differed markedly from the then prevailing chemical theory originally proposed by E.C. (Bill) Slater in 1953, which by 1961 was failing to account for a number of experimental observations. Immediately following the publication of Williams’s hypothesis and before his own was published, Mitchell initiated a correspondence. Examination of the letters shows the development of a dispute based on the validity of the proposals, who should have priority and particularly whether Mitchell had drawn on Williams’s work without acknowledgement. We have concluded that Mitchell’s proposals were original (a view still questioned by Williams) although it is evident that prior to the correspondence Williams had considered and rejected a proposition similar to Mitchell’s theory. However, a major cause of the dispute was the difference in disciplinary backgrounds of Mitchell, a microbial biochemist and Williams, a chemist.