Electrochemical gradients and energy coupling in photosynthetic bacteria

The electrochemical proton gradient formed during light-induced electron transport in bacterial chromatophores is composed of both a proton concentration gradient and a membrane potential that can interchange under appropriate conditions. Both components, whether light-induced or imposed artificially in the dark, can drive ATP synthesis.     

Metabolic regulation by pH gradients. Inhibition of photosynthesis by indirect proton transfer across the chloroplast envelope

Anions of several weak acids inhibited photosynthesis in isolated spinach chloroplasts. Inhibition was drastic at low pH and weak or absent at high pH. Glyoxylate was particularly effective and inhibition decreased in the order: glyoxylate, nitrite, glycerate, formate, hydroxypyruvate, glycolate, propionate, acetate, pyruvate. These anions operated as indirect proton shuttles across the chloroplast envelope. They compensated active proton fluxes into the medium, minimized gradients in proton activity across the chloroplast envelope, and so prevented light-dependent stroma alkalization. This caused inhibition of sugar bisphosphatases which are known to be pH-regulated. At concentrations that caused potosynthesis inhibition, the proton shuttles were not effective in decreasing the proton gradient across the thylakoids. Some anions also inhibited fructose-bisphosphatase directly, when present at concentratins higher than needed for photosynthesis inhibition.     

ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. II. Two modes of post-illumination phosphorylation driven by either delocalized or localized proton gradient coupling

Two modes of chloroplast membrane post-illumination phosphorylation were detected, using the luciferin-luciferase ATP assay, one of which was not influenced by added permeable buffer (pyridine). That finding provides a powerful new tool for studying proton-membrane interactions during energy coupling. When ADP and P_i were added to the thylakoid suspension after a train of flashes [similar to the traditional post-illumination phosphorylation protocol (termed PIP^– here)], the post-illumination ATP yield was influenced by pyridine as expected, in a manner consistent with the ATP formation, in part, being driven by protons present in the bulk inner aqueous phase, i.e., through a delocalized protonmotive force. However, when ADP and P_i were present during the flash train (referred to as PIP⁺), and ATP formation occurred during the flash train, the post-illumination ATP yield was unaffected by the presence of pyridine, consistent with the hypothesis that localized proton gradients were driving ATP formation. To test this hypothesis further, the pH and flash number dependence of the PIP^– and PIP⁺ ATP yields were measured, the results being consistent with the above hypothesis of dual compartment origins of protons driving post-illumination ATP formation.Measuring proton accumulation during the attainment of the threshold energization level when no component was allowed to form (+ valinomycin, K⁺), and testing for pyridine effects on the proton uptake, reveals that the onset of ATP formation requires the accumulation of about 60 nmol H⁺ (mg Chl)^–1. Between that level and about 110–150 nmol H⁺ (mg Chl)^–1, the accumulation appears to be absorbed by localized-domain membrane buffering groups, the protons of which do not equilibrate readily with the inner aqueous (lumen) phase. Post-illumination phosphorylation driven by the dissipation of the domain protons was not affected by pyridine (present in the lumen), even though the effective pH in the domains must have been well into the buffering range of the pyridine. That finding provides additional insight into the localized domains, namely that protons can be absorbed by endogenous low pK buffering groups, and released at a low enough pH (5.7 when the external pH was 8, 4.7 at pH 7 external) to drive significant ATP formation when no further proton production occurs due to the redox turnovers. We propose that proton accumulation beyond the 110–150 nmol (mg Chl)^–1 level spills over into the lumen, interacting with additional, lumenal endogenous buffering groups and with pyridine, and subsequent efflux of those lumenal protons can also drive ATP formation. Such a dual-compartment thylakoid model for the accumulation of protons competent to drive ATP formation would require a gating mechanism to switch the proton flux from the localized pathway into the lumen, as discussed by R. A. Dilley, S. M. Theg, and W. A. Beard (1987)Annu. Rev. Plant Physiol. 38, 348–389, and recently suggested by R. D. Horner and E. N. Moudrianakis (1986)J. Biol. Chem. 261, 13408–13414. The model can explain conflicting data from past work showing either localized or delocalized gradient coupling patterns.     

Structural map of the dicyclohexylcarbodiimide site of chloroplast coupling factor determined by resonance energy transfer

Fluorescence resonance energy-transfer measurements were made on the membrane-bound chloroplast coupling factor. The distances from the N,N'-dicyclohexylcarbodiimide-binding site on the membrane-bound portion of the enzyme (CF0) to the vesicle surface and to two sulfhydryl sites on the gamma-polypeptide were determined. The dicyclohexylcarbodiimide-binding site was labeled with the fluorescent species N-cyclohexyl-N'-pyrenylcarbodiimide. The vesicle surface was labeled with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine. Steady-state energy transfer between the fluorescent-labeled enzyme (energy donor) and varying concentrations of the ethanolamine derivative (energy acceptor) indicated that the distance of closest approach between the energy donor and the outer vesicle surface is 16-24 A. Two specific sites on the gamma-polypeptide were reacted with a coumarinylmaleimide derivative; one is a sulfhydryl that can be labeled only on the thylakoids under energized conditions (the "light" site), while the other is the disulfide site that regulates enzymatic activity. Energy-transfer measurements utilizing steady-state fluorescence and fluorescence lifetime methods indicated that the dicyclohexylcarbodiimide site is approximately 41 A from the light site and approximately 50 A from the gamma-disulfide site. These distances are used to extend the current structural model of the chloroplast coupling factor.     

ATP synthesis and hydrolysis in chloroplast membranes. Differential inhibition by antibodies to chloroplast coupling factor 1.

1. Divalent antibodies against chloroplast coupling factor 1 inhibited the factor ATPase, ATP synthesis, hydrolysis and Pi-ATP exchange in chloroplasts. These antibodies also inhibited coupled electron flow rates but not the basal or uncoupled rates. 2. Several types of non-precipitating, modified antibodies prepared from the original antibody preparation strongly inhibited the ATPase and Pi-ATP exchange reaction but had little effect on ATP formation. 3. It is suggested that the inhibition of ATP synthesis by the divalent antibodies is probably due to an indirect blocking of the active site, while the inhibition of ATP-utilizing reactions by the modified antibodies is related to their effect on the transfer of ATP from a non-catalytic to a catalytic site on coupling factor 1, via an energy-dependent conformational change.     

A protein oxidase catalysing disulfide bond formation is localized to the chloroplast thylakoids

In chloroplasts, thiol/disulfide-redox-regulated proteins have been linked to numerous metabolic pathways. However, the biochemical system for disulfide bond formation in chloroplasts remains undetermined. In the present study, we characterized an oxidoreductase, AtVKOR-DsbA, encoded by the gene At4g35760 as a potential disulfide bond oxidant in Arabidopsis. The gene product contains two distinct domains: an integral membrane domain homologous to the catalytic subunit of mammalian vitamin K epoxide reductase (VKOR) and a soluble DsbA-like domain. Transient expression of green fluorescent protein fusion in Arabidopsis protoplasts indicated that AtVKOR-DsbA is located in the chloroplast. The first 45 amino acids from the N-terminus were found to act as a transit peptide targeting the protein to the chloroplast. An immunoblot assay of chloroplast fractions revealed that AtVKOR-DsbA was localized in the thylakoid. A motility complementation assay showed that the full-length of AtVKOR-DsbA, if lacking its transit peptide, could catalyze the formation of disulfide bonds. Among the 10 cysteine residues present in the mature protein, eight cysteines (four in the AtVKOR domain and four in the AtDsbA domain) were found to be essential for promoting disulfide bond formation. The topological arrangement of AtVKOR-DsbA was assayed using alkaline phosphatase sandwich fusions. From these results, we developed a possible topology model of AtVKOR-DsbA in chloroplasts. We propose that the integral membrane domain of AtVKOR-DsbA contains four transmembrane helices, and that both termini and the cysteines involved in catalyzing the formation of disulfide bonds face the oxidative thylakoid lumen. These studies may help to resolve some of the issues surrounding the structure and function of AtVKOR-DsbA in Arabidopsis chloroplasts.     

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.     

ATP synthesis by ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716 by ionophore-induced electric potentials and proton gradients

ATP synthesis driven by low pre-established electric potentials and pH gradients is studied in large ATPase proteoliposomes, prepared from the ATPase complex and native lipids from the thermophilic cyanobacterium Synechococcus 6716. Electric potentials and pH gradients were achieved by valinomycin and nigericin, respectively, in the presence of a K⁺ gradient across the membrane. External base-pulses were also applied. In this system ATP synthesis driven by valinomycin-induced K⁺ influx, nigericin-induced internal acidification and by external base-pulses is demonstrated. Electric potentials and pH gradients of equivalent size lead to roughly similar ATP synthesis activities. ATP synthesis is optimal at 80–100 nM valinomycin and at 0.75−1 μM nigericin at the proper pre-set ion gradients. Uncoupler and DCCD inhibit ATP synthesis. Prior activation of the complex by thiol agents or trypsin was not required for synthesis activity. The ATP synthesis rate increases with the size of electric potential or pH gradient. The threshold value of the electrochemical gradient for significant ATP synthesis is about 30 mV. ATP production proceeds for more than 60 min. The generation of ionophore-induced electric potentials and pH gradients have been followed by oxonol VI and intraliposomal Neutral red, respectively. The extent of the absorbance changes of both probes is proportional to the size of electric potential or pH gradient. Ionophore-induced oxonol VI and Neutral red responses are stable for at least 30 min. The results are discussed in terms of membrane permeability and vesicle size.     

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents

1. o-Iodosobenzoate and 2,2′-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol.2. The inhibition of the Ca²⁺-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2′-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide.3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher K_m for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents.

1. O-Iodosobenzoate and 2,2'-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol. 2. The inhibition of the Ca2+-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2'-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide. 3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher Km for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

Studies on the relation between the fast phase of millisecond delayed light emission and the proton released from oxidation of water in spinach chloroplast

The relation between the fast phase of ms-DLE (delayed light emission measured with a phosphoroscope) and the proton released from water oxidation in spinach chloroplasts was studied in several aspects. When photophosphorylation was allowed to be coupled to the Hill reaction the intensity of the fast phase of ms-DLE of chloroplast was lowered more at 1 °C than at 25 °C, and the photophosphorylation rate within 40 ms of flashing light was higher at 1 °C than at 25 °C. Adding the subunit of ATP synthase to the chloroplast preparation to block the leakage of protons through ATP synthase, the intensity of the fast phase of ms-DLE was enhanced, to a larger extent at 1 °C than at 25°C. When the ms-DLE was measured under isotonic conditions, the intensity of fast phase of ms-DLE enhanced by proton released from oxidation of water was more pronounced. The above results support the suggestion that under lower temperature and isotonic conditions, the proton released from water oxidation was liable to be localized and could enhance the intensity of the fast phase of ms-DLE more effectively.     

Mechanics of coupling proton movements to c-ring rotation in ATP synthase

F1F0 ATP synthases generate ATP by a rotary catalytic mechanism in which H+ transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Subunit a of the F0 sector is thought to provide proton access channels to and from aspartyl-61. Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and fifth transmembrane helices (TMHs). Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface. We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle. The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.     

Damage to mitochondrial electron transport and energy coupling by visible light

The effect of treating mitochondria with visible light above 400 nm on electron transport and coupled reactions was examined. The temporal sequence of changes was: stimulation of respiration coupled to ATP synthesis, a decline in ATP synthesis, inactivation of respiration, increased ATPase activity and, later, loss of the membrane potential. Loss of respiration was principally due to inactivation of dehydrogenases. Of the components of dehydrogenase systems, flavins and quinones were most susceptible to illumination, the iron-sulfur centers were remarkably resistant to being damaged. Succinate dehydrogenase was inactivated before choline and NADH dehydrogenase. Redox reactions of cytochromes and cytochrome c oxidase activity were unaffected.Inactivation was O₂-dependent and prevented by anaerobiosis or the presence of substrates for the dehydrogenases. Light in the range 400–500 nm was most effective and the presence of free flavins greatly enhanced inactivation of all of the above mitochondrial activities. This suggests that visible light mediates a flavin-photosensitized reaction that initiates damage involving participation of an activated species of oxygen in the damage propagation.     

Relations between divalent cation binding and ATPase activity in coupling factor from chloroplast.

Although 150 individual samples of milk from Italian water buffalo (Bubalus arnee) were examined by acid and alkaline gel electrophoresis, no polymorphism was observed for α-lactalbumin and β-lactoglobulin. After isolation and purification of these two proteins their amino acid compositions were determined and compared with those of the corresponding bovine proteins. The sequence alignments of 36 and 17 amino-acids from the N-terminal ends and 2 amino-acids from the C-terminal ends of buffalo α-lactalbumin and β-lactoglobulin, respectively, have been established. Our results indicate that buffalo α-lactalbumin differs from its cow B counterpart by a substitution Asn/Gly at position 17 and by another substitution, likely Glu/Gln or Asp/Asn, at an unknown position. Buffalo β-lactoglobulin is homologous to the bovine B variant. Three substitutions differentiate the two proteins: Ile/Leu and Val/Ile at positions 1 and 162 respectively; a further one, Gln/Ile, has not yet been located. According to these results the B variant of bovine β-lactoglobulin might be the wild type of the Bos genus.     

A twofold role for global energy gradients in marine biodiversity trends

Explanations for major biodiversity patterns have not achieved a consensus, even for the latitudinal diversity gradient (LDG), but most relate to patterns of solar energy influx into Earth systems, and its effects on temperature (as biochemical activity rates are temperature sensitive) and photosynthesis (which drives nearly all of the productivity that fuels ecosystems). Marine systems break some of the confounding correlations among temperature, latitude and biodiversity that typify the terrestrial systems that have dominated theoretical discussions and large‐scale analyses. High marine diversities occur not only in warm shallow seas where productivity may be either low or high, depending on regional features, but also in very cold deep‐sea regions, indicating that diversity is promoted by stability in temperature and in trophic resources (nutrients and food items), and more specifically by their interaction, rather than by high mean values of either variable. The common association of high diversity with stable but low to moderate annual productivity suggests that ecological specialization underlies the similarly high diversities in the shallow tropics and deep sea. Recent work on shallow‐marine bivalves is consistent with this view of decreasing specialization in less stable habitats. Lower diversities in shallow seas are associated with either high thermal seasonality (chiefly in temperate latitudes) or highly seasonal trophic supplies (at any latitude), which exclude species that are adapted to narrow ranges of those variables.     

Regulation of coupling factor in field-grown sunflower: A Redox model relating coupling factor activity to the activities of other thioredoxin-dependent chloroplast enzymes

Simultaneous, non-invasive measurements were made of the rate of photosynthetic CO₂ fixation and the state of activation of the chloroplast CF₁CF₀-ATP synthase (CF) in field-grown sunflower (Helianthus annuus L.) during the dark-to-light transition at sunrise. CO₂ fixation showed a linear response with light intensity from zero to about 500–700 E m^-2 s^-1. However, at light intensities of only 5–22 E m^-2 s^-1, the energetic threshold for activation of the CF was found to be significantly lowered (as compared to the pre-dawn state), presumably through reduction of the regulatory sulfhdryl groups of the -subunit of the CF. When these studies were extended to chamber-grown plants, it was found that as little as 5 seconds of illumination at 4 E m^-2 s^-1 caused apparently full CF reduction. It is clear, therefore, that the catalytic activation of CF is not rate limiting to the induction of carbon assimilation under field conditions during a natural dark-to-light transition at sunrise. A model, based on the redox properties of the regulatory sulfhydryls, was developed to examine the significance of sulfhydryl midpoint potential in explaining the differences in light sensitivity and oxidation and reduction kinetics, between the CF and other thioredoxin-modulated chloroplast enzymes. Computer simulations of the light-induced regulation of three representative thioredoxin-modulated enzymes are presented.Abbreviations CF chloroplast CF₁-CF₀ ATP synthease or coupling factor - E_a active form of CF - E_a ⁰ active, oxidized form of CF - E_a ^r active, reduced form of CF - E_i inactive form of CF - E_i ⁰ inactive, oxidized form of CF - E_i ^r inactive, reduced form of CF - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin oxidoreductase - G6PDH glucose-6-phosphate dehydrogenase - MDH NADP-malate dehydrogenase - pmf protonmotive force - pmf_T threshold pmf required to activate CF - pmf_T ⁰ threshold pmf required to active the oxidized form of CF - pmf_T ^r threshold pmf required to activate the reduced form of CF - TR thioredoxin