BBA - Bioenergetics (v.1706, #1-2)

Mitochondrial mechanistic P/O ratios are still in question. The major studies since 1937 are summarized and various systematic errors are discussed. Values of about 2.5 with NADH-linked substrates and 1.5 with succinate are consistent with most reports after apparent contradictions are explained. Variability of coupling may occur under some conditions but is generally not significant. The fractional values result from the coupling ratios of proton transport. An additional revision of P/O ratios may be required because of a report of the structure of ATP synthase (D. Stock, A.G.W. Leslie, J.E. Walker, Science 286 (1999) 1700–1705) which suggests that the H+/ATP ratio is 10/3, rather than 3, consistent with P/O ratios of 2.3 with NADH and 1.4 with succinate, values that are also possible.
Keywords: Oxidation phosphorylation; P/O ratio; Proton leak; Slip; ATP synthase;

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein–protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment–protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.
Keywords: Photosynthesis; Photosystem I; Photosystem II; Light-harvesting complex; Cytochrome b 6 /f complex; Thylakoid membrane; Supercomplex; Grana; Cyclic electron transport; State transition; Nonphotochemical quenching;

Mitochondrial permeability transitions: how many doors to the house? by Mario Zoratti; Ildikò Szabò; Umberto De Marchi (40-52).
The inner mitochondrial membrane is famously impermeable to solutes not provided with a specific carrier. When this impermeability is lost, either in a developmental context or under stress, the consequences for the cell can be far-reaching. Permeabilization of isolated mitochondria, studied since the early days of the field, is often discussed as if it were a biochemically well-defined phenomenon, occurring by a unique mechanism. On the contrary, evidence has been accumulating that it may be the common outcome of several distinct processes, involving different proteins or protein complexes, depending on circumstances. A clear definition of this putative variety is a prerequisite for an understanding of mitochondrial permeabilization within cells, of its roles in the life of organisms, and of the possibilities for pharmacological intervention.
Keywords: Permeability transition; Adenine nucleotide transporter; Bax; Cyclosporin A; Protein import; Mitochondria;

P700+- and 3P700-induced quenching of the fluorescence at 760 nm in trimeric Photosystem I complexes from the cyanobacterium Arthrospira platensis by Eberhard Schlodder; Marianne Çetin; Martin Byrdin; Irina V. Terekhova; Navassard V. Karapetyan (53-67).
The 5 K absorption spectrum of Photosystem I (PS I) trimers from Arthrospira platensis (old name: Spirulina platensis) exhibits long-wavelength antenna (exciton) states absorbing at 707 nm (called C707) and at 740 nm (called C740). The lowest energy state (C740) fluoresces around 760 nm (F760) at low temperature. The analysis of the spectral properties (peak position and line width) of the lowest energy transition (C740) as a function of temperature within the linear electron–phonon approximation indicates a large optical reorganization energy of ∼110 cm−1 and a broad inhomogeneous site distribution characterized by a line width of ∼115 cm−1. Linear dichroism (LD) measurements indicate that the transition dipole moment of the red-most state is virtually parallel to the membrane plane.The relative fluorescence yield at 760 nm of PS I with P700 oxidized increases only slightly when the temperature is lowered to 77 K, whereas in the presence of reduced P700 the fluorescence yield increases nearly 40-fold at 77 K as compared to that at room temperature (RT). A fluorescence induction effect could not be resolved at RT. At 77 K the fluorescence yield of PS I trimers frozen in the dark in the presence of sodium ascorbate decreases during illumination by about a factor of 5 due to the irreversible formation of P700+FA/B in about 60% of the centers and the reversible accumulation of the longer-lived state P700+FX .The quenching efficiency of different functionally relevant intermediate states of the photochemistry in PS I has been studied. The redox state of the acceptors beyond A0 does not affect F760. Direct kinetic evidence is presented that the fluorescence at 760 nm is strongly quenched not only by P700+ but also by 3P700. Similar kinetics were observed for flash-induced absorbance changes attributed to the decay of 3P700 or P700+, respectively, and flash-induced fluorescence changes at 760 nm measured under identical conditions.A nonlinear relationship between the variable fluorescence around 760 nm and the [P700red]/[P700total] ratio was derived from titration curves of the absorbance change at 826 nm and the variable fluorescence at 760 nm as a function of the redox potential imposed on the sample solution at room temperature before freezing. The result indicates that the energy exchange between the antennae of different monomers within a PS I trimer stimulates quenching of F760 by P700+.
Keywords: Photosystem I; P700; Energy transfer; Fluorescence quenching; Transient absorbance spectroscopy; Long-wavelength antenna chlorophyll; Arthrospira platensis;

Evidence for the role of the oxygen-evolving manganese complex in photoinhibition of Photosystem II by Marja Hakala; Ilona Tuominen; Mika Keränen; Taina Tyystjärvi; Esa Tyystjärvi (68-80).
Photoinhibition of PSII occurs at the same quantum efficiency from very low to very high light, which raises a question about how important is the rate of photosynthetic electron transfer in photoinhibition. We modulated electron transfer rate and light intensity independently of each other in lincomycin-treated pea leaves and in isolated thylakoids, in order to elucidate the specific effects of light and PSII electron transport on photoinhibition. Major changes in the rate of electron transport caused only small changes in the rate of photoinhibition, suggesting the existence of a significant photoinhibitory pathway that contains an electron-transfer-independent phase. We compared the action spectrum of photoinhibition with absorption spectra of PSII components that could function as photoreceptors of the electron-transfer-independent phase of photoinhibition and found that the absorption spectra of Mn(III) and Mn(IV) compounds resemble the action spectrum of photoinhibition, showing a steep decrease from UV-C to blue light and a low visible-light tail. Our results show that the release of a Mn ion to the thylakoid lumen is the earliest detectable step of both UV- and visible-light-induced photoinhibition. After Mn release from the oxygen-evolving complex, oxidative damage to the PSII reaction center occurs because the Mn-depleted oxygen-evolving complex cannot reduce P680 + normally.
Keywords: Photoinhibition; Photosystem II; Manganese; Action spectrum;

Reconstitution of phycobilisome core–membrane linker, LCM, by autocatalytic chromophore binding to ApcE by Kai-Hong Zhao; Ping Su; Stephan Böhm; Bo Song; Ming Zhou; Claudia Bubenzer; Hugo Scheer (81-87).
The core–membrane linker, LCM, connects functionally the extramembraneous light-harvesting complex of cyanobacteria, the phycobilisome, to the chlorophyll-containing core-complexes in the photosynthetic membrane. Genes coding for the apoprotein, ApcE, from Nostoc sp. PCC 7120 and for a C-terminally truncated fragment ApcE(1–240) containing the chromophore binding cysteine-195 were overexpressed in Escherichia coli. Both bind covalently phycocyanobilin (PCB) in an autocatalytic reaction, in the presence of 4M urea necessary to solubilize the proteins. If judged from the intense, red-shifted absorption and fluorescence, both products have the features of the native core-membrane linker LCM, demonstrating that the lyase function, the dimerization motif, and the capacity to extremely red-shift the chromophore are all contained in the N-terminal phycobilin domain of ApcE. The red-shift is, however, not the result of excitonic interactions: Although the chromoprotein dimerizes, the circular dichroism shows no indication of excitonic coupling. The lack of homologies with the autocatalytically chromophorylating phytochromes, as well as with the heterodimeric cysteine-α84 lyases, indicates that ApcE constitutes a third type of bilin:biliprotein lyase.
Keywords: Allophycocyanin; ApcE; Biosynthesis; Core–membrane linker (LCM); Cyanobacteria; Energy transfer; Fluorescence labeling; Photosynthesis; Phycobilisome; Phycocyanobilin attachment;

Cd2+ transport and storage in the chloroplast of Euglena gracilis by David G. Mendoza-Cózatl; Rafael Moreno-Sánchez (88-97).
Euglena gracilis lacks a plant-like vacuole and, when grown in Cd2+-containing medium, 60% of the accumulated Cd2+ is located inside the chloroplast. Hence, the biochemical mechanisms involved in Cd2+ accumulation in chloroplast were examined. Percoll-purified chloroplasts showed a temperature-sensitive uptake of the free 109Cd2+ ion. Kinetics of the uptake initial rate was resolved in two components, one hyperbolic and saturable (V max 11 nmol 109Cd2+ min−1 mg protein −1, K m 13 μM) and the other, linear and non-saturable. 109Cd2+ uptake was not affected by metabolic inhibitors or illumination. Zn2+ competitively inhibited 109Cd2+ uptake (K i 8.2 μM); internal Cd2+ slightly inhibited 109Cd2+ uptake. Cadmium was partially and rapidly released from chloroplasts. These data suggested the involvement of a cation diffusion facilitator-like protein. Chloroplasts isolated from cells grown with 50 μM CdCl2 (ZCd50 chloroplasts) showed a 1.6 times increase in the uptake V max, whereas the K m and the non-saturable component did not change. In addition, Cd2+ retention in chloroplasts correlated with the amount of internal sulfur compounds. ZCd50 chloroplasts, which contained 4.4 times more thiol-compounds and sulfide than control chloroplasts, retained six times more Cd2+. The Cd2+ storage-inactivation mechanism was specific for Cd2+, since Zn2+ and Fe3+ were not preferentially accumulated into chloroplasts.
Keywords: Heavy metal transport; Phytochelatin; Sulfide; Cd2+ compartmentation; Cd2+ inactivation;

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria by Zemfira Z. Gizatullina; Ying Chen; Stephan Zierz; Frank Norbert Gellerich (98-104).
Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 μM CAT and 75 μM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 μM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 μM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP.We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.
Keywords: Mitochondria; Permeability transition pore; Adenine nucleotide translocator; Carboxyatractyloside; Bongkrekic acid;

Equilibration between cytochrome f and P700 in intact leaves by Alison J. Golding; Pierre Joliot; Giles N. Johnson (105-109).
Electron transport between the two photosynthetic reaction centres of high plants is mediated by plastoquinone, a rieske iron–sulfur centre, cytochrome f and plastocyanin. Measurements of redox equilibration amongst these have produced confusing results, with apparent equilibrium constants being estimated that are inconsistent with in vitro measurements of redox midpoint potentials of the components concerned.We have critically reexamined methods for deconvoluting cytochrome f absorbance signals in intact leaves. We have determined the decay of cytochrome f + following light to dark transitions from steady state and compared this with the decay of the oxidised photosystem I primary donor, P700+. Measurements across a wide range of different irradiances and CO2 concentrations were all consistent with cyt f and P700 existing in redox equilibrium, with a potential difference of around 117 mV. These results are discussed in relation to our understanding of the organisation of the photosynthetic electron transport. They also have implications for measurements of PSI electron flux—provided more than about 20% of P700+ is oxidised in the light, then the initial decay in the concentration of P700+ following a light to dark transition provides a good estimate of electron flux through PSI. Where P700 is largely reduced in the light, net reduction of cyt f + might need to be corrected for.
Keywords: Equilibration; Cytochrome f; P700;

Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase by Robert R. Ishmukhametov; Mikhail A. Galkin; Steven B. Vik (110-116).
His-tagged cysteine-less F1Fo ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 °C), and was sensitive to N,N′-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.
Keywords: Escherichia coli F1Fo ATP synthase; Protein purification; Ni-NTA resin; Reconstitution; ACMA; Steady-state kinetics;

The yeast cAMP protein kinase Tpk3p is involved in the regulation of mitochondrial enzymatic content during growth by Cyrille Chevtzoff; Julie Vallortigara; Nicole Avéret; Michel Rigoulet; Anne Devin (117-125).
During aerobic cell growth, mitochondria must meet energy demand either by adjusting cellular mitochondrial content or by adjusting ATP production flux, allowing a constant growth yield. On respiratory substrate, the Ras/cAMP pathway has been shown to be involved in this process in the yeast Saccharomyces cerevisiae. We show that of the three cAMP protein kinase catalytic subunits, Tpk3p is the one specifically involved in the regulation of cellular mitochondrial content when energy demand decreases. In decreased energy demand, the Δtpk3 mitochondrial enzymatic content decreases leading to a subsequent decrease in the cellular growth rate. Moreover, enzymatic content decreases in the Δtpk3 isolated mitochondria, suggesting that the amount of cellular mitochondria is not affected, but rather that the mitochondria are modified. Our study points to an important decrease in the cytochrome c content in the Δtpk3 mitochondria, which leads to a decrease in the slipping process at the level of cytochrome-c-oxidase.
Keywords: Yeast; Mitochondria; Oxidative phosphorylation; cAMP protein kinase; Slipping;

Characterization of steady-state activities of cytochrome c oxidase at alkaline pH: mimicking the effect of K-channel mutations in the bovine enzyme by David Riegler; Lois Shroyer; Christine Pokalsky; Dmitry Zaslavsky; Robert Gennis; Lawrence J. Prochaska (126-133).
The cytochrome c oxidase activity of the bovine heart enzyme decreases substantially at alkaline pH, from 650 s−1 at pH 7.0 to less than 10 s−1 at pH 9.75. In contrast, the cytochrome c peroxidase activity of the enzyme shows little or no pH dependence (30–50 s−1) at pH values greater than 8.5. Under the conditions employed, it is demonstrated that the dramatic decrease in oxidase activity at pH 9.75 is fully reversible and not due to a major alkaline-induced conformational change in the enzyme. Furthermore, the K m values for cytochrome c interaction with the enzyme were also not significantly different at pH 7.8 and pH 9.75, suggesting that the pH dependence of the activity is not due to an altered interaction with cytochrome c at alkaline pH. However, at alkaline pH, the steady-state reduction level of the hemes increased, consistent with a slower rate of electron transfer from heme a to heme a 3 at alkaline pH. Since it is well established that the rate of electron transfer from heme a to heme a 3 is proton-coupled, it is reasonable to postulate that at alkaline pH, proton uptake becomes rate-limiting. The fact that this is not observed when hydrogen peroxide is used as a substrate in place of O2 suggests that the rate-limiting step is proton uptake via the K-channel associated with the reduction of the heme a 3/CuB center prior to the reaction with O2. This step is not required for the reaction with H2O2, as shown previously in the examination of mutants of bacterial oxidases in which the K-channel was blocked. It is concluded that at pH values near 10, the delivery of protons via the K-channel becomes the rate-limiting step in the catalytic cycle with O2, so that the behavior of the bovine enzyme resembles that of the K-channel mutants in the bacterial enzymes.
Keywords: Cytochrome c oxidase; Cytochrome c; Peroxidase; Steady-state kinetics; Circular dichroism; Alkaline pH value;

The formation of water chains in cytochrome c oxidase (CcO) is studied by molecular dynamics (MD). Focus is on water chains in the K channel that can supply a proton to the binuclear center (the heme a3 Fe/CuB region), the site of O2 reduction. By assessing the presence of chains of any length on a short time scale (0.1 ps), a view of the kinds of chains and their persistence is obtained. Chains from the entry of the channel on the inner membrane to Thr359 (Rhodobacter sphaeroides numbering) are often present but are blocked at that point until a rotation of the Thr359 side chain occurs, permitting formation of chains from Thr359 towards the binuclear center. No continuous hydrogen-bonded water chains are found connecting Thr359 and the binuclear center. Instead, waters hydrogen bond from Thr359 to the hydroxyl of the heme a3 farnesyl and then continue to the binuclear center via Tyr288, which has been identified as a source of a proton for O2 reduction. Three hydrogen-bonded waters are found to be present in the binuclear center after a sufficiently long simulation time. One is ligated to the CuB and could be associated with a water (or hydroxyl) identified in the crystal structure as the fourth ligand of CuB. The water hydrogen-bonded to the hydroxyl of Tyr288 is extremely persistent and well positioned to participate in O2 reduction. The third water is located where O2 is often suggested to reside in mechanistic studies of O2 reduction.
Keywords: Cytochrome c oxidase; Proton transfer and translocation; Molecular dynamics;

Purification, characterisation and crystallisation of photosystem II from Thermosynechococcus elongatus cultivated in a new type of photobioreactor by J. Kern; B. Loll; C. Lüneberg; D. DiFiore; J. Biesiadka; K.-D. Irrgang; A. Zouni (147-157).
The thermophilic cyanobacterium Thermosynechococcus elongatus was cultivated under controlled growth conditions using a new type of photobioreactor, allowing us to optimise growth conditions and the biomass yield. A fast large-scale purification method for monomeric and dimeric photosystem II (PSII) solubilized from thylakoid membranes of this cyanobacterium was developed using fast protein liquid chromatography (FPLC). The obtained PSII core complexes (PSIIcc) were analysed for their pigment stoichiometry, photochemical and oxygen evolution activities, as well as lipid and detergent composition. Thirty-six chlorophyll a (Chla), 2 pheophytin a (Pheoa), 9± 1 β-carotene (Car), 2.9±0.8 plastoquinone 9 (PQ9) and 3.8±0.5 Mn were found per active centre. For the monomeric and dimeric PSIIcc, 18 and 20 lipid as well as 145 and 220 detergent molecules were found in the detergent shell, respectively. The monomeric and dimeric complexes showed high oxygen evolution activity with 1/4 O2 released per 37–38 Chla and flash in the best cases. Crystals were obtained from dimeric PSIIcc by a micro-batch method. They diffract synchrotron X-rays to a maximum resolution of 2.9-Å, resulting in complete data sets of 3.2 Å resolution.
Keywords: Photosystem II; Crystallisation; Thermosynechococcus elongatus; Cofactor composition; Oxygen evolution activity; Cell cultivation;

Cadmium (Cd2+) is a well-known highly toxic element. The molecular mechanisms of the Cd2+ toxicity are not well understood. In photosynthetic organisms, toxic Cd2+ concentrations are often in the low-μM range. It has been proposed that low-μM Cd2+ concentrations affect photosynthesis at the level of Photosystem II by inhibiting oxygen evolution. However, in vitro studies on isolated, functional Photosystem II showed that much higher Cd2+ concentrations (mM range) were needed for inhibition. Here we show that Cd2+ in the low-μM range inhibited photoactivation (i.e., assembly of the water splitting complex) in Chlamydomonas reinhardtii and in isolated Photosystem II. Photoactivation is the last step in the assembly of Photosystem II before it becomes functional. The exact Cd2+ concentration necessary for inhibition depended on the concentration of calcium. It is proposed that Cd2+ binds competitively to the essential Ca2+ site in Photosystem II during photoactivation. The low Cd2+ concentration needed to inhibit photoactivation suggests that this event is also involved in the Cd2+-induced inhibition of photosynthesis in vivo. This mechanism is likely to be important for Cd2+ toxicity towards photosynthetic organisms in general, at least in unicellular like C. reinhardtii where Cd2+ has easy access to the photosynthetic apparatus.
Keywords: Photosynthesis; Photosystem II; Photoactivation; Cadmium toxicity;

Excited-state dynamics of bacteriorhodopsin probed by broadband femtosecond fluorescence spectroscopy by B. Schmidt; C. Sobotta; B. Heinz; S. Laimgruber; M. Braun; P. Gilch (165-173).
The impact of varying excitation densities (∼0.3 to ∼40 photons per molecule) on the ultrafast fluorescence dynamics of bacteriorhodopsin has been studied in a wide spectral range (630–900 nm). For low excitation densities, the fluorescence dynamics can be approximated biexponentially with time constants of <0.15 and ∼0.45 ps. The spectrum associated with the fastest time constant peaks at 650 nm, while the 0.45 ps component is most prominent at 750 nm. Superimposed on these kinetics is a shift of the fluorescence maximum with time (dynamic Stokes shift). Higher excitation densities alter the time constants and their amplitudes. These changes are assigned to multi-photon absorptions.
Keywords: Excited state; Bacteriorhodopsin; Femtosecond fluorescence;

Specificity of coenzyme Q10 for a balanced function of respiratory chain and endogenous ubiquinone biosynthesis in human cells by Daniel J.M. Fernández-Ayala; Guillermo López-Lluch; Macarena García-Valdés; Antonio Arroyo; Plácido Navas (174-183).
Coenzyme Q (Q) is an obligatory component of both respiratory chain and uncoupling proteins. Also, Q acts as an antioxidant in cellular membranes. Several neurodegenerative diseases are associated with modifications of Q10 levels. For these reasons, therapies based on Q supplementation in the diet are currently studied in order to mitigate the symptoms of these diseases. However, the incorporation of exogenous Q also affects aging process in nematodes probably affecting reactive oxygen species (ROS) production. The aim of the present work is to clarify if supplementation with both Q10 and Q6 isoforms affects mitochondrial Q10 content, respiratory chain activity and ROS levels in human cells. Cells incorporated exogenously added Q10 and Q6 isoforms into mitochondria that produced changes in mitochondrial activity depending on the side chain length. Supplementation with Q10, but not with Q6, increased mitochondrial Q-dependent activities. However, Q6 affected the mitochondrial membrane potential, ROS production, and increased the protein levels of both catalase and Mn-superoxide dismutase (Mn-SOD). Also, Q6 induced a transient decrease in endogenous mitochondrial Q10 levels by increasing its catabolism. These results show that human cells supplemented with Q6 undergo a mitochondrial impairment, which is not observed with Q10 supplementation.
Keywords: Coenzyme Q; Mitochondria; ROS; Respiratory chain; CoQ uptake;

FOF1-ATP synthase converts two energetic “currencies” of the cell (ATP and protonmotive force, pmf) by coupling two rotary motors/generators. Their coupling efficiency is usually very high. Uncoupled proton leakage (slip) has only been observed in chloroplast enzyme at unphysiologically low nucleotide concentration.We investigated the properties of proton slip in chromatophores (sub-bacterial vesicles) from Rhodobacter capsulatus in the single-enzyme-per-vesicle mode. The membrane was energized by excitation with flashing light and the relaxation of the transmembrane voltage and pH difference was photometrically detected. We found that: (1) Proton slip occurred only at low nucleotide concentration (<1 μM) and after pre-illumination over several seconds. (2) Slip induction by pmf was accompanied by the release of ≈0.25 mol ADP per mole of enzyme. There was no detectable detachment of F1 from FO. (3) The transmembrane voltage and the pH difference were both efficient in slip induction. Once induced, slip persisted for hours, and was only partially reverted by the addition of ADP or ATP (>1 μM). (4) There was no pmf threshold for the proton transfer through the slipping enzyme; slip could be driven both by voltage and pH difference. (5) The conduction was ohmic and weakly pH-dependent in the range from 5.5 to 9.5. The rate constant of proton transfer under slip conditions was 185 s−1 at pH 8.Proton slip probably presents the free-wheeling of the central rotary shaft, subunit γ, in an open structure of the (αβ)3 hexagon with no nucleotides in the catalytic sites.
Keywords: ATP synthase; Proton slip; Regulation; Rhodobacter capsulatus;