Exciton interactions of chlorophyll tetramer in water-soluble chlorophyll-binding protein BoWSCP.

The exciton interaction of four chlorophyll a (Chl a) molecules in a symmetrical tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP was analyzed in the pH range of 3-11. Exciton splitting ΔE = 232 ± 2 cm-1 of the Qy band of Chl a into two subcomponents with relative intensities of 78.1 ± 0.7 % and 21.9 ± 0.7 % was determined by a joint decomposition of the absorption and circular dichroism spectra into Gaussian functions. The exciton coupling parameters were calculated based on the BoWSCP atomic structure in three approximations: the point dipole model, the distributed atomic monopoles, and direct ab initio calculations in the TDDFT/PCM approximation. The Coulomb interactions of monomers were calculated within the continuum model using three values of optical permittivity. The models based on the properties of free Chl a in solution suffer from significant errors both in estimating the absolute value of the exciton interaction and in the relative intensity of exciton transitions. Calculations within the TDDFT/PCM approximation reproduce the experimentally determined parameters of the exciton splitting and the relative intensities of the exciton bands. The following factors of pigment-protein and pigment-pigment interactions were examined: deviation of the macrocycle geometry from the planar conformation of free Chl; the formation of hydrogen bonds between the macrocycle and water molecules; the overlap of wave functions of monomers at close distances. The most significant factor is the geometrical deformation of the porphyrin macrocycle, which leads to an increase in the dipole moment of Chl monomer from 5.5 to 6.9 D and to a rotation of the dipole moment by 15° towards the cyclopentane ring. The contributions of resonant charge-transfer states to the wave functions of the Chl dimer were determined and the transition dipole moments of the symmetric and antisymmetric charge-transfer states were estimated.

Expansion of the "Sodium World" through Evolutionary Time and Taxonomic Space.

In 1986, Vladimir Skulachev and his colleagues coined the term "Sodium World" for the group of diverse organisms with sodium (Na)-based bioenergetics. Albeit only few such organisms had been discovered by that time, the authors insightfully noted that "the great taxonomic variety of organisms employing the Na-cycle points to the ubiquitous distribution of this novel type of membrane-linked energy transductions". Here we used tools of bioinformatics to follow expansion of the Sodium World through the evolutionary time and taxonomic space. We searched for those membrane protein families in prokaryotic genomes that correlate with the use of the Na-potential for ATP synthesis by different organisms. In addition to the known Na-translocators, we found a plethora of uncharacterized protein families; most of them show no homology with studied proteins. In addition, we traced the presence of Na-based energetics in many novel archaeal and bacterial clades, which were recently identified by metagenomic techniques. The data obtained support the view that the Na-based energetics preceded the proton-dependent energetics in evolution and prevailed during the first two billion years of the Earth history before the oxygenation of atmosphere. Hence, the full capacity of Na-based energetics in prokaryotes remains largely unexplored. The Sodium World expanded owing to the acquisition of new functions by Na-translocating systems. Specifically, most classes of G-protein-coupled receptors (GPCRs), which are targeted by almost half of the known drugs, appear to evolve from the Na-translocating microbial rhodopsins. Thereby the GPCRs of class A, with 700 representatives in human genome, retained the Na-binding site in the center of the transmembrane heptahelical bundle together with the capacity of Na-translocation. Mathematical modeling showed that the class A GPCRs could use the energy of transmembrane Na-potential for increasing both their sensitivity and selectivity. Thus, GPCRs, the largest protein family coded by human genome, stem from the Sodium World, which encourages exploration of other Na-dependent enzymes of eukaryotes.

Electron Transfer through the Acceptor Side of Photosystem I: Interaction with Exogenous Acceptors and Molecular Oxygen.

This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin-Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A1A/A1B in the symmetric redox cofactor branches of PS I and iron-sulfur clusters FX, FA, and FB have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants.

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273-1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.

Mechanism of primary and secondary ion-radical pair formation in photosystem I complexes.

The mechanisms of the ultrafast charge separation in reaction centers of photosystem I (PS I) complexes are discussed. A kinetic model of the primary reactions in PS I complexes is presented. The model takes into account previously calculated values of redox potentials of cofactors, reorganization energies of the primary P700(+)A0(-) and secondary P700(+)A1(-) ion-radical pairs formation, and the possibility of electron transfer via both symmetric branches A and B of redox-cofactors. The model assumes that the primary electron acceptor A0 in PS I is represented by a dimer of chlorophyll molecules Chl2A/Chl3A and Chl2B/Chl3B in branches A and B of the cofactors. The characteristic times of formation of P700(+)A0(-) and P700(+)A1(-) calculated on the basis of the model are close to the experimental values obtained by pump-probe femtosecond absorption spectroscopy. It is demonstrated that a small difference in the values of redox potentials between the primary electron acceptors A0A and A0B in branches A and B leads to asymmetry of the electron transfer in a ratio of 70 : 30 in favor of branch A. The secondary charge separation is thermodynamically irreversible in the submicrosecond range and is accompanied by additional increase in asymmetry between the branches of cofactors of PS I.

Interaction of tetraphenylphosphonium and dodecyltriphenylphosphonium with lipid membranes and mitochondria.

The permeability of a planar lipid membrane (composed of diphytanoylphosphatidylcholine) for tetraphenylphosphonium (TPP) was investigated. The observed level of the diffusion potential generated as a function of the TPP concentration gradient differed from the theoretically expected value, possibly due to proton leakage of the membrane mediated by the traces of fatty acids in the phospholipid forming the membrane. Using the molecular dynamics approach to study movement of TPP and dodecyltriphenylphosphonium (C(12)TPP) with different affinity to the lipid bilayer through a bilayer lipid membrane, it was found that C(12)TPP has a greater affinity to the membrane surface than TPP. However, the two cations have the same activation energy for transmembrane transfer. Interaction of TPP and C(12)TPP with tightly-coupled mitochondria from the yeast Yarrowia lipolytica was also investigated. At low, micromolar concentrations, both cations are "relatively weak, mild uncouplers", do not shunt electron transfer along the respiratory chain, do not disturb (damage) the inner mitochondrial membrane, and profoundly promote the uncoupling effect of fatty acids. At higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their detergent action.

Mitochondrial-targeted plastoquinone derivatives. Effect on senescence and acute age-related pathologies.

Plastoquinone, a very effective electron carrier and antioxidant of chloroplasts, was conjugated with decyltriphenylphosphonium to obtain a cation easily penetrating through membranes. This cation, called SkQ1, is specifically targeted to mitochondria by electrophoresis in the electric field formed by the mitochondrial respiratory chain. The respiratory chain also regenerates reduced SkQ1H(2) from its oxidized form that appears as a result of the antioxidant activity of SkQ1H(2). SkQ1H(2) prevents oxidation of cardiolipin, a mitochondrial phospholipid that is especially sensitive to attack by reactive oxygen species (ROS). In cell cultures, SkQ1 and its analog plastoquinonyl decylrhodamine 19 (SkQR1) arrest H(2)O(2)-induced apoptosis. When tested in vivo, SkQs (i) prolong the lifespan of fungi, crustaceans, insects, fish, and mice, (ii) suppress appearance of a large number of traits typical for age-related senescence (cataract, retinopathies, achromotrichia, osteoporosis, lordokyphosis, decline of the immune system, myeloid shift of blood cells, activation of apoptosis, induction of ß-galactosidase, phosphorylation of H2AX histones, etc.) and (iii) lower tissue damage and save the lives of young animals after treatments resulting in kidney ischemia, rhabdomyolysis, heart attack, arrhythmia, and stroke. We suggest that the SkQs reduce mitochondrial ROS and, as a consequence, inhibit mitochondria-mediated apoptosis, an obligatory step of execution of programs responsible for both senescence and fast "biochemical suicide" of an organism after a severe metabolic crisis.

Ubiquinone reduction in the photosynthetic reaction centre of Rhodobacter sphaeroides: interplay between electron transfer, proton binding and flips of the quinone ring.

This review is focused on reactions that gate (control) the electron transfer between the primary quinone Q(A) and secondary quinone Q(B) in the photosynthetic reaction centre of Rhodobacter sphaeroides. The results on electron and proton transfer are discussed in relation to structural information and to the steered molecular dynamics simulations of the Q(B) ring flip in its binding pocket. Depending on the initial position of Q(B) in the pocket and on certain conditions, the rate of electron transfer is suggested to be limited either by the quinone ring flip or by the charge-compensating proton equilibration between the surface and the buried Q(B) site.

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 > or =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 microsec. 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" protons 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.

Inter-subunit rotation and elastic power transmission in F0F1-ATPase.

ATP synthase (F-ATPase) produces ATP at the expense of ion-motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360 degrees rotary motion of subunits against each other. The rotor, subunits gamma(epsilon)C10-14, moves against the stator, (alphabeta)3delta(ab2). The enzyme copes with symmetry mismatch (C3 versus C10-14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8.10(-26) N x m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three-fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators.

Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: angular torque profile of the enzyme.

ATP synthase (F(O)F(1)) operates as two rotary motor/generators coupled by a common shaft. Both portions, F(1) and F(O), are rotary steppers. Their symmetries are mismatched (C(3) versus C(10-14)). We used the curvature of fluorescent actin filaments, attached to the rotating c-ring, as a spring balance (flexural rigidity of 8. 10(-26) Nm(2)) to gauge the angular profile of the output torque at F(O) during ATP hydrolysis by F(1) (see theoretical companion article (. Biophys. J. 81:1234-1244.)). The large average output torque (50 +/- 6 pN. nm) proved the absence of any slip. Variations of the torque were small, and the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the threefold stepping and high activation barrier of the driving motor proper, the rather constant output torque implied a soft elastic power transmission between F(1) and F(O). It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate of the two counteracting and stepping motor/generators.

Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: curvature as an indicator of the torque.

ATP synthase (F-ATPase) operates as an electrochemical-to-mechanical-to-chemical energy transducer with an astounding 360 degrees rotary motion of subunits epsilongammac(10-14) (rotor) against delta(alphabeta)(3)ab(2) (stator). The enzyme's torque as a function of the angular reaction coordinate in relation to ATP-synthesis/hydrolysis, internal elasticity, and external load has remained an important issue. Fluorescent actin filaments of micrometer length have been used to detect the rotation as driven by ATP hydrolysis. We evaluated the viscoelastic dynamics of actin filaments under the influence of enzyme-generated torque, stochastic Langevin force, and viscous drag. Modeling with realistic parameters revealed the dominance of the lowest normal mode. Because of its slow relaxation (approximately 100 ms), power strokes of the enzyme were expected to appear strongly damped in recordings of the angular velocity of the filament. This article describes the theoretical background for the alternative use of the filament as a spring balance. The enzyme's angular torque profile under load can be gauged by measuring the average curvature and the stochastic fluctuations of actin filaments. Pertinent experiments were analyzed in the companion paper.

Proton transfer in Azotobacter vinelandii ferredoxin I: entatic Lys84 operates as elastic counterbalance for the proton-carrying Asp15.

In ferredoxin I from Azotobacter vinelandii, the reduction of a [3Fe-4S] iron-sulphur cluster is coupled with the protonation of the mu2S sulphur atom that is approx. 6 A away from the protein boundary. The recent study of the site-specific mutants of ferredoxin I led to the conclusion that a particular surface aspartic residue (Asp15) is solely responsible for the proton transfer to the mu2S atom by 'rapid penetrative excursions' (K. Chen, J. Hirst, R. Camba, C.A. Bonagura, C.D. Stout, B.K. Burgess, F.A. Armstrong, Nature 405 (2000) 814-817). In the same paper it has been reported that the replacement of Asp15 by glutamate led to the blockage of the enzyme, although glutamate, with its longer and more flexible side chain, should apparently do even better as a mobile proton carrier than aspartate. We tackled this puzzling incompetence of Glu15 by molecular dynamics simulations. It was revealed that the conformational alterations of Asp15 are energetically balanced by the straining of the nearby Lys84 side chain in wild-type ferredoxin I but not in the Asp15-->Glu mutant. Lys84 in ferredoxin I of A. vinelandii seems to represent the first case where the strained (entatic) conformation of a particular amino acid side chain could be directly identified in the ground state of an enzyme and assigned to a distinct mechanism of energy balance during the catalytic transition.

Photosynthetic electron transfer controlled by protein relaxation: analysis by Langevin stochastic approach.

Relaxation processes in proteins range in time from picoseconds to seconds. Correspondingly, biological electron transfer (ET) could be controlled by slow protein relaxation. We used the Langevin stochastic approach to describe this type of ET dynamics. Two different types of kinetic behavior were revealed, namely: oscillating ET (that could occur at picoseconds) and monotonically relaxing ET. On a longer time scale, the ET dynamics can include two different kinetic components. The faster one reflects the initial, nonadiabatic ET, whereas the slower one is governed by the medium relaxation. We derived a simple relation between the relative extents of these components, the change in the free energy (DeltaG), and the energy of the slow reorganization Lambda. The rate of ET was found to be determined by slow relaxation at -DeltaG < or = Lambda. The application of the developed approach to experimental data on ET in the bacterial photosynthetic reaction centers allowed a quantitative description of the oscillating features in the primary charge separation and yielded values of Lambda for the slower low-exothermic ET reactions. In all cases but one, the obtained estimates of Lambda varied in the range of 70-100 meV. Because the vast majority of the biological ET reactions are only slightly exothermic (DeltaG > or = -100 meV), the relaxationally controlled ET is likely to prevail in proteins.

Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F(0)F(1)-ATP synthase is absent from about half of chromatophores.

F(0)F(1)-ATP synthase (H(+)-ATP synthase, F(0)F(1)) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (P(i)). Structurally the enzyme consists of a membrane-embedded proton-translocating F(0) portion and a protruding hydrophilic F(1) part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F(0)F(1) (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F(1) (efrapeptin) and of F(0) (venturicidin), we decomposed the kinetics of the total proton flow through F(0)F(1) into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F(0). Taking the coupled proton flow, we calculated the H(+)/ATP ratio; it was found to be 3.3+/-0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1+/-0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F(0)F(1) dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and P(i) led to an increase in the fraction of chromatophores with at least one de-coupled F(0)(F(1)). The disclosed fraction of chromatophores that lacked proton-conducting F(0)(F(1)) (approx. 40% of the total amount) remained constant upon these treatments.

Reduction and protonation of the secondary quinone acceptor of Rhodobacter sphaeroides photosynthetic reaction center: kinetic model based on a comparison of wild-type chromatophores with mutants carrying Arg-->Ile substitution at sites 207 and 217 in the L-subunit.

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone QA, the loosely bound ubiquinone Q(B) After two subsequent flashes of light, Q(B) is reduced to ubiquinol Q(B)H2, with a semiquinone anion Q-(B) formed as an intermediate after the first flash. We studied Q(B)H2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg-->Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 A away from Q(B), Arg-L217 is closer (9 A) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated deltaG(AB), the free energy difference between the Q-(A)Q(B) and Q(A)Q-(B) states, and pK212, the apparent pK of Glu-L212, a residue that is only 4 A away from Q(B). As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q-(B) state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, pK212 increased by approximately 0.4 pH units in the L207RI mutant. The value of pK212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical' affinity of the QB site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established pK2J2 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the QB turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.

Proton transfer from the bulk to the bound ubiquinone Q(B) of the reaction center in chromatophores of Rhodobacter sphaeroides: retarded conveyance by neutral water.

The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule Q(B) by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (i) the electron transfer to Q(B) (at 450 nm), (ii) the electrogenic proton delivery from the surface to the Q(B) site (by electrochromic carotenoid response at 524 nm), and (iii) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to Q(B)(-) and the proton-related electrogenesis proceeded with the same time constant of approximately 100 microseconds (at pH 6.2), whereas the alkalinization in the bulk was distinctly delayed (tau approximately 400 microseconds). We investigated the latter reaction as a function of the pH indicator concentration, the added pH buffers, and the temperature. The results led us to the following conclusions: (i) proton transfer from the surface-located acidic groups into the Q(B) site followed the reduction of Q(B) without measurable delay; (ii) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (iii) as a result, the protons were slowly donated by neutral water to refill the proton vacancies at the surface. It is conceivable that the same mechanism accounts for the delayed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling between proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface.

Transient accumulation of elastic energy in proton translocating ATP synthase.

ATP synthase is conceived as a rotatory engine with two reversible drives, the proton-transporting membrane portion, F0, and the catalytic peripheral portion, F1. They are mounted on a central shaft (subunit gamma) and held together by an eccentric bearing. It is established that the hydrolysis of three molecules of ATP in F1 drives the shaft over a full circle in three steps of 120 degrees each. Proton flow through F0 probably generates a 12-stepped rotation of the shaft so that four proton-translocating steps of 30 degrees each drive the synthesis of one molecule of ATP. We addressed the elasticity of the transmission between F0 and F1 in a model where the four smaller steps in F0 load a torsional spring which is only released under liberation of ATP from F1. The kinetic model of an elastic ATP synthase described a wealth of published data on the synthesis/hydrolysis of ATP by F0F1 and on proton conduction by F0 as function of the pH and the protonmotive force. The pK values of the proton-carrying group interacting with the acidic and basic sides of the membrane were estimated as 5.3-6.4 and 8.0-8.3, respectively.

ATP-synthase of Rhodobacter capsulatus: coupling of proton flow through F0 to reactions in F1 under the ATP synthesis and slip conditions.

A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by pH-indicators) and ATP release (measured by luminescence of luciferin-luciferase) were monitored. The ratio between the amount of protons translocated by F0F1 and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F0F1. The proton transfer through F0F1 after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATP-synthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons.

Contraction transitions of F1-F0 ATPase during catalytic turnover.

Strong acoustic pressure was applied to submitochondrial particles (SMP) from bovine heart in order to drive ATP synthesis by F1-F0 complex for the account of sound waves. We observed a net ATP production at two narrow frequency ranges, about 170 Hz and about 340 Hz, that corresponds to the resonance oscillations of experimental cuvette when the acoustic pressure had a magnitude of 100 kPa. The results can be explained quantitatively by contractive conformational changes of F1-F0 complex during catalytic turnover. Negative staining electron microscopy of SMP preparations was used to visualize the ADP(Mg2+)-induced conformational changes of F1-F0 complex. In the particles with high ATPase activity in the presence of phosphate the factors F1 and F0 formed a congregated domain plunged into the membrane without any observable stalk in between. The presence of ADP(Mg2+) caused a structural rearrangement of F1-F0 to the essentially different conformation: the domains F1 and F0 were dislodged distinctly from each other and connected by a long thin stalk. The latter conformation resembled well the usual bipartite profile of ATPase. The data indicate that besides rotation, the catalytic turnover of ATP synthase is also accompanied by stretch transitions of F1-F0 complex.