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.     

Electrogenic reactions and dielectric properties of photosystem II

This review is focused on the mechanism of photovoltage generation involving the photosystem II turnover. This large integral membrane enzyme catalyzes the light-driven oxidation of water and reduction of plastoquinone. The data discussed in this work show that there are four main electrogenic steps in native complexes: (i) light-induced charge separation between special pair chlorophylls P(680) and primary quinone acceptor Q(A); (ii) P(680)(+) reduction by the redox-active tyrosine Y(Z) of polypeptide D1; (iii) oxidation of Mn cluster by Y(Z)(ox) followed by proton release, and (iv) protonation of double reduced secondary quinone acceptor Q(B). The electrogenicity related to (i) proton-coupled electron transfer between Q(A)(-) and preoxidized non-heme iron (Fe(3+)) in native and (ii) electron transfer between protein-water boundary and Y(Z)(ox) in the presence of redox-dye(s) in Mn-depleted samples, respectively, were also considered. Evaluation of the dielectric properties using the electrometric data and the polarity profiles of reaction center from purple bacteria Blastochloris viridis and photosystem II are presented. The knowledge of the profile of dielectric permittivity along the photosynthetic reaction center is important for understanding of the mechanism of electron transfer between redox cofactors.     

Structural Basis for Functional Tetramerization of Lentiviral Integrase

Experimental evidence suggests that a tetramer of integrase (IN) is the protagonist of the concerted strand transfer reaction, whereby both ends of retroviral DNA are inserted into a host cell chromosome. Herein we present two crystal structures containing the N-terminal and the catalytic core domains of maedi-visna virus IN in complex with the IN binding domain of the common lentiviral integration co-factor LEDGF. The structures reveal that the dimer-of-dimers architecture of the IN tetramer is stabilized by swapping N-terminal domains between the inner pair of monomers poised to execute catalytic function. Comparison of four independent IN tetramers in our crystal structures elucidate the basis for the closure of the highly flexible dimer-dimer interface, allowing us to model how a pair of active sites become situated for concerted integration. Using a range of complementary approaches, we demonstrate that the dimer-dimer interface is essential for HIV-1 IN tetramerization, concerted integration in vitro, and virus infectivity. Our structures moreover highlight adaptable changes at the interfaces of individual IN dimers that allow divergent lentiviruses to utilize a highly-conserved, common integration co-factor.     

Leukocyte elastase induces epithelial apoptosis: role of mitochondial permeability changes and Akt

During acute inflammation, neutrophil-mediated injury to epithelium may lead to disruption of epithelial function, including the induction of epithelial apoptosis. Herein, we report the effects of neutrophil transmigration and of purified leukocyte elastase on epithelial cell survival. Neutrophil transmigration induced apoptosis of epithelial cells [control monolayers: 5 +/- 1 cells/25 high-power fields (HPF) vs. neutrophil-treated monolayers: 29 +/- 10 cells/HPF, P < 0.05, n = 3 as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay] as did low concentrations (0.1 U/ml) of purified leukocyte elastase (control monolayers: 6.4 +/- 2.5% apoptotic vs. elastase: 26.2 +/- 2.9% apoptotic, P < 0.05, as determined by cytokeratin 18 cleavage). Treatment with elastase resulted in decreased mitochondrial membrane potential, release of cytochrome c to the cytosol, and cleavage of caspases-9 and -3 as determined by Western blot analysis, implicating altered mitochondrial membrane permeability as a primary mechanism for elastase-induced apoptosis. Additionally, incubation of epithelial cells with leukocyte elastase resulted in an early increase followed by a decrease in the phosphorylation of epithelial Akt, a serine/threonine kinase important in cell survival. Inhibition of epithelial Akt before elastase treatment potentiated epithelial cell apoptosis, suggesting that the initial activation of Akt represents a protective response by the epithelial cells to the proapoptotic effects of leukocyte elastase. Taken together, these observations suggest that epithelial cells exhibit a dual response to cellular stress imposed by leukocyte elastase with a proapoptotic response mediated via early alterations in mitochondrial membrane permeability countered by activation of the survival pathway involving Akt.     

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.     

Approaches to laser speckle reduction for uniform illumination of the field of view of a microscope during biophysical studies

Biophysics - A comparative study of various approaches to speckle reduction showed that the use of a liquid crystal-based speckle reducer did not allow complete elimination of speckles. The use of...     

Structures, dynamic behaviour in solution and cross-coupling reactions of the α-palladiated (η-alkylarene)tricarbonylchromium complexes containing a palladium-chromium bond

Structures of the complexes (η³-C₃H₅)Pd(μ-η^6:1-CH₂PhCr(CO)₃ and (η³-C₃H₅)Pd[μ-η^6:1-CH(Ph)Ph]Cr(CO)₃ in solution were evaluated by NMR (¹H and ¹³C) and IR spectroscopy. The dynamic behaviour of the complexes was investigated. Quick rotation (on the NMR time scale) of the tricarbonylchromium groups around the axis passing through the centre of the η⁶-coordinated phenyl ring and the chromium atom takes place at room temperature and becomes slow on cooling. The η³-allylic ligand was proved to undergo no dynamic changes in solution. Unlike the solid state, the semi-bridging carbonyl groups between chromium and palladium atoms are absent or very weak in solution. Cross-coupling reactions of the complexes with organohalides are described.     

Rotary F1-ATPase. Is the C-terminus of subunit gamma fixed or mobile?

F-ATP synthase synthesizes ATP at the expense of ion motive force by a rotary coupling mechanism. A central shaft, subunit gamma, functionally connects the ion-driven rotary motor, F(O), with the rotary chemical reactor, F(1). Using polarized spectrophotometry we have demonstrated previously the functional rotation of the C-terminal alpha-helical portion of gamma in the supposed 'hydrophobic bearing' formed by the (alpha beta)(3) hexagon. In apparent contradiction with these spectroscopic results, an engineered disulfide bridge between the alpha-helix of gamma and subunit alpha did not impair enzyme activity. Molecular dynamics simulations revealed the possibility of a 'functional unwinding' of the alpha-helix to form a swivel joint. Furthermore, they suggested a firm clamping of that part of gamma even without the engineered cross-link, i.e. in the wild-type enzyme. Here, we rechecked the rotational mobility of the C-terminal portion of gamma relative to (alpha beta)(3). Non-fluorescent, engineered F(1) (alpha P280C/gamma A285C) was oxidized to form a (nonfluorescent) alpha gamma heterodimer. In a second mutant, containing just the point mutation within alpha, all subunits were labelled with a fluorescent dye. Following disassembly and reassembly of the combined preparations and cystine reduction, the enzyme was exposed to ATP or 5'-adenylyl-imidodiphosphate (AMP-PNP). After reoxidation, we found fluorescent alpha gamma dimers in all cases in accordance with rotary motion of the entire gamma subunit under these conditions. Molecular dynamics simulations covering a time range of nanoseconds therefore do not necessarily account for motional freedom in microseconds. The rotation of gamma within hours is compatible with the spectroscopically detected blockade of rotation in the AMP-PNP-inhibited enzyme in the time-range of seconds.     

Proton transfer dynamics at the membrane/water interface: dependence on the fixed and mobile pH buffers, on the size and form of membrane particles, and on the interfacial potential barrier

Crossing the membrane/water interface is an indispensable step in the transmembrane proton transfer. Elsewhere we have shown that the low dielectric permittivity of the surface water gives rise to a potential barrier for ions, so that the surface pH can deviate from that in the bulk water at steady operation of proton pumps. Here we addressed the retardation in the pulsed proton transfer across the interface as observed when light-triggered membrane proton pumps ejected or captured protons. By solving the system of diffusion equations we analyzed how the proton relaxation depends on the concentration of mobile pH buffers, on the surface buffer capacity, on the form and size of membrane particles, and on the height of the potential barrier. The fit of experimental data on proton relaxation in chromatophore vesicles from phototropic bacteria and in bacteriorhodopsin-containing membranes yielded estimates for the interfacial potential barrier for H(+)/OH(-) ions of approximately 120 meV. We analyzed published data on the acceleration of proton equilibration by anionic pH buffers and found that the height of the interfacial barrier correlated with their electric charge ranging from 90 to 120 meV for the singly charged species to >360 meV for the tetra-charged pyranine.