Biomedical subjects

Armen Y Mulkidjanian

Publications and source records attributed to Armen Y Mulkidjanian.

14 recordsLinked to original sources

PubMed2006

Proton translocation by the cytochrome bc1 complexes of phototrophic bacteria: introducing the activated Q-cycle.

The cytochrome bc1 complexes are proton-translocating, dimeric membrane ubiquinol:cytochrome c oxidoreductases that serve as "hubs" in the vast majority of electron transfer chains. After each ubiquinol molecule is oxidized in the catalytic center P at the positively charged membrane side, the two liberated electrons head out, according to the Mitchell's Q-cycle mechanism, to different acceptors. One is taken by the [2Fe-2S] iron-sulfur Rieske protein to be passed further to cytochrome c1. The other electron goes across the membrane, via the low- and high-potential hemes of cytochrome b, to another ubiquinone-binding site N at the opposite membrane side. It has been assumed that two ubiquinol molecules have to be oxidized by center P to yield first a semiquinone in center N and then to reduce this semiquinone to ubiquinol. This review is focused on the operation of cytochrome bc1 complexes in phototrophic purple bacteria. Their membranes provide a unique system where the generation of membrane voltage by light-driven, energy-converting enzymes can be traced via spectral shifts of native carotenoids and correlated with the electron and proton transfer reactions. An "activated Q-cycle" is proposed as a novel mechanism that is consistent with the available experimental data on the electron/proton coupling. Under physiological conditions, the dimeric cytochrome bc1 complex is suggested to be continually primed by prompt oxidation of membrane ubiquinol via center N yielding a bound semiquinone in this center and a reduced, high-potential heme b in the other monomer of the enzyme. Then the oxidation of each ubiquinol molecule in center P is followed by ubiquinol formation in center N, proton translocation and generation of membrane voltage.

Bacterial Chromatophores↗

PubMed2006

The cyanobacterial genome core and the origin of photosynthesis.

Comparative analysis of 15 complete cyanobacterial genome sequences, including "near minimal" genomes of five strains of Prochlorococcus spp., revealed 1,054 protein families [core cyanobacterial clusters of orthologous groups of proteins (core CyOGs)] encoded in at least 14 of them. The majority of the core CyOGs are involved in central cellular functions that are shared with other bacteria; 50 core CyOGs are specific for cyanobacteria, whereas 84 are exclusively shared by cyanobacteria and plants and/or other plastid-carrying eukaryotes, such as diatoms or apicomplexans. The latter group includes 35 families of uncharacterized proteins, which could also be involved in photosynthesis. Only a few components of cyanobacterial photosynthetic machinery are represented in the genomes of the anoxygenic phototrophic bacteria Chlorobium tepidum, Rhodopseudomonas palustris, Chloroflexus aurantiacus, or Heliobacillus mobilis. These observations, coupled with recent geological data on the properties of the ancient phototrophs, suggest that photosynthesis originated in the cyanobacterial lineage under the selective pressures of UV light and depletion of electron donors. We propose that the first phototrophs were anaerobic ancestors of cyanobacteria ("procyanobacteria") that conducted anoxygenic photosynthesis using a photosystem I-like reaction center, somewhat similar to the heterocysts of modern filamentous cyanobacteria. From procyanobacteria, photosynthesis spread to other phyla by way of lateral gene transfer.

Bacterial Proteins↗

PubMed2006

Proton in the well and through the desolvation barrier.

The concept of the membrane proton well was suggested by Peter Mitchell to account for the energetic equivalence of the chemical (DeltapH) and electrical (Deltapsi) components of the proton-motive force. The proton well was defined as a proton-conducting crevice passing down into the membrane dielectric and able to accumulate protons in response to the generation either of Deltapsi or of DeltapH. In this review, the concept of proton well is contrasted to the desolvation penalty of > 500 meV for transferring protons into the membrane core. The magnitude of the desolvation penalty argues against deep proton wells in the energy-transducing enzymes. The shallow DeltapH- and Deltapsi-sensitive proton traps, mechanistically linked to the functional groups in the membrane interior, seem more realistic. In such constructs, the draw of a trapped proton into the membrane core can happen at the expense of some exergonic reaction, e.g., release of another proton from the membrane into the aqueous phase. It is argued that the proton transfer in the ATP synthase and the cytochrome bc complex could proceed in this way.

Biological Transport↗

PubMed2006

Probing biological interfaces by tracing proton passage across them.

The properties of water at the surface, especially at an electrically charged one, differ essentially from those in the bulk phase. Here we survey the traits of surface water as inferred from proton pulse experiments with membrane enzymes. In such experiments, protons that are ejected (or captured) by light-triggered enzymes are traced on their way between the membrane surface and the bulk aqueous phase. In several laboratories it has been shown that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with increase in their electric charge, it was suggested that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. In terms of ordinary electrostatics, the barrier could be ascribed to dielectric saturation of water at a charged surface. In terms of nonlocal electrostatics, the barrier could result from the dielectric overscreening in the surface water layers. It is discussed how the interfacial potential barrier can affect the reactions at interface, especially those coupled with biological energy conversion and membrane transport.

Ion Transport↗

PubMed2006

Protons @ interfaces: implications for biological energy conversion.

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from "pulsed" experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the "sources" and "sinks", the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.

Biological Transport↗

PubMed2005

Ubiquinol oxidation in the cytochrome bc1 complex: reaction mechanism and prevention of short-circuiting.

This review is focused on the mechanism of ubiquinol oxidation by the cytochrome bc1 complex (bc1). This integral membrane complex serves as a "hub" in the vast majority of electron transfer chains. The bc1 oxidizes a ubiquinol molecule to ubiquinone by a unique "bifurcated" reaction where the two released electrons go to different acceptors: one is accepted by the mobile redox active domain of the [2Fe-2S] iron-sulfur Rieske protein (FeS protein) and the other goes to cytochrome b. The nature of intermediates in this reaction remains unclear. It is also debatable how the enzyme prevents short-circuiting that could happen if both electrons escape to the FeS protein. Here, I consider a reaction mechanism that (i) agrees with the available experimental data, (ii) entails three traits preventing the short-circuiting in bc1, and (iii) exploits the evident structural similarity of the ubiquinone binding sites in the bc1 and the bacterial photosynthetic reaction center (RC). Based on the latter congruence, it is suggested that the reaction route of ubiquinol oxidation by bc1 is a reversal of that leading to the ubiquinol formation in the RC. The rate-limiting step of ubiquinol oxidation is then the re-location of a ubiquinol molecule from its stand-by site within cytochrome b into a catalytic site, which is formed only transiently, after docking of the mobile redox domain of the FeS protein to cytochrome b. In the catalytic site, the quinone ring is stabilized by Glu-272 of cytochrome b and His-161 of the FeS protein. The short circuiting is prevented as long as: (i) the formed semiquinone anion remains bound to the reduced FeS domain and impedes its undocking, so that the second electron is forced to go to cytochrome b; (ii) even after ubiquinol is fully oxidized, the reduced FeS domain remains docked to cytochrome b until electron(s) pass through cytochrome b; (iii) if cytochrome b becomes (over)reduced, the binding and oxidation of further ubiquinol molecules is hampered; the reason is that the Glu-272 residue is turned towards the reduced hemes of cytochrome b and is protonated to stabilize the surplus negative charge; in this state, this residue cannot participate in the binding/stabilization of a ubiquinol molecule.

Catalytic Domain↗

PubMed2005

Proton slip in the ATP synthase of Rhodobacter capsulatus: induction, proton conduction, and nucleotide dependence.

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 microM) and after pre-illumination over several seconds. (2) Slip induction by pmf was accompanied by the release of approximately 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 microM). (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 gamma, in an open structure of the (alphabeta)3 hexagon with no nucleotides in the catalytic sites.

Bacterial Chromatophores↗

PubMed2004

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.

Bacterial Chromatophores↗

PubMed2004

The proton-driven rotor of ATP synthase: ohmic conductance (10 fS), and absence of voltage gating.

The membrane portion of F(0)F(1)-ATP synthase, F(0), translocates protons by a rotary mechanism. Proton conduction by F(0) was studied in chromatophores of the photosynthetic bacterium Rhodobacter capsulatus. The discharge of a light-induced voltage jump was monitored by electrochromic absorption transients to yield the unitary conductance of F(0). The current-voltage relationship of F(0) was linear from 7 to 70 mV. The current was extremely proton-specific (>10(7)) and varied only slightly ( approximately threefold) from pH 6 to 10. The maximum conductance was approximately 10 fS at pH 8, equivalent to 6240 H(+) s(-1) at 100-mV driving force, which is an order-of-magnitude greater than of coupled F(0)F(1). There was no voltage-gating of F(0) even at low voltage, and proton translocation could be driven by deltapH alone, without voltage. The reported voltage gating in F(0)F(1) is thus attributable to the interaction of F(0) with F(1) but not to F(0) proper. We simulated proton conduction by a minimal rotary model including the rotating c-ring and two relay groups mediating proton exchange between the ring and the respective membrane surface. The data fit attributed pK values of approximately 6 and approximately 10 to these relays, and placed them close to the membrane/electrolyte interface.

ATP Synthetase Complexes↗

PubMed2003

Survival of the fittest before the beginning of life: selection of the first oligonucleotide-like polymers by UV light.

BACKGROUND: A key event in the origin of life on this planet has been formation of self-replicating RNA-type molecules, which were complex enough to undergo a Darwinian-type evolution (origin of the "RNA world"). However, so far there has been no explanation of how the first RNA-like biopolymers could originate and survive on the primordial Earth. RESULTS: As condensation of sugar phosphates and nitrogenous bases is thermodynamically unfavorable, these compounds, if ever formed, should have undergone rapid hydrolysis. Thus, formation of oligonucleotide-like structures could have happened only if and when these structures had some selective advantage over simpler compounds. It is well known that nitrogenous bases are powerful quenchers of UV quanta and effectively protect the pentose-phosphate backbones of RNA and DNA from UV cleavage. To check if such a protection could play a role in abiogenic evolution on the primordial Earth (in the absence of the UV-protecting ozone layer), we simulated, by using Monte Carlo approach, the formation of the first oligonucleotides under continuous UV illumination. The simulations confirmed that UV irradiation could have worked as a selective factor leading to a relative enrichment of the system in longer sugar-phosphate polymers carrying nitrogenous bases as UV-protectors. Partial funneling of the UV energy into the condensation reactions could provide a further boost for the oligomerization. CONCLUSION: These results suggest that accumulation of the first polynucleotides could be explained by their abiogenic selection as the most UV-resistant biopolymers.

Biopolymers↗

PubMed2003

Low dielectric permittivity of water at the membrane interface: effect on the energy coupling mechanism in biological membranes.

Protonmotive force (the transmembrane difference in electrochemical potential of protons, ) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of relates to the difference in the proton activity between two bulk water phases (deltapH(B)) or between two membrane surfaces (deltapH(S)). To scrutinize whether deltapH(S) can deviate from deltapH(B), we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, PHYS: Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5-1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2-0.3 eV) than for monovalent cations (0.1-0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton "pumps" at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pH(S) of approximately 6.0; the value of pH(S) was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria.

Cell Membrane↗

PubMed2003

MASE1 and MASE2: two novel integral membrane sensory domains.

Escherichia coli proteins YegE and YaiC contain N-terminal integral membrane regions, followed by the putative diguanylate cyclase (GGDEF, DUF1) domains. The membrane domains of these proteins, named MASE1 (membrane-associated sensor) and MASE2, respectively, were found in other bacterial signaling proteins, such as histidine kinases (MASE1) and an adenylate cyclase (MASE2). Although the nature of the signals sensed by MASE1 and MASE2 is still unknown, MASE1-containing receptors appear to play important roles in bacteria, including iron and/or oxygen sensing by hemerythrine-containing proteins in the sulfate-reducing bacterium Desulfovibrio vulgaris.

Adenylyl Cyclases↗

PubMed2002

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn2+ decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation.

The effect of Zn2+ on the rates of electron transfer and of voltage generation in the cytochrome bc1 complex (bc1) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn2+ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme b(h) (in the absence of Zn2+ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c(1) reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn2+ <100 microM the reduction of heme b(h) remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc1 turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b(h) is the main electrogenic reaction in the cytochrome bc1 complex, (2) imply an allosteric link between the site of heme b(h) oxidation and the site of cytochrome c1 reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc1 complex.

Bacterial Chromatophores↗

PubMed2002

Chromatophore vesicles of Rhodobacter capsulatus contain on average one F(O)F(1)-ATP synthase each.

ATP synthase is a unique rotary machine that uses the transmembrane electrochemical potential difference of proton (Delta(H(+))) to synthesize ATP from ADP and inorganic phosphate. Charge translocation by the enzyme can be most conveniently followed in chromatophores of phototrophic bacteria (vesicles derived from invaginations of the cytoplasmic membrane). Excitation of chromatophores by a short flash of light generates a step of the proton-motive force, and the charge transfer, which is coupled to ATP synthesis, can be spectrophotometrically monitored by electrochromic absorption transients of intrinsic carotenoids in the coupling membrane. We assessed the average number of functional enzyme molecules per chromatophore vesicle. Kinetic analysis of the electrochromic transients plus/minus specific ATP synthase inhibitors (efrapeptin and venturicidin) showed that the extent of the enzyme-related proton transfer dropped as a function of the inhibitor concentration, whereas the time constant of the proton transfer changed only marginally. Statistical analysis of the kinetic data revealed that the average number of proton-conducting F(O)F(1)-molecules per chromatophore was approximately one. Thereby chromatophores of Rhodobacter capsulatus provide a system where the coupling of proton transfer to ATP synthesis can be studied in a single enzyme/single vesicle mode.

Adenosine Diphosphate↗