Photoelectric currents across planar bilayer membranes containing bacterial reaction centers: the response under conditions of multiple reaction-center turnovers

The characteristics of the photocurrent response activated by continuous illumination of planar bilayer membranes containing bacterial reaction centers have been resolved by voltage clamp methods. The photocurrent response to a long light pulse consists of an initial spike arising from the fast, quasi-synchronous electron transfer from the reaction center bacteriochlorophyll dimer, BChl2, to the primary quinone QA. This is followed by a slow relaxation of the current to that promoted by secondary, asynchronous multiple electron transfers from the reduced cytochrome c through the reaction centers to the ubiquinone-10 pool. Currents derived from cytochrome c oxidation that occurs when cytochrome c is associated with the reaction center or when limited by diffusional interaction from solution are recognized. Changes of the ionic strength and pH in the aqueous phase, and the clamped membrane potential (+/- 150 mV), affect the electron-transfer rate between cytochrome c and BChl2. In contrast, the primary light-induced charge separation between BChl2 and QA, or electron transfer between QA on the ubiquinone pool are unaffected. During illumination of reaction center membranes supplemented with cytochrome c and a ubiquinone pool, there is a small but significant steady-state current which is considered to be caused by the re-oxidation of photoreduced quinone by molecular oxygen. In the dark, after illumination of reaction centers supplemented with cytochrome c and a ubiquinone pool, there is a small amount of reverse current resulting from the movement of charges back across the membrane. This reverse current is observed maximally after 400 ms illumination while prolonged illumination diminishes the effect. The source of this current is uncertain, but it is considered to be due to the flux of anionic semiquinone within the membrane profile; this may also be the species that interacts with oxygen giving rise to the steady-state current. It is postulated that when the reaction centers are contained in an alkane-containing phospholipid membrane, in contrast to the in vivo situation, the semiquinone anion formed in the QB site is not tightly bound to the site and can, by exchange-diffusion with the membrane-quinone pool, move away from the site and accumulate in the membrane. However, in the absence, more quantitative work superoxide anion, resulting from O2 interaction with semiquinone of QA, QB or pool cannot be excluded.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers. Response under conditions of single electron turnover.

Light-induced electric current and potential responses have been measured across planar phospholipid membranes containing reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides. Under conditions in which the reaction centers are restricted to a single electron turnover, the responses can be correlated with the light-induced electron transfer reactions associated with the reaction center. The results indicate that electron transfer from the bacteriochlorophyll dimer to the primary ubiquinone molecule, and from ferrocytochrome c to the oxidized dimer occur in series across the planar membrane. Electron transfer from the primary to secondary ubiquinone molecule is not electrogenic.     

Light-induced conformational changes in photosynthetic reaction centers: redox-regulated proton pathway near the dimer

The influence of the hydrogen bonds on the light-induced structural changes were studied in the wild type and 11 mutants with different hydrogen bonding patterns of the primary electron donor of reaction centers from Rhodobacter sphaeroides. Previously, using the same set of mutants at pH 8, a marked light-induced change of the local dielectric constant in the vicinity of the dimer was reported in wild type and in mutants retaining Leu L131 that correlated with the recovery kinetics of the charge-separated state [ Deshmukh et al. (2011) Biochemistry, 50, 340-348]. In this work after prolonged illumination the recovery of the oxidized dimer was found to be multiphasic in all mutants. The fraction of the slowest phase, assigned to a recovery from a conformationally altered state, was strongly pH dependent and found to be extremely long at room temperature, at pH 6, with rate constants of ～10(-3) s(-1). In wild type and in mutants with Leu at L131 the very long recovery kinetics was coupled to a large proton release at pH 6 and a decrease of up to 79 mV of the oxidation potential of the dimer. In contrast, in the mutants carrying the Leu to His mutation at the L131 position, only a negligible fraction of the dimer exhibited lowered potential, the large proton release was not observed, the oxidized dimer recovered 1 or 2 orders of magnitude faster depending on the pH, and the very long-lived state was not or barely detectable. These results are modeled as arising from the loss of a proton pathway from the bacteriochlorophyll dimer to the solvent when His is present at the L131 position.     

Light-induced conformational changes in photosynthetic reaction centers: dielectric relaxation in the vicinity of the dimer

Conformational changes near the bacteriochlorophyll dimer induced by continuous illumination were identified in the wild type and 11 different mutants of reaction centers from Rhodobacter sphaeroides. The properties of the bacteriochlorophyll dimer, which has a different hydrogen bonding pattern with the surrounding protein in each mutant, were characterized by steady-state and transient optical spectroscopy. After illumination for 1 min, in the absence of the secondary quinone, the recovery of the charge-separated states was nearly 1 order of magnitude slower in one group of mutants including the wild type than in the mutants carrying the Leu to His mutation at the L131 position. The slower recovery was accompanied by a substantial decrease in the electrochromic absorption changes associated with the Q(y) bands of the nearby monomers during the illumination. The other set of mutants containing the Leu L131 to His substitution exhibited slightly altered electrochromic changes that decreased only half as much during the illumination as in the other family of mutants. The correlation between the recovery of the charge-separated states in the light-induced conformation and the electrochromic absorption changes suggests a dielectric relaxation of the protein that stabilizes the charge on the dimer.     

Primary electron transfer in Rhodobacter sphaeroides R-26 reaction centers under dehydration conditions

The photoinduced charge separation in Q_B-depleted reaction centers (RCs) from Rhodobacter sphaeroides R-26 in solid air-dried and vacuum-dried (~10⁻² Torr) films, obtained in the presence of detergent n-dodecyl-β-D-maltoside (DM), is characterized using ultrafast transient absorption spectroscopy. It is shown that drying of RC-DM complexes is accompanied by reversible blue shifts of the ground-state absorption bands of the pigment ensemble, which suggest that no dehydration-induced structural destruction of RCs occurs in both types of films. In air-dried films, electron transfer from the excited primary electron donor P^⁎ to the photoactive bacteriopheophytin H_A proceeds in 4.7 ps to form the P⁺H_A⁻ state with essentially 100% yield. P⁺H_A⁻ decays in 260 ps both by electron transfer to the primary quinone Q_A to give the state P⁺Q_A⁻ (87% yield) and by charge recombination to the ground state (13% yield). In vacuum-dried films, P^⁎ decay is characterized by two kinetic components with time constants of 4.1 and 46 ps in a proportion of ~55%/45%, and P⁺H_A⁻ decays about 2-fold slower (462 ps) than in air-dried films. Deactivation of both P^⁎ and P⁺H_A⁻ to the ground state effectively competes with the corresponding forward electron-transfer reactions in vacuum-dried RCs, reducing the yield of P⁺Q_A⁻ to 68%. The results are compared with the data obtained for fully hydrated RCs in solution and are discussed in terms of the presence in the RC complexes of different water molecules, the removal/displacement of which affects spectral properties of pigment cofactors and rates and yields of the electron-transfer reactions.     

Oxidation of cytochromes c and c2 by bacterial photosynthetic reaction centers in phospholipid vesicles. 1. Studies with neutral membranes

The oxidation of cytochrome c2 by photosynthetic reaction center isolated from Rhodopseudomonas sphaeroides and incorporated into unilamellar phosphatidylcholine vesicles was found to be kinetically similar to that observed earlier for reaction centers in low detergent solution [Overfield, R.E., Wraight, C.A., & DeVault, D. (1979) FEBS Lett. 105, 137-142]. At low ionic strength the kinetics were biphasic. The fast phase indicated the formation of a cytochrome-reaction center complex with an apparent binding constant, KB, of about 10(5) M-1. However, KB decreased dramatically with increasing salt concentration, and no fast oxidation was detectable in 0.1 M NaCl. The slow cytochrome oxidation was first order in both cytochrome and reaction centers and, thus, second order overall. Deviations from theoretical second-order behavior were observed when the rate of the first-order back reaction of the primary photoproducts was significant compared to the cytochrome oxidation. This can cause serious overestimation of the second-order rate constant. The slow oxidation of cytochrome c2 by reaction centers in phosphatidylcholine vesicles exhibited a 40% lower encounter frequency than with the solubilized reaction center. This was attributed to the much lower diffusion coefficient of the reaction center in the vesicle membrane than in solution. No effects of diminished dimensionality were detected with neutral vesicles. An activation energy of 8.0 +/- 0.4 kcal x mol-1 was determined for the slow phase of cytochrome c2 oxidation by reaction centers in solution and in vesicles of several different phosphatidylcholines, including dimyristoylphosphatidylcholine above and below its phase transition temperature. Thus, the physical state of the lipid did not appear to affect any rate-limiting steps leading to cytochrome oxidation. The ionic strength dependence of the slow kinetics of oxidation of cytochromes c and c2 confirmed the electrostatic nature of the cytochrome-reaction center interaction, and the pH dependence indicated the titration of a group or groups, important to this interaction, at pH 9.5.     

Light-induced charge separation in Rhodopseudomonas viridis reaction centers monitored by Fourier-transform infrared difference spectroscopy: the quinone vibrations.

Static FTIR light-induced difference spectra have been recorded for reaction centers from Rhodopseudomonas viridis in the following charge-separated states: P+QA(-)-PQA, P+QB(-)-PQB, I(-)-I, I-QA(-)-IQA, and I-QA(2-)-IQA. A comparison of the I(-)-I difference spectra with the I-QA(-)-IQA difference spectra reveals new bands which can be assigned to QA- vibrations; these vibrations are also observed in the P+QA(-)-PQA and P+QB(-)-PQB difference spectra. Through an analysis of all of the static difference spectra, the electron-transfer pathway can be monitored in the infrared from the primary donor, P, to the secondary acceptor, QB, via the intermediate acceptor, I, and the primary acceptor, QA. The difference spectra are dominated by absorbance changes of prosthetic groups, with very few identifiable contributions from amino acids and little overall structural change in the protein backbone, involving only one or two residues for the various charge-separated states. Oxidation of the primary donor in the reaction center shows the characteristic absorbance changes of the 9-keto and 10-ester carbonyl groups observed upon oxidation of bacteriochlorophyll b in a non-hydrogen-bonded environment [Ballschmiter, K. H., & Katz, J. J. (1969) J. Am. Chem. Soc. 91, 2661-2677]. Reduction of the quinones in the reaction center yields absorbance changes of the carbonyls observed during reduction of quinones in a hydrogen-bonded environment [Bauscher, M., Nabedryk, E., Bagley, K., Breton, J., & M?ntele, W. (1990) FEBS Lett. 261, 191-195].(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-induced conformational changes in photosynthetic reaction centers: impact of detergents and lipids on the electronic structure of the primary electron donor

Light-induced hypsochromic shifts of the Q(y) absorption band of the bacteriochlorophyll dimer (P) from 865 to 850 nm were identified using continuous illumination of dark-adapted reaction centers (RCs) from Rhodobacter capsulatus when dispersed in the most commonly used detergent, the zwitterionic lauryl N-dimethylamine-N-oxide. Such a shift is known to be the consequence of the decreased degree of delocalization of P. A 2-fold acceleration of the recovery kinetics of P(+) was found in RCs that underwent light-induced structural changes compared to those where the P-band position did not change. The light-induced shift was irreversible except in the presence of a secondary electron donor. Prolonged (15 min) illumination resulted in a shift in the position of the P-band even in neutral or negatively charged detergents. In contrast, RCs reconstituted into liposomes made from lipids with different headgroup charges showed light-induced shifts only if shorter fatty acid chains were used. The light-induced conformational changes caused a prominent decrease of the redox potential of P ranging from 120 to 160 mV depending on the detergent compared to the potential of P in dark-adapted reaction centers. The measured light-induced potential decreases were 55 to 85 mV larger than those reported for reaction centers where the P-band position remained at 865 nm. The influence of structural factors, such as the delocalization of the electron hole on P(+), the involvement of Tyr M210, and the hydrophobic mismatch between the thickness of the hydrophobic belt of the detergent micelles or the lipid bilayer and the RC protein, on the spectral features and electron transfer kinetics is discussed.     

Proton flux induced by free fatty acids across phospholipid bilayers: New evidences based on short-circuit measurements in planar lipid membranes

Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance () was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes.     

A hydroxide ion carrier in planar phospholipid bilayer membranes: (C6F5)2Hg (dipentafluorophenylmercury)

Although a number of molecules are known to function as current-carrying proton carriers across lipid bilayer membranes, no such hydroxide ion carriers have been found to date. We report that (C6F5)2Hg, which can function as a chloride ion carrier, can also carry a hydroxide ion. In 100 mM Na2SO4 solutions, membranes treated with (C6F5)2Hg are almost ideally selective for H+/OH- between pH 6.0 and 9.5. Membrane conductance varies linearly with [OH-] over this pH range and with the square of the (C6F5)2Hg concentration. The presumed current-carrying species is the dimer [(C6F5)2Hg]2OH-, which, along with the neutral molecule (C6F5)2Hg, shuttles back and forth within the bilayer. In 0.2 M NaCl at pH 9.5, the OH- and Cl- conductances are approximately equal. Thus, the carrier displays an approximately 10(4)-fold preference for OH- over Cl-.     

Photoreduction of pheophytin in reaction centers of the photosystem II in intact cells of green algae and cyanobacteria under anaerobic conditions

Reversible photoreduction of pheophytin (Pheo) accompanied by a decrease in the chlorophyll fluorescence yield is observed in Photosystem 2 of the intact cells of green algae and cyanobacteria under anaerobic conditions. The photoreaction is inhibited by DCMU and reactivated upon subsequent addition of either ascorbate of dithionite. It is suggested that as a result of electron donation from the water splitting system being in the state S(3), to the reaction centre of Photosystem 2 in the state [P(+)(680)Pheo(-)] Q(-) after the primary photoreaction there occurs formation of the long-living state [P(680)Pheo(-)] Q(-). It was found that oxidized NADP, benzyl viologen and methyl viologen accelerate oxidation of Pheo reduced int he Photosystem 2 in the light indicating that these electron acceptors (typical for Photosystem 1) can accept an election from Pheo in Photosystem 2.     

Coupling optical and electrical measurements in artificial membranes: Lateral diffusion of lipids and channel forming peptides in planar bilayers

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K⁺ channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.     

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.     

Intermediates in the aerobic autoxidation of 6-hydroxydopamine: relative importance under different reaction conditions

Autoxidation of 6-hydroxydopamine (6-OHDA) proceeds through a balanced network of: transition metal ions, superoxide, hydrogen peroxide, hydroxyl radicals, and other species. The contribution of each to the reaction mechanism varies dramatically depending upon which scavengers are present. The contribution of each propagating intermediate increases when the involvement of others is diminished. Thus, superoxide (which is relatively unimportant when metal ions can participate) dominates the reaction when transition metal ions are bound (especially at higher pH), and it becomes essential in the simultaneous presence of catalase plus chelators. Transition metal ions participate more if superoxide is excluded; hydrogen peroxide becomes more important if both .O2- and metal ions are excluded; and hydroxyl radicals contribute more to the reaction mechanism if both H2O2 and .O2- are excluded. Superoxide dismutase inhibited strongly, by two distinct mechanisms: a high affinity mechanism (less than 13% inhibition) at catalytically effective concentrations, and a low affinity mechanism (almost complete inhibition at the highest concentrations) which depends upon both metal binding and catalytic actions. In the presence of DETAPAC catalytic concentrations of superoxide dismutase inhibited by over 98%. Conversely, metal chelating agents inhibited strongly in the presence of superoxide dismutase. When present alone they stimulated (like EDTA), inhibited (like desferrioxamine), or had little effect (like DETAPAC). Catalase which stimulated slightly but consistently (less than 5%) when added alone, inhibited 100% in the presence of superoxide dismutase + DETAPAC. However, in the absence of DETAPAC, catalase decreased inhibition by superoxide dismutase, yielding a 100% increase in reaction rate. Hydroxyl scavengers (formate, mannitol or glucose) alone produced little or no (less than 10%) inhibition, but inhibited by 30% in the presence of catalase + superoxide dismutase. Paradoxically, they stimulated the reaction in the presence of catalase + superoxide dismutase + DETAPAC.     

A hydroxide ion carrier in planar phospholipid bilayer membranes: (C6F5)2Hg (dipentafluorophenylmercury)

Although a number of molecules are known to function as current-carrying proton carriers across lipid bilayer membranes, no such hydroxide ion carriers have been found to date. We report that (C₆F₅)₂ Hg, which can function as a chloride ion carrier, can also carry a hydroxide ion. In 100 mM Na₂SO₄ solutions, membranes treated with (C₆F₅)₂Hg are almost ideally selective for H⁺/OH⁻ between pH 6.0 and 9.5. Membrane conductance varies linearly with [OH⁻] over this pH range and with the square of the (C₆F₅)₂Hg concentration. The presumed current-carrying species is the dimer [(C₆F₅)₂Hg]₂OH⁻, which, along with the neutral molecule (C₆F₅)₂Hg, shuttles back and forth within the bilayer. In 0.2 M NaCl at pH 9.5, the OH⁻ and Cl⁻ conductances are approximately equal. Thus, the carrier displays an approximately 10⁴-fold preference for OH⁻ over Cl⁻.     

Ammonia toxicity under hyponatremic conditions in astrocytes: de novo synthesis of amino acids for the osmoregulatory response

This study investigates how the metabolic activity and de novo synthesis of amino acids from glucose correlate with changes in intracellular organic osmolytes involved in astrocytic volume regulation during hyperammonemia and hyponatremia. Multinuclear (1H-, 31P-, 13C-) NMR spectra were recorded to quantify water-soluble metabolites, the cellular energy state, as well as the incorporation of [1-(13)C]glucose into amino acids of primary astrocyte cultures. Myo-inositol levels were strongly decreased already at 3h after treatment with NH4Cl; other intracellular osmolytes, such as hypotaurine and choline-containing compounds were also decreased, along with a concomitant increase of both the total concentration and the amount of newly synthesized glutamine, alanine, and glutathione. During ammonia stress, the decrease of organic osmolytes compensated in part for increased intracellular osmolarity caused by amino acid synthesis. Hypotonic conditions alone also lowered the content of organic osmolytes including cellular amino acids, but much less than in hyperammonemia. This was due to impaired mitochondrial metabolic activity via the Krebs cycle, which also enhanced ammonia-induced ATP decrease. However, the changes in the sum of organic osmolytes were not significantly different after ammonia-treatment in hypoosmotic compared to anisoosmotic media, suggesting that the decrease of cellular organic osmolytes may not adequately compensate for the increased intracellular osmolarity caused by amino acids under hyponatremia. Therefore, the ammonia-induced release of osmolytes is an early process in response to increased intracellular osmolarity evoked by increased glutamine and alanine as a consequence of stimulated metabolic activity. The imperfect correlation of changes in astrocytic glutamine, other organic osmolytes, and the cellular energy state under hyperammonemic stress in isoosmotic and hypoosmotic media, however, point to additional mechanisms contributing to astrocyte dysfunction in hyperammonemic states, which are independent from glutamine formation.     

Interaction of biologically active molecules with phospholipid membranes: I. Fluorescence depolarization studies on the effect of polymeric biocide bearing biguanide groups in the main chain

Interaction of poly(hexamethylene biguanide hydrochloride) (PHMB), which is a polymeric biocide bearing biguanide groups in its main chain, with phospholipid bilayers was studied by the fluorescence depolarization method. A strong interaction of PHMB with negatively charged bilayers composed of phosphatidylglycerol(PG) alone or of PG and phosphatidylcholine (PC) was observed, whereas neutral PC bilayers were not affected. On adding PHMB, the fluorescence polarization of diphenylhexatriene embedded in the negatively charged bilayers was reduced to a great extent, especially in the gel phase. This was interpreted in terms of PHMB-induced expansion and fluidization of the bilayer, which enables the probe molecule to undergo less-hindered torsional motion. Similarity between PHMB and polymyxin B in the structure, the mode of action against bacteria and the interaction with lipid membranes is discussed.     

Organization of the photosystem II centers and their associated antennae in the thylakoid membranes: a comparative ultrastructural,biochemical, and biophysical study of Chlamydomonas wild type and mutants lacking in photosystem II reaction centers

We investigated the ultrastructure of thylakoid membranes that lacked either some or all of their Photosystem II centers in the F34SU3 and F34 mutants of Chlamydomonas reinhardtii. We obtained the following results: (a) There are no particles of the 160-A size class on the EF faces of the thylakoids in the absence of Photosystem II centers (as in F34); the F34SU3 contains 50% of the wild-type number of PSII centers and EF particles. (b) The density of the particles on the PF faces of the thylakoids is higher in the mutants than in the wild type. (c) The fluorescence analysis shows that the organization of the pigments is the same regardless of whether 50% of the PSII centers are temporarily inactivated (by preilluminating the wild type) or are actually missing from the thylakoid membrane (F34SU3). Our results, therefore, support a model in which: (a) each 160-A EF particle has only one PSII center surrounded by light-harvesting complexes and (b) part of the PSH antenna is associated with 80-A PF particles in both of the mutants and the wild type.     

Proton cycles through membranes in bacteria: Relationship between proton passive and active fluxes and their dependence on some external physico-chemical factors under fermentation

This paper represents H⁺ circles through the bacterial membranes, their peculiarities and relationship with ATP synthesis or hydrolysis, utilization or accumulation of energy are considered. Data on passive and active proton (H⁺) fluxes through the bacterial membranes are analyzed and their relationship with membrane H⁺ conductance $\left( {G_m^{H^ + } } \right)$ and permeability for H⁺ $\left( {P_{H^ + } } \right)$ is discussed. Methods for determination of bacterial membrane $G_m^{H^ + }$ are presented and some difficulties in obtaining and interpreting data are pointed out. Different ways and mechanisms of passive and active H⁺ fluxes, including a role of membrane lipids in H⁺ transfer, importance of phase transitions in lipid bilayers, operation of protonophores as well as H⁺ translocation via the F₀ factor of the F₀F₁-ATPase, are discussed. Dependence of $G_m^{H^ + }$ for Escherichia coli, Enterococcus hirae, Streptococcus lactis and other bacteria on some external physico-chemical growth factors, particularly, on pH and oxidation reduction potential as well as influence of osmotic stress on $G_m^{H^ + }$ and H⁺ active fluxes through the bacterial membrane under fermentation have been shown. The relationship between $G_m^{H^ + }$ , $P_{H^ + }$ and active H⁺ fluxes through a membrane is proposed, possible mechanisms of relationship between their alterations depending on pH and oxidation reduction potential are discussed. The results are important for understanding the structural and functional properties of bacterial membranes determining H⁺ cycles operation and mechanisms of H⁺ fluxes essential in adaptation of bacteria to altered environment conditions.