The structure of chromatophores from purple photosynthetic bacteria fused with lipid-impregnated collodion films determined by near-field scanning optical microscopy. (1/166)

Lipid-impregnated collodion (nitrocellulose) films have been frequently used as a fusion substrate in the measurement and analysis of electrogenic activity in biological membranes and proteoliposomes. While the method of fusion of biological membranes or proteoliposomes with such films has found a wide application, little is known about the structures formed after the fusion. Yet, knowledge of this structure is important for the interpretation of the measured electric potential. To characterize structures formed after fusion of membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides with lipid-impregnated collodion films, we used near-field scanning optical microscopy. It is shown here that structures formed from chromatophores on the collodion film can be distinguished from the lipid-impregnated background by measuring the fluorescence originating either from endogenous fluorophores of the chromatophores or from fluorescent dyes trapped inside the chromatophores. The structures formed after fusion of chromatophores to the collodion film look like isolated (or sometimes aggregated, depending on the conditions) blisters, with diameters ranging from 0.3 to 10 microm (average approximately 1 microm) and heights from 0.01 to 1 microm (average approximately 0.03 microm). These large sizes indicate that the blisters are formed by the fusion of many chromatophores. Results with dyes trapped inside chromatophores reveal that chromatophores fused with lipid-impregnated films retain a distinct internal water phase.  (+info)

Escape probability and trapping mechanism in purple bacteria: revisited. (2/166)

Despite intensive research for decades, the trapping mechanism in the core complex of purple bacteria is still under discussion. In this article, it is attempted to derive a conceptionally simple model that is consistent with all basic experimental observations and that allows definite conclusions on the trapping mechanism. Some experimental data reported in the literature are conflicting or incomplete. Therefore we repeated two already published experiments like the time-resolved fluorescence decay in LH1-only purple bacteria Rhodospirillum rubrum and Rhodopseudomonas viridis chromatophores with open and closed (Q(A)(-)) reaction centers. Furthermore, we measured fluorescence excitation spectra for both species under the two redox-conditions. These data, all measured at room temperature, were analyzed by a target analysis based on a three-state model (antenna, primary donor, and radical pair). All states were allowed to react reversibly and their decay channels were taken into consideration. This leads to seven rate constants to be determined. It turns out that a unique set of numerical values of these rate constants can be found, when further experimental constraints are met simultaneously, i.e. the ratio of the fluorescence yields in the open and closed (Q(A)(-)) states F(m)/F(o) approximately 2 and the P(+)H(-)-recombination kinetics of 3-6 ns. The model allows to define and to quantify escape probabilities and the transfer equilibrium. We conclude that trapping in LH1-only purple bacteria is largely transfer-to-the-trap-limited. Furthermore, the model predicts properties of the reaction center (RC) in its native LH1-environment. Within the framework of our model, the predicted P(+)H(-)-recombination kinetics are nearly indistinguishable for a hypothetically isolated RC and an antenna-RC complex, which is in contrast to published experimental data for physically isolated RCs. Therefore RC preparations may display modified kinetic properties.  (+info)

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. (3/166)

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.  (+info)

DCCD inhibits the reactions of the iron-sulfur protein in Rhodobacter sphaeroides chromatophores. (4/166)

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1).  (+info)

Fusion of chromatophores from photosynthetic bacteria with a supported lipid layer: characterization of the electric units. (5/166)

Direct electrometric measurements of membrane potential changes are a valuable tool for study of vectorial transfer of electrons, protons, and ions. Commonly model membrane systems are created by fusion of lipid/protein vesicles with lipid-coated thin films. We characterized the electric units resulting from this process using chromatophores from the purple bacterium Rhodobacter sphaeroides and either a Mylar film or a planar modified gold electrode as support. Investigation of the shunting activity of the ionophore gramicidin on the flash-induced potential change demonstrates fusion of individual chromatophores to form independent 'blisters', which preserve an interior aqueous compartment. Under current-clamp conditions the photovoltage follows the change of the membrane potential of the individual blisters.  (+info)

Electrogenic proton transfer in Rhodobacter sphaeroides reaction centers: effect of coenzyme Q(10) substitution by decylubiquinone in the Q(B) binding site. (6/166)

An electrometric technique was used to investigate the effect of coenzyme Q(10) (UQ), substitution by decylubiquinone (dQ) at the Q(B) binding site of reaction centers (UQ-RC and dQ-RC, respectively) on the electrogenic proton transfer kinetics upon Q(B) reduction in Rhodobacter sphaeroides chromatophores. Unlike dQ-RC, the kinetics of the second flash-induced proton uptake in UQ-RC clearly deviated from the mono-exponential one. The activation energy (about 30 kJ/mol) and the pH profile of the kinetics in dQ-RC were similar to those in UQ-RC, with the power law approximation used in the latter case. The interpretation of the data presumed the quinone translocation between the two binding positions within the Q(B) site. It is proposed that the native isoprenyl side chain (in contrast to decyl chain) favors the equilibrium binding of neutral quinone at the redox-active 'proximal' position, but causes a higher barrier for the hydroquinone movement from 'proximal' to 'distal' position.  (+info)

Changes in the acyl lipid composition of photosynthetic bacteria grown under photosynthetic and non-photosynthetic conditions. (7/166)

The acyl lipids and their constituent fatty acids were studied in the photosynthetic bacteria Rhodospirillum rubrum, Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides, which were grown under photosynthetic and non-photosynthetic conditions. The major lipids were found to be phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in each bacterium. The two Rhodopseudomonas species also contained significant quantities of phosphatidylcholine. Other acyl lipids accounted for less than 10% of the total. On changing growth conditions from non-photosynthetic to photosynthetic a large increase in the relative proportion of phosphatidylglycerol was seen at the expense of phosphatidyl-ethanolamine. In Rhodospirillum rubrum the fatty acids of the major phospholipids showed an increase in the proportion of palmitate and stearate and a decrease in palmitoleate and vaccenate on changing growth conditions to photosynthetic. In contrast, the exceptionally high levels (>80%) of vaccenate in individual phospholipids of Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides were unaffected by changing growth conditions to photosynthetic. Analysis of the lipids of chromatophores, isolated from the three bacteria, showed that these preparations were enriched in phosphatidylglycerol. The large increase in this phospholipid, seen during growth under photosynthetic conditions, appeared, therefore, to be due to a proliferation of chromatophore membranes. Possible roles for acyl lipids in the formation and function of the photosynthetic apparatus of bacteria are discussed.  (+info)

Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F(0)F(1)-ATP synthase is absent from about half of chromatophores. (8/166)

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.  (+info)

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