Carotenoid Shift Research Articles

Rhodobacter sphaeroides as a model to study the ecotoxicity of 1-alkyl-3-methylimidazolium bromide.

The purple non-sulfur bacterium Rhodobacter sphaeroides was selected as a biological model to investigate its response to the toxicity of 1-alkyl-3-methylimidazolium bromide ([Cnmim]Br), a type of ionic liquid (IL), with different alkyl chain lengths (n describes the number of carbon atoms in the alkyl chain). The inhibition of bacterial growth by [Cnmim]Br was positively correlated with n. Morphological characterization revealed that [Cnmim]Br caused cell membrane perforation. The signal amplitude of the electrochromic absorption band shift of endogenous carotenoids showed a negatively linear correlation with n, and the amplitude of the blue-shift of the B850 band in light-harvesting complex 2 showed a positively linear correlation with n. Furthermore, an increase in blocked ATP synthesis and increase in antioxidant enzyme activity were observed in chromatophores treated with ILs containing longer alkyl chains. In summary, the purple bacterium can be developed as a model to monitor ecotoxicity and examine the mechanism of IL toxicity.

Surfactant Effects on the Permeability of Photosynthetic Membrane from Rhodobacter sphaeroides 2.4.1 Probed by Electrochromic Shift of Endogenous Carotenoids

Four surfactants, sodium cholate(SC), n-dodecyl-β-D-maltopyranoside(DDM), lauryldimethylamine oxide(LDAO) and Triton X-100(TX), which are generally used in photosynthetic pigment-protein complexes preparation, were studied on their interaction with photosynthetic membrane from Rhodobacter sphaeroides 2.4.1 by electrochromic absorption band-shift of endogenous carotenoids and by vesicle size measurements as well. The surfactant critical micelle concentration(cmc) was found to be negatively correlated with the capability of enhancing the permeability of photosynthetic membranes to proton, and more elaborated model of surfactants interacting with membranes was obtained. The electrochromic absorption band-shift measurement might develop into a useful tool to evaluate the effects of surfactants on various membranes.

Ionic liquids effects on the permeability of photosynthetic membranes probed by the electrochromic shift of endogenous carotenoids

Ionic liquids (ILs) are promising materials exploited as solvents and media in many innovative applications, some already used at the industrial scale. The chemical structure and physicochemical properties of ILs can differ significantly according to the specific applications for which they have been synthesized. As a consequence, their interaction with biological entities and toxicity can vary substantially. To select highly effective and minimally harmful ILs, these properties need to be investigated. Here we use the so called chromatophores – protein-phospholipid membrane vesicles obtained from the photosynthetic bacterium Rhodobacter sphaeroides– to assess the effects of imidazolinium and pyrrolidinium ILs, with chloride or dicyanamide as counter anions, on the ionic permeability of a native biological membrane. The extent and modalities by which these ILs affect the ionic conductivity can be studied in chromatophores by analyzing the electrochromic response of endogenous carotenoids, acting as an intramembrane voltmeter at the molecular level. We show that chromatophores represent an in vitro experimental model suitable to probe permeability changes induced in cell membranes by ILs differing in chemical nature, degree of oxygenation of the cationic moiety and counter anion.

Protein-Induced Color Shift of Carotenoids in β-Crustacyanin.

β-Crustacyanin (β-CR) is a pigment protein responsible for the blue color of lobsters. We show using correlated ab initio calculations how the protein environment tunes the chromophores of β-CR through electrostatic and steric effects.

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

The Modified Q-cycle Explains the Apparent Mismatch between the Kinetics of Reduction of Cytochromes c1 and bH in the bc1 Complex

Crystallographic structures of the bc1 complex from different sources have provided evidence that a movement of the Rieske iron-sulfur protein (ISP) extrinsic domain is essential for catalysis. This dynamic feature has opened up the question of what limits electron transfer, and several authors have suggested that movement of the ISP head, or gating of such movement, is rate-limiting. Measurements of the kinetics of cytochromes and of the electrochromic shift of carotenoids, following flash activation through the reaction center in chromatophore membranes from Rhodobacter sphaeroides, have allowed us to demonstrate that: (i) ubiquinol oxidation at the Qo-site of the bc1 complex has the same rate in the absence or presence of antimycin bound at the Qi-site, and is the reaction limiting turnover. (ii) Activation energies for transient processes to which movement of the ISP must contribute are much lower than that of the rate-limiting step. (iii) Comparison of experimental data with a simple mathematical model demonstrates that the kinetics of reduction of cytochromes c1 and bH are fully explained by the modified Q-cycle. (iv) All rates for processes associated with movement of the ISP are more rapid by at least an order of magnitude than the rate of ubiquinol oxidation. (v) Movement of the ISP head does not introduce a significant delay in reduction of the high potential chain by quinol, and it is not necessary to invoke such a delay to explain the kinetic disparity between the kinetics of reduction of cytochromes c1 and bH.

Light-Induced Ascorbate-Dependent Electron Transport and Membrane Energization in Chloroplasts of Bundle Sheath Cells of the C4 Plant Maize

Chloroplasts in bundle sheath cells (BSC) of maize perform photosystem I (PSI)-mediated production of ATP. In this study, the participation of ascorbate (Asc) as an electron donor to PSI in light-induced electron transport in isolated maize BSC was demonstrated. It was found that Asc, at physiological concentrations, rapidly reduced photooxidized reaction center chlorophyll of PSI (P700). The rate of Asc donation of electrons to P700+ reached rates of 50–100 μequivalents (mg Chl)−1 h−1 at 70–80 mM ascorbate with methyl viologen as an electron acceptor. Electron transport supported by Asc was coupled with membrane energization, as demonstrated by the light-induced formation of a trans-thylakoid electric field measured by the electrochromic shift of carotenoids. The possible physiological function of Asc-dependent electron transport in bundle sheath chloroplasts of maize, as an electron donor for linear electron flow versus sustaining cyclic electron transport, is discussed.

Aspartate-187 of cytochrome b is not needed for DCCD inhibition of ubiquinol: cytochrome c oxidoreductase in Rhodobacter sphaeroides chromatophores.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-state 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. In chromatophores of the purple bacterium Rhodobacter sphaeroides, this has been associated with the specific labeling of a surface-exposed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang et al. (1998) Arch. Biochem. Biophys. 352, 193-198]. To explore the possible role of this amino acid residue in the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and the electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores from wild type (WT) and mutant cells, in which aspartate-187 of cytochrome b (Asp(B187)) has been changed to asparagine (mutant B187 DN). The kinetics and amplitude of phase III of the electrochromic shift of carotenoids, reflecting electrogenic reactions in the bc(1) complex, and of the redox changes of cytochromes and reaction center, were similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chromatophores. The dependence of the kinetics and amplitude of phase III of the electrochromic shift on DCCD concentration was identical in WT and B187DN chromatophores, indicating that covalent modification of Asp(B187) is not specifically responsible for the effect of DCCD-induced effects of cytochrome bc(1) complex. Furthermore, no evidence for differential inhibition of electrogenesis and electron transport was found in either strain. We conclude that Asp(B187) plays no crucial role in the protonogenic reactions of bc(1) complex, since its replacement by asparagine does not lead to any significant effects on either the electrogenic reactions of bc(1) complex, as revealed by phase III of the electrochromic shift of carotenoids, or sensitivity of turnover to DCCD.

Electron and proton transfer on the acceptor side of the reaction center in chromatophores of Rhodobacter capsulatus: evidence for direct protonation of the semiquinone state of QB.

1. The absorption changes associated with the formation of P+QBred (QBred stands for the semiquinone state of the secondary quinone acceptor) were investigated in chromatophores of Rhodobacter capsulatus. Marked modifications of the semiquinone spectrum were observed when the pH was lowered from 7 to 5. These modifications match those expected for a complete conversion of QBred from the anionic state QB- at pH 7 to the neutral protonated state QBH at pH 5. Similar modifications were observed in chromatophores from Rb. sphaeroides, but not in purified reaction centers from Rb. capsulatus, suggesting that the environment of the reaction center (native membrane vs detergent micelle) is the crucial parameter. 2. The recombination reaction P+QBred --> PQB was investigated as a function of pH. No particular kinetic heterogeneity was observed at low pH, showing that QBH remains mostly bound to the reaction center. The rate constant reaches a minimum value of 0.08 s-1 at pH 6, suggesting that the direct route for recombination prevails in chromatophores below this pH, instead of the usual pathway via QA-. 3. The proton uptake caused by QBred is about 1 below pH 7 and decreases at higher pH. It is suggested that the pH dependence of the conversion of QB- to QBH, occurring in a range where the uptake is constant, cannot be accommodated by a purely electrostatic model, but probably involves a conformational change. 4. The kinetics of the electron-transfer reaction QA-QB-->QAQBred were investigated. A 2-fold acceleration was observed between pH 7 and pH 5 (t1/2 approximately 30 and 15 microseconds, respectively). A fast (<<10 microseconds) unresolved phase appears to be present at both pHs. The second electron-transfer QA-QBred-->QAQBH2 proceeds with a similar rate as the first electron transfer (15-30 microseconds phase). Consequences for the rate-limiting step are discussed. 5. The carotenoid shift, indicative of the membrane potential, displays a rising phase concomitant with the QA-QB-->QAQBred electron transfer. Its relative extent is markedly increased at pH 5, with part of the kinetics occurring during the unresolved fast phase. 6. The extent of the electrochromic shift of bacteriopheophytin around 750 nm associated with formation of QBred decreases toward acidic pH, reflecting the charge compensation due to proton uptake and the formation of neutral QBH.

Reconstitution of electrochromically active pigment-protein complexes from Rhodobacter sphaeroides into liposomes

Light-harvesting complex II (B800/850, LHC II complexes) and reaction centers with both light-harvesting complex I and II (B875 and B800/850; i.e.,RC LHC I-LHC II complexes) have been isolated from Rhodobacter sphaeroides . Both complexes have been incorporated into liposomes made from phospholipids purified from Escherichia coli. The electrochromic band shift of carotenoids, present in these complexes, could stillbe observed in these liposomes upon generation of a potassium diffusion potential. For RC LHC I-LHC II complexes the characteristics of the liposomes were studied in more detail: the extent of the absorbance changes increased with the amount of pigment protein complex incorporated and was maximal at a ratio of 70 nmol bacteriochlorophyll per mg lipid. Higher amounts of incorporated complexes led to a decrease in carotenoid signal due to an increasing membrane leakage. The carotenoid absorbance change at 503–487 nm showed a linear dependency on the diffusion potentials both in the negative as well as in the positive potential range. The spectrum of the absorbance changes at a fixed diffusion potential for RC LHC I-LHC II liposomes had asimilar shape as the spectrum found for chromatophores of Rb. Sphaeroides , however, with shifted maxima and minima. The spectrum foundfor LHC II liposomes was the inverted spectrum of the RC LHC I-LHC II liposomes. Electron micrographs of both type of liposomes showed distinct protein particles with a diameter of 15 nmfor RC LHC I-LHC II and 11 nm for RC LHC II complexes.

Flash-induced electrogenic events in the photosynthetic reaction center and bc1 complexes of Rhodobacter sphaeroides chromatophores

Electrogenic events in Rb. Sphaeroides chromatophores have been studied (i) electrometrically in the chromatophore/phospholipid-impregnated collodion film system and (ii) spectrophotometrically by measuring the electrochromic spectral shift of carotenoids. Under the conditions when the bc 1 complex and ubiquinone pool were oxidized at pH 7.5, the second flashwas shown to give rise to at least two additional electrogenic phases of τ values approx. 0.2 and approx. 20 ms, which were not induced by the first flash. The fast phase was resistant to the inhibitors of the bc 1 complex, antimycin A and myxothiazol. It seems to be due to the protonation of reduced Q B in the RC complex. The slow phase was partly inhibited by antimycin A and completely by subsequent addition of myxothiazol. The antimycin-sensitive constituent of the slow phase was τ ≈ 40 ms and its rise was non-exponential. The antimycin-insensitive, myxothiazol-sensitive constituent was τ ≈ 7 ms. A comparison of (i) the kinetics of cytochrome b h redox conversion induced by the first and second flashes and (ii) the electrogenic reactions sensitive to the Q-cycle inhibitors suggests that the myxothiazol-sensitive electrogenic phase is associated with the reduction of cytochrome b h ( b -561). The antimycin-sensitive electrogenic phase apparently results from the protonation of reduced Q in the quinone-reducing center of the bct complex. Reduction of ubiquinone to ubisemiquinone by b h seems to be electrically silent, since there is no electrogenic phase to follow the kinetics of this process. Myxothiazol addedin the absence of antimycin A induced a negative electrogenic phase with an opposite polarity (τ ≈ 2.5 ms) after the second flash. This phase, completely abolished by the addition of antimycin A, is assumed to be due to the electrogenic deprotonation of the RC-reduced QH 2 which combines with center C in the bc 1 complex. The data obtained by the electrometric and spectrophotometric methods appear to be very similar, though the electrometric method is more sensitive because of the much higher signal-to-noise ratio.

The effect of the size of the quinone pool on the electrogenic reactions in the ubiquinol-cytochrome c2 oxidoreductase of Rhodobacter capsulatus. Pool behaviour at the quinone reductase site

(1) Electron transfer within the ubiquinol-cytochrome c 2 oxidoreductase ( bc 1) complex produces a charge separation across the membrane which, in some bacterial chromatophores, can be monitored by the electrochromic band shift of carotenoids. The electrogenic steps are believed to coincide with the electron transfer through the cytochrome b chain to the quinone reductase site (Q c) of the complex. (2) The kinetics of these electrogenic reactions have been studied in chromatophores from Rhodobacter capsulatus, activated by a single turnover flash, as a function of the ambient redox potential, and of the size of the ubiquinone pool in isooctane-extracted membranes. (3) In chromatophores containing the native ubiquinone (UQ) pool the rate of charge transfer, monitored by the electrochromic red shift of caretenoids, is progressively increased by lowering the E h below 160 mV. At 100 mV the rate, when normalized to the number of reaction centers turning over, reaches a maximum and declines significantly when the E h is lowered further (down to 40 mV). This rate remains constant for E h values between 40 mV and −40 mV. (4) When the size of the UQ pool is decreased by isoctane extraction, the stimulation in the rate of charge separation takes place only at more negative E h values and the value of the maximum rate is decreased. Also at negative E h values, however, when no pre-oxidized quinone from the pool is present in the membrane, the rate does not decline significantly from its maximum value. (5) The size of the UQ pool affects also the number of charges translocated per flash by the bc 1 complex, since it affects the probability of rapid multiple turnover of the complex, which occurs at E h < 160 mV in unextracted membranes. (6) These data are interpreted as evidence that oxidized UQ from the pool interacts collisionally with the Q c site. At E h < 40 mV, when the pool is totally reduced before the flash, the oxidized UQ molecule produced at every turnover at the Q z site of the bc 1 complex is sufficient for sustaining a rather high reaction rate at the Q c site. The apparent K m estimated for quinone reduction at the Q c site is approx. 1 mM, less than one order of magnitude smaller than that estimated for QH 2 at the Q z site. The rate of transfer of ubiquinone from Q z to Q c required by such a Q cycle mechanism does not appear incompatible with the experimental data.

A slow phase of the carotenoid shift induced by pH jump or by continuous illumination. An electron absorption and resonance Raman study on chromatophores of Rhodopseudomonas spheroides G1C

A slow phase of the carotenoid shift induced by pH jump or by continuous illumination. An electron absorption and resonance Raman study on chromatophores of Rhodopseudomonas spheroides G1C

Redox potential dependence of photophosphorylation and electron transfer in continuous illumination of Rhodopseudomonas sphaeroides chromatophores

The dependence on redox potential ( E h) of the steady-state photophosphorylation rate in chromatophores of Rhodopseudomonas sphaeroides Ga was measured using slowly equilibrating (and hence less interfering) redox mediators. The remaining interference of the mediators was taken into account by extrapolating to zero mediator concentration. The extents of cytochrome redox reactions (in the presence of antimycin) and of the carotenoid shift were similarly measured. The redox titration of cytochrome c oxidation is consistent with a requirement for prior cytochrome c 2 reduction and prior Q I (primary electron acceptor) oxidation, while the titration of cytochrome b reduction is consistent with a requirement for prior (BChl) 2 reduction and prior cytochrome b 560 oxidation. The steady-state carotenoid shift extent is a much more broadly peaked function of E h than is the extent following a single-turnover flash, indicating that the transmembrane electrical potential difference can be built up to significant levels by minimal rates of electron flow. The photophosphorylation rate, in contrast, is much more strongly E h dependent, supporting the concept of a threshold membrane energization below which phosphorylation cannot occur. Earlier work by others showing a requirement for equilibrium cytochrome c 2 reduction and Q I oxidation is clearly confirmed. A much greater enhancement in phosphorylation rate was found at low E h than has heretofore been reported. This threefold enhancement is discussed in relation to the equilibrium redox states of the ubiquinone-10 complement of the chromatophore membrane.

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata

1. When cytochrome c 2 is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10- ortho-phenanthroline) and coincides with the photosynthetic back reaction. 2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux. 3. At high redox potentials, where cytochrome c 2 is unavailable for photo-oxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H + efflux is nevertheless decreased by valinomycin/K +; demonstrating that the [BChl] 2 → Q electron transfer generates a delocalised membrane potential.

Kinetic factors limiting the synthesis of ATP by chromatophores exposed to short flash excitation

ATP synthesis was measured after chromatophores from Rhodopseudomonas capsulata had been subjected to illumination by single turnover flashes fired at variable frequencies. Three processes were examined, which under different conditions can limit the net yield of ATP. (1) A process with an apparent relaxation time of 10–20 ms. This reaction probably limits the rate of ATP synthesis in continuous illumination. It has a similar time dependence to the stimulation of the carotenoid shift decay by ADP after a single flash. (2) An active state of the ATPase only persists when the chromatophores are excited more often than once in 10 s. This state decays with similar kinetics to the entire carotenoid shift decay. Full activation is achieved after two flashes. (1) and (2) are not significantly affected by concentrations of antimycin A sufficient to block electron flow through the cytochrome b c 2 oxidoreductase and abolish phase III in the generation of the carotenoid shift. (3) In the presence of antimycin A, after the third, fourth and subsequent flashes ATP synthesis is limited by the quantity of reducing equivalents transported through the reaction centre rather than by the level of the electrochemical proton gradient.

Correlation between ATP synthesis and the decay of the carotenoid band shift after single flash activation of chromatophores from Rhodopseudomonas capsulata

ATP synthesis and the acceleration of the decay of the carotenoid absorption band shift after single flash excitation of Rhodopseudomonas capsulata chromatophores were compared. The two processes behave similarly with respect to: (1) ADP and P i concentration; (2) inhibition by efrapeptin and venturicidin, and (3) inhibition by valinomycin/K + and by ionophores. Taken together with earlier evidence for the electrochromic nature of the carotenoid band shift the data support the contention that positive charge moves outwards across the chromatophore membrane during ATP synthesis and justify the method for determination of the H +/ATP ratio (Petty, K.M. and Jackson, J.B. (1979) FEBS Lett. 97, 367–372). The ability of nucleotide diphosphates in the presence of P i and Mg 2+ to give rise to the acceleration of the carotenoid shift decay closely correlates with the rate of phosphorylation of the nucleotides in steady-state light. Nucleotide triphosphates enhance the decay in parallel with their rate of hydrolysis. Adenylyl imidodiphosphate is itself without effect on the decay of the carotenoid shift and it does not prevent the ADP-induced acceleration. The analogue does prevent the ATP effect but only after repeated flashes.

Comparison of permeant ion uptake and carotenoid band shift as methods for determining the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga

1. A comparison was made of two methods for estimating the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga. Illuminated chromatophores generated a potential that is apparently much larger when estimated on the basis of the red-band shift of carotenoids rather than from the extent of uptake of the permeant SCN- ion. 2. In contrast, when the chromatophores were oxidizing NADH or succinate the uptake of SCN- indicated a larger membrane potential than was estimated from the carotenoid band shift. 3. The extent of SCN- uptake and the carotenoid-band shift respond differently to changes in the ionic composition of the reaction medium. 4. The effects of antimycin on the carotenoid band shift and SCN- uptake are reported. 5. It is concluded that the carotenoid band shift and the uptake of SCN- are responding to different aspects of the energized state.

Transport of local anaesthetics across chromatophore membranes

1. 1. Both simple amines and tertiary amino local anaesthetics give rise to an accelerated decay of the absorption change of added pH indicator dyes and a decelerated decay of the endogenous carotenoid absorption band shift, following short flash excitation of Rhodopseudomonas sphaeroides chromatophores. 2. 2. With increasing medium pH, lower concentrations of amine or local anaesthetics are effective. 3. 3. The order of potency of the local anaesthetics concurs with their reported membrane/buffer partition coefficients and concentrations required for action potential blockade in nerve fibres. 4. 4. The data are taken as evidence for rapid transport of the free base across the chromatophore membrane and relatively slow penetration of the protonated local anaesthetic. Protolytic reactions complete the effective dissipation of the trans-membrane pH gradient. 5. 5. Benzocaine, with its unusually low p K a and the quaternary derivative, chloropromazine methiodide do not display this type of behaviour. 6. 6. In the presence of membrane potential-collapsing agents, such as valinomycin/K + or thiocyanate ions, local anaesthetics decelerate the decay of the cresol red change but have no effect on the carotenoid shift decay. It appears that transport of the unprotonated local anaesthetic although electrically neutral, requires the presence of a membrane potential. 7. 7. In contrast, the non-anaesthetic amines act independently of the membrane potential. 8. 8. Ca 2+ interferes with the mechanism of local anaesthetic deceleration of the cresol red change decay in the presence of valinomycin/K + or thiocyanate but not with other anaesthetic or amine reactions.

Electrogenic events in the ubiquinone-cytochrome [formula omitted] oxidoreductase of rhodopseudomonas sphaeroides

The reductant of ferricytochrome c 2 in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c 2 by ZH 2 is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c 2 reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH 2 and ferricytochrome c 2, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c 2 reduction at pH 7.0 and at 24°C was 2 · 109 M −1 · s −1, but this varied with pH, being 5.1 · 10 8 M −1 · s −1 at pH 5.2 and 4.3 · 10 9 M −1 · s −1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.