Energy tranduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the co-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata.

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.     

Energy transduction in photosynthetic bacteria IV. Light-dependent ATPase in photosynthetic membranes from Rhodopseudomonas capsulata

ATPase activity of photosynthetic membrane fragments from the bacterium Rhodopseudomonas capsulata can be stimulated by continuous illumination under conditions of active cyclic electron flow. The activation corresponds to an increase in the maximum velocity of the reaction and does not affect the apparent K_m for ATP (0.11 mM). No stimulation in the light is observed in the presence of classical uncouplers or oxidized 2,6-dichlorophenolindophenol (DCIP), which, per se, stimulate ATPase in the dark. It is demonstrated, however, that oxidized DCIP acts as an uncoupler of bacterial photophosphorylation.

The effect of light is elicited after a few minutes of preillumination, or in a much shorter time if an ADP trapping system is supplied. Activation does not occur if ADP is added during the preillumination (apparent K_m for inhibition by ADP = 1 μM). The effect of ADP is not related to competitive inhibition with ATP, which can be observed at higher concentrations (apparent K_i = 0.26 mM). ADP, however, is not effective if added after some minutes of preillumination.     

Energy transduction in photosynthetic bacteria VI. Respiratory sites of energy conservation in membranes from dark-grown cells of Rhodopseudomonas capsulata

Membranes prepared from Rhodopseudomonas capsulata grown heterotrophically in the dark perform phosphorylation linked to oxidation of NADH and succinate, with P/2e⁻ ratios of about 0.5 and 0.15, respectively. The localization of the sites of energy conservation was investigated by observing the respiration-induced quenching of the fluorescence of atebrine.

Energization of the membrane can be demonstrated when NADH is oxidized by O₂, ferricyanide or Q₁, when succinate is oxidized by O₂ or by oxidized diaminodurene, and during the oxidation of reduced diaminodurene.

Antimycin A completely inhibits energization between succinate and O₂ or succinate and diaminodurene; however, it only inhibits partially NADH or succinate oxidases and energization between NADH and O₂. KCN inhibits NADH oxidase in a biphasic way: the first level of inhibition is observed at concentrations which block the oxidation of exogenous cytochrome c or of diaminodurene and energization between succinate or ascorbate-diaminodurene and O₂. The second level corresponds to the inhibition of the antimycin-insensitive oxidase.

The results are interpreted as evidence of the presence in these bacteria of a respiratory chain branching after the dehydrogenase system, one arm of the chain being sensitive to antimycin A and low concentrations of KCN and capable of energy conservation, the other being represented by a completely uncoupled system.     

Transposon Tn5 mutagenesis of genes for the photosynthetic apparatus in Rhodopseudomonas capsulata

Summary Three plasmids containing the transposon Tn5, i.e. pSUP201::Tn5, pACYC184::Tn5 and pJB4JI were transferred from Escherichia coli to Rhodopseudomonas capsulata in order to mutagenize the genome. Mutants defective in bacteriochlorophyll and carotenoid synthesis and mutants unable to form the photochemical reaction center or one of the light-harvesting complexes were isolated. Of special interest were mutants that could not form the light-harvesting complex B800-850. Two of these mutants synthesized only two of the three polypeptides of this complex whereas the corresponding near infrared absorbance bands were not observed. Complementation analysis with the Rprime plasmid pRPS404, which contains a 50 kb region of the genome of R. capsulata carrying most genes responsible for expression of photosynthetic apparatus, revealed that some genes of the B800-850 light-harvesting complex lie outside this photosynthetic gene cluster.Abbreviations Bchl Bacteriochlorophyll - Cm chloramphenicol - Km kanamycin - Tc tetracycline - Ap ampicillin - Gm gentamicin - Spc spectinomycin     

Molecular cloning of the ribosomal RNA genes of the photosynthetic bacterium Rhodopseudomonas capsulata

Summary Chromosomal segments of Rhodopseudomonas capsulata carrying the ribosomal operons and cloned with the cosmid vector pHC79 have been identified by cross hybridization with ³²P-ATP labeled rRNAs. At least seven rRNA operons are present in the R. capsulata chromosome. By R-loop analyses of DNA-RNA hybrids, two distinct loop structures of sizes 1.50 kb and 2.52 kb corresponding to the 16S and 23S RNA molecules, respectively, were detected. Intact 23S RNA molecules can be isolated from R. capsulata ribosomes by sucrose density centrifugation. However, fragmentation of the 23S RNA molecule into a 16S-like molecule was observed during gel electrophoresis. Restriction mapping and hybridization of a 9 kb PstI fragment that contained one copy of the rRNA operon showed the following sequence of the RNA genes in R. capsulata 16S, 23S, and 5S. A spacer region of 0.91 kb was found between the 16S and the 23S RNA genes.     

Isolation and amino-terminal sequences of subunits from the photosynthetic reaction center of Rhodopseudomonas capsulata

The amino-terminal sequences have been determined by Edman degradation for the reaction center polypeptides from a carotenoidless mutant of Rhodopseudomonas capsulata. Individual polypeptides were isolated by preparative electrophoresis and electroelution. By comparison with the sequences deduced from the DNA (Youvan, D.C., Alberti, M., Begush, H., Bylina, E.J. and Hearst, J.E. (1984) Proc. Natl. Acad. Sci. USA 81, 189–192) we conclude that the M and L subunits are processed so as to remove the amino-terminal methionine, whereas the H subunit is not processed at the amino-terminus after translation. None of the subunits is synthesized with a significant amino-terminal extension peptide.     

Rapid proton release accompanying photosynthetic electron transport in intact cells of Rhodopseudomonas capsulata

The release of protons from intact cells of Rhodopseudomonas capsulata after either 4μs flashes or during brief periods of continuous illumination has been measured with the indicator, cresol red. The half-time for H⁺-release after a flash was 35 ms and the extent, 1H⁺ per 134 bacteriochlorophyll. Myxothiazol completely inhibited the flash-induced H⁺-release and antimycin A reduced it by 37%. The proton-releasing reaction is discussed with reference to the protonmotive Q-cycle. During continuous illumination the rapid phase of H⁺ release is followed by a lag and then by another period of acidification, suggesting that other protolytic reactions may be in operation.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

Plasmid pU29, a vehicle for mutagenesis of the photosynthetic puf operon in Rhodopseudomonas capsulata

Plasmid pU21, which carries the reaction center and light-harvesting genes (puf operon) of Rhodopseudomonas capsulata, has been redesigned by site-specific mutagenesis. Five restriction sites have been removed and three unique restriction sites have been introduced into this 11,589-bp pBR322 derivative. The modifications divide the puf structural genes into four regions separated by five unique and nonmutagenic restriction sites. These four fragments have been subcloned into the M13-mp series of vectors to facilitate oligonucleotide-mediated site-specific mutagenesis experiments on the photosynthetic apparatus structural genes. The inserts can then be returned from the M13 replicative form to the redesigned pU21 derivative. The modified plasmid, pU29, greatly facilitates in vitro mutagenesis experiments since previously described techniques and screening procedures are more efficient with M13 derivatives carrying smaller inserts. Additionally, tandem homologous sequences (the reaction center L and M subunits) within the puf operon are now separated on different phage vectors, eliminating problems encountered in the targeting of mutagenic oligonucleotides to only one of the two homologous sites.     

The adaptation of the electron transfer chain of Rhodopseudomonas capsulata to different light intensities

(1) Cells of Rhodopseudomonas capsulata (wild-type) were grown photoheterotrophically in a turbidostat under very high and very low light intensity. Membranes were isolated from cells adapted to the respective light conditions and fractionated by sucrose density centrifugation. The molar ratios of ubiquinone and cytochromes c₂, c₁, b-561 and b-566 per reaction center were 3-fold to 5-fold higher in high-light than in low-light membranes. (2) Most of the Cyt(c₁ + c₂) and Cyt b-561 detected in dark redox titrations undergoes light-induced redox changes, both in high- and in low-light membranes. (3) The fractions of the total photooxidizable reaction center and Cyt(c₁ + c₂) oxidized under continuous light in the absence of antimycin are higher in membranes from low-light- than from high-light-grown cells. (4) From these data and results of kinetic studies it is proposed that cyclic electron flow under saturating light intensities is faster in high-light-grown cells.     

Evidence for regulation in vivo of the ATP synthase in intact cells of the photosynthetic bacterium Rhodopseudomonas capsulata

(1) The cytoplasmic membrane potential (Δψ) of intact cells of Rhodopseudomonas capsulata, measured either from the uptake of butyltriphenylphosphonium cation or from the electrochromic carotenoid band shift, increased upon illumination (negative on the cytoplasmic side) and then, within the next 20 s, partly declined while the light was still on. In the presence of the F₀ inhibitor venturicidin the light-induced Δψ was increased by 30% and the partial decline was abolished. (2) From the ionic current/Δψ curves for the bacterial membranes it was concluded that the slow, partial decline of Δψ after the onset of illumination was the result of an increase in membrane conductance. The conductance increase seen in the ionic current/Δψ curves was blocked by venturicidin suggesting that it was caused by increased proton flux through the ATP synthase. (3) Analysis of the light-induced changes in adenine nucleotide levels in intact bacterial cells showed that the apparent increase in ATP synthase activity was not the result of a decrease in phosphorylation potential. The data were consistent with either an increase in the catalytic activity of the ATP synthase or with an increase in H⁺ flux through the enzyme without a proportionate increase in the rate of phosphorylation (increased ‘slip’). (4) This slow change in the properties of the ATP synthase, as judged by the venturicidin-sensitive partial decline of Δψ, required a minimum initial value of Δψ. When Δψ was reduced, either by decreasing the actinic light intensity or by adding carbonylcyanide trifluoromethoxyphenylhydrazone the partial decline in Δψ was abolished. (5) The slow change in ATP synthase properties reversed upon darkening the bacterial cell suspension. A second illumination period shortly after the first elicited a smaller initial Δψ and a smaller Δψ decline. The relaxation of the ATP synthase in the dark was measured from the dependence of the initial increase in Δψ after the second illumination period upon the dark-time between the two illumination periods.     

Mitochondrial respiratory chain of Tetrahymena pyriformis The properties of submitochondrial particles and the soluble b and c type pigments

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 °C there are two b-type pigments with half-reduction potentials of −0.04 and −0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a₂ with E_m7.2 of 0.245 and 0.345 V.

EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spin ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g = 6, and a g = 2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed.

Cytochrome c₅₅₃ is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c₅₅₃ is a negatively charged protein at neutral pH with an E_m7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with a K_D of 0.8 · 10⁻⁶ M. Reduced cytochrome c₅₅₃ serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c₅₅₃ shows the presence of a low spin ferric heme with the dominant resonance signal at g = 3.28.

A pigment with an absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and E_m7.2 of −0.17 V and exhibits a high spin ferric heme signal at g = 6.     

Energy transduction in photosynthetic bacteria. X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata.

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.     

Cytochrome c1 and b-type cytochrome with two heme centers in the photosynthetic membranes from Rhodopseudomonas palustris

The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c₁, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c₁ were 30000 and 275 mV, respectively. The peak of the reduced-minus-oxidized difference spectrum of cytochrome c₁ was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b-c₁ complex in Rps. palustris.     

Energy dependent changes in the cytochromes of the mitochondrial respiratory chain

In intact mitochondria a stoichiometric coupling exists between cytochrome a₃ and the hydrolysis of adenosine triphosphate (ATP). In each case the modification of one cytochrome a₃ (measured as a spectral change) is coupled to the hydrolysis of one ATP molecule. When both cytochromes a₃ and a are reduced the measured equilibrium constant is 0.06 m^?1 but this constant is 10³ M^?1 when both cytochromes are oxidized. When the sixth ligand for cytochrome a₃ is an externally added ligand (HCN, H₂S, CO, NO) the equilibrium constant is different for each ligand, suggesting that the ATP induced modification is of the fifth ligand but that it is energetically dependent on the chemical nature of the sixth ligand. The measured half-reduction potentials for cytochromes a₃ and b_T are dependent on the concentrations of added ATP, adenosine diphosphate (ADP), and orthophosphate. The relationship is consistent with a ligand exchange mechanism in which the ligand on the cytochrome is dependent on the phosphate potential ($\text{ATP}\text{ADP}$ × P_i). The equilibrium constants obtained by the ligand exchange treatment of the E_m values for cytochrome a₃ are consistent with those obtained by direct measurement of the equilibrium constants for the spectrally measured changes.     

Spectral and functional comparisons between the carotenoids of the two antenna complexes of Rhodopseudomonas capsulata

The spectral and functional properties of carotenoids associated with each of the two light-harvesting complexes of the Rhodopseudomonas capsulata photosynthetic antenna system have been distinguished by studying mutants lacking one or the other complex. In mutants containing only the light-harvesting I complex (LH-I), the absorption spectrum of the carotenoids is blue-shifted compared to wild type. Carotenoid absorption in mutants possessing only the light-harvesing II complex (LH-II) complex is red-shifted. The circular dichroism spectrum of carotenoids in each complex is also distinctive. Although carotenoids in each complex function with approximately the same efficiency in harvesting and transmitting light energy for photosynethesis, only the carotenoids associated with LH-II undergo an electrochromic bandshift upon generation of a transmembrane potential. These observations are interpreted to indicate that both the orientation of carotenoid molecules with respect to the plane of the membrane, and the immediate electrochemical environment of these molecules differ in the two light-harvesting complexes.     

A kinetic completion of the cyclic photosynthetic electron pathway of Rhodopseudomonas sphaeroides: Cytochrome b-cytochrome c2 oxidation-reduction

In Rhodopseudomonas sphaeroides, following a single-turnover flash of light, cytochrome c₂ is oxidized by reaction center bacteriochlorophyll, and a cytochrome b is reduced by the primary electron acceptor, probably via ubiquinone. In this report we show that, in the uncoupled state, the rate of re-oxidation of the cytochrome b is identical to the rate of reduction of the cytochrome c₂, a kinetic completion of the cyclic photosynthetic electron transport system.     

Spectral study of b cytochromes in yeast mitochondria and intact cells

Three types of b cytochromes are demonstrated in Candida utilis mitochondria. One of these b cytochromes has a symmetrical -band at 561.5 nm at room temperature. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate both in coupled and uncoupled mitochondria. The second b cytochrome has a double -band at 565 nm and 558 nm. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate in coupled mitochondria, but in uncoupled mitochondria it is slowly reduced after anaerobiosis and this reduction rate is enhanced by antimycin A addition. Thus the oxidation-reduction state of this cytochrome is energy dependent. The first cytochrome is spectroscopically identified as cytochrome b_K and the second as cytochrome b_T. The third b cytochrome has an -band around 563 nm (b₅₆₃) and is reduced slowly after anaerobiosis in uncoupled mitochondria but faster than the b_T. Further properties of this component are not known. Midpoint potentials of cytochromes b_T, b₅₆₃ and b_K are approximately −50 mV, +5 mV, and +65 mV, respectively.

In intact cells, cytochrome b_T is reduced immediately after anaerobiosis or cyanide treatment, and rapidly oxidized when uncoupler is added. Addition of antimycin A instead of uncoupler to the anaerobic cells causes oxidation of mainly cytochrome b_T while addition of antimycin A to the aerobic cells results in a reduction of the cytochrome b_T.     

Relations between pigments and proteins in the photosynthetic membranes of Rhodopseudomonas spheroides

We have isolated from Rhodopseudomonas spheroides a pigment-protein complex of apparent weight 9 kdaltons that bears more than 60% of the light harvesting bacteriochlorophyll. The isolation procedure involved exposure to 1% lauryl dimethyl amine oxide (LDAO). The purified 9-kdalton fraction showed the light harvesting bacteriochlorophyll components B800 and B850, plus carotenoids. The ratio of bacteriochlorophyll to protein was 17%. This protein is probably the same as the “band 15” protein of Fraker and Kaplan. It may exist in vivo as characteristic aggregates of higher molecular weight. LDAO added to Rps. spheroides chromatophores converted the bacteriochlorophyll component B870 to a form absorbing at 770 nm but had little effect on the “B800 + B850” system, causing only a reversible shift of the 850-nm band to 845 nm. Anti-reaction center serum, added to subcellular fractions from Rps. spheroides with 1% LDAO, precipitated reaction center chromoprotein unaccompanied by light harvesting bacteriocholorophyll. Other antisera precipitated light harvesting components and left the reaction center chromophores in solution. A major protein of apparent weight 45 kdaltons was found in relatively nonpigmented fractions from Rps. spheroides, associated with cell wall fragments. The 45-kdalton protein showed considerable interstrain variability, whereas the 9-kdalton and reaction center proteins appeared constant.