Properties of the cytochrome a-like material developed in the photosynthetic bacterium Rhodopseudomonas spheroides when grown aerobically

A photosynthetically-incompetent mutant Rhodopseudomonas spheroides that lacks bacteriochlorophyll was isolated. Spectroscopic evidence from CO difference spectra and cyanide difference spectra suggested that a cytochrome oxidase was present in this mutant that contained two components, corresponding to cytochromes a and a₃ of mitochondria. Potentiometric titration at 607 nm also showed the presence of two components with oxidation-reduction mid-point potentials of +375 mV and +200 mV. They were present in a ratio close to unity. No cytochrome of the the c-type corresponding to mitochondrial cytochrome c was detected, but a minor c component (near 10% of the total cytochrome c) with an oxidation-reduction mid-point potential of +120 mV was found

Growth of the mutant in medium with low aeration or lacking added copper diminished the concentration of the a-type cytochrome but not the concentrations of cytochromes of the b and c-type.     

Occurrence of both a-type and o-type cytochromes as the functional terminal oxidases in Rhodopseudomonas spheroides

1. 1. The functional terminal oxidase of the light-anaerobically grown Rhodopseudomonas spheroides cells was found to be the o-type cytochrome, whereas that of the dark-aerobically grown cells was the a-type cytochrome. When the dark-aerobically grown cells were further incubated under a semianaerobic condition in the dark, the content of the o-type cytochrome was increased in these cells, while the synthesis of the a-type cytochrome appeared to be repressed. In Rhodospirillum rubrum cells, grown either aerobically in the dark or anaerobically in the light, cytochrome o was the sole functional terminal oxidase.

2. 2. Reactions with the a-type and o-type cytochromes from Rhodopseudomonas spheroides and also with the o-type cytochrome from Rhodospirillum rubrum were compared using reduced yeast cytochrome c as substrate. The reaction with the a-type cytochrome was far less sensitive to NaN₃ and hydroxylamine than those with the o-type cytochromes, whereas all the reactions were inhibited by KCN in apparently the same manner.

Mutant strains of Rhodopseudomonas spheroides which form photosynthetic pigments aerobically in the dark. Growth characteristics and enzymic activities

Mutant strains of Rhodopseudomonas spheroides have been isolated which contain 5–50 times more bacteriochlorophyll and carotenoids than the wild type when grown under highly aerobic conditions in the dark. Their pigment content is similar to the wild type when grown in the light. One of the mutants (TA-R) grew more slowly than its parent strain under aerobic conditions but formed pigments at about 60% of the rate observed under photosynthetic conditions. The other mutants grew at rates similar to the wild type under all conditions. Synthesis of bacteriochlorophyll by suspensions of the mutants began without delay upon transfer from conditions of high to low aeration. In contrast to the wild type, magnesium protoporphyrin-S-adenosylmethionine methyltransferase (EC 2.1.1.11) activity in particulate preparations from the mutants was not repressed by growth under aerobic conditions in the light or dark. Ribulose diphosphate carboxylase (EC 4.1.1.39) activity was repressed by O₂ in the mutants as in the wild type. Other enzyme activities were compared in mutant TA-R and its parent strain grown under the same conditions. NADH oxidase activity in particles from aerobically grown TA-R was about one third that found in the parent strain. However, the respiration rates of the intact cells did not differ. Light inhibited the respiration of aerobically grown TA-R, indicating that the bacteriochlorophyll formed under these conditions had photochemical activity. It is concluded that the insensitivity of the mutants to O₂ repression is due to defects in the regulatory system which controls formation of the enzymes concerned in pigment synthesis.     

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.     

Electron transport and cytochromes in aerobically grown Proteus mirabilis

The function of the cytochromes in electron transport from NADH to oxygen in aerobically grown Proteus mirabilis has been determined. 77K-Spectra of cytoplasmic membrane suspensions, frozen while catalyzing electron transport from NADH to oxygen, in the presence as well as in the absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide, have been recorded. Analysis of these 77K-spectra revealed that cytochrome b-563 (E'0 = +140 mV), cytochrome b-556 (E'0 = +140 mV) [or alternatively cytochrome b-563/556 (E'0 = +140 mV)] and cytochrome b-557 (E'0 = +50 mV) may function in a Q or b-cycle. The function of cytochrome c-549 (E'0 = +75 mV), which seems to be present only in a very low concentration, and cytochrome b-556 (E'0 = -105 mV), which reacts very slowly to the addition of NADH and oxygen, remains unclear. Cytochrome o, the main oxidase of aerobically grown P. mirabilis cells, can not be detected by the methods described above. Only when the reduced form of cytochrome o is liganded with carbon monoxide a specific alpha-band can be detected at 569 nm at 25 degrees C and 565 nm at 77K.     

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients.

Two independent sets of measurements using a variety of methods show that a sample with A_{800 nm} = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1.

Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77.

These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: _{802 nm} = 288 (± 14) mM⁻¹ · cm⁻¹ and _{865 nm} = 128 (± 6) mM⁻¹ · cm⁻¹. For reversible bleaching of the 865-nm band, Δ^{red - ox}_865nm = 112 (± 6) mM⁻¹ · cm⁻¹ (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.     

The subcellular localization of the pteridines in strain R-26 of Rhodopseudomonas spheroides

Pteridines were detected in strain R-26 of Rhodopseudomonas spheroides and the pteridine concentrations in subcellular fractions were measured by a sensitive fluorimetric method. The whole cell fraction contained nearly equal concentrations of pteridine and reaction center bacteriochlorophyll. The soluble subcellular fraction contained the majority of the pteridine and the photochemically active chromatophore preparations contained very little. No pteridine was detected in purified reaction center complex preparations. This subcellular localization of pteridines was not consistent with a pteridine functioning as the primary electron acceptor in this photosynthetic system.     

The membrane-bound electron-transfer components of aerobically grown Chromatium vinosum

The electron-transfer chain components of aerobically grown Chromatium vinosum have been characterized. Membranes isolated from aerobically grown C. vinosum have been shown to contain a Rieske iron-sulfur protein, at least three c-type cytochromes and at least one b-type cytochrome. Two cytochromes that bind CO appear to be present, one of which may function as a terminal oxidase. Membranes isolated from these cells appear to lack a photochemical reaction center and the high potential (E_m = +340 _mV) cytochrome c-555 that serves as the immediate donor to the reaction center in photosynthetically grown C. vinosum. In addition, the b-cytochrome(s) of aerobically grown C. vinosum has (have) been shown to be considerably more electronegative (E′_m = -90 _mV) than that of photosynthetically grown cells (E′_m = +30 _mV).     

Membrane cytochromes of Escherichia coli grown aerobically and anaerobically with nitrate

Redox titration has been coupled to spectroscopic techniques, enzyme fractionation, and the use of mutants to examine the cytochrome composition of the membranes from cells grown aerobically and anaerobically with nitrate. A combination of techniques was found to be necessary to resolve the cytochromes. At least six b-type cytochromes were present. Besides cytochromes bfdh and bnr, components of the formate dehydrogenase-nitrate reductase pathway, cytochromes b556, b555, b562, and o, characteristic of aerobic respiratory pathways, were present. The midpoint oxidation-reduction potentials of the aerobic b-type cytochromes suggested that the sequence of electron transfer is: cytochrome b556 leads to b555 leads to b562 leads to O2.     

Comparison of cytochromes from anaerobically and aerobically grown cells of Pseudomonas perfectomarinus.

Pseudomonas perfectomarinus (ATCC 14405) is a facultative anaerobe capable of either oxygen respiration or anaerobic nitrate respiration, i.e., denitrification. A comparative study of the electron transfer components of cells revealed five c-type cytochromes and cytochrome cd in the soluble fraction from anaerobically grown cells and four c-type cytochromes in the soluble fraction from aerobically grown cells. Purification procedures yielded three c-type cytochromes (designated c-551, c-554, and acidic c-type) from both kinds of cells as indicated by similarities in absorption spectra, molecular weight, and electrophoretic mobility. Cytochrome cd, a diheme c-type cytochrome (cytochrome c-552), and a split-alpha c-type cytochrome were recovered only from anaerobically grown cells. A c-type cytochrome with a low ratio of alpha to beta absorption peak heights was uniquely present in the aerobically grown cells. Liquid N2 temperature absorption spectroscopy on the membrane fraction from anaerobically grown cells revealed residual cytochrome cd as well as differences in the relative amounts of c-type and b-type cytochromes in membranes prepared from cells grown under the two different conditions.     

Detection of two further b-type cytochromes in Rhodopseudomonas spheroides

The photosynthetically-incompetent mutant V-2 of Rhodopseudomonas spheroides which is incapable of synthesising bacteriochlorophyll was grown aerobically under conditions of both high and low aeration. Potentiometric titration at 560 nm minus 570 nm revealed the presence of several different components tentatively identified as b-type cytochromes. Two such components of oxidation-reduction midpoint potentials of +390 mV ± 10 mV and +255 mV ± 7 mV have not previously been detected in membranes of Rps. spheroides. These components have also been resolved by difference spectra at controlled oxidation-reduction potentials and fourth derivative spectra. Neither component appeared to react with CO. With increasing aeration of the culture medium the relative concentration of these two b-type cytochromes diminished, whilst that of the a-type oxidase increased.     

Microsomal cytochromes of Candida tropicalis grown on alkanes

A comparison of methods used in isolating microsomes and in measuring microsomal cytochrome P-450 demonstrated that separation following protoplast lysis gave the best results. By this latter technique a high amount of cytochrome P-450 (0.2–0.3 nmol/mg) was recovered but cytochrome P-420, considered as the denatured form, was absent.The alkanes specifically induce cytochromes P-450 and b₅ localized on the microsomes. The denaturation in vivo of cytochrome P-450 into cytochrome P-420 even occurs during storage at 1 °C. This degradation is increased during preparation of subcellular fractions if no preventive measures are taken.     

Changes in the cytochrome composition of Rhodopseudomonas sphaeroides grown aerobically, photosynthetically and on dimethyl sulphoxide.

Several strains and mutants of Rhodopseudomonas sphaeroides can be grown anaerobically in the dark in the presence of dimethyl sulphoxide as an electron acceptor. During adaptation to this fermentative mode of growth, two major c-type cytochromes are synthesized, one with Mr 45 000 and the second with Mr 20 000 and a midpoint potential of +120 mV. These cytochromes are barely detectable in membranes prepared from cells grown in aerobic or photosynthetic conditions. An electrophoretic method is presented for the detection of the b-type and c-type cytochromes of pigmented or unpigmented membranes. The method resolves three b-type cytochromes and four c-type cytochromes in membranes from aerobically and photosynthetically grown cells.     

The electrostatic interaction between the reaction-center bacteriochlorophyll derived from Rhodopseudomonas spheroides and mammalian cytochrome c and its effect on light-activated electron transport

1. By means of Q-switched ruby-laser flash excitation, the photooxidation of P870 in the reaction-center complex isolated from Rhodopseudomonas spheroides takes place within 1 μsec. The reduction of photooxidized P870 in the dark follows a first-order kinetics, with a pseudo first-order rate constant of 1.85×10⁸ l×mole^-1×sec^-1 and an activation energy of 6 kcal/mole.

2. Through an electrostatic interaction of the bacteriochlorophyll reaction-center complex and mammalian cytochrome c, an intimate contact between the two components resulted, and a collision-independent electron-transfer with a halftime of 25 μsec can be attained by laser-flash excitation. The absorbance changes at 870 and 550 nm indicated a good stoichiometry of the reaction. The oxidation of the c-type cytochrome in cells of Rps. spheroides (R-26 mutant) has a halftime of 12 μsec.

3. The portion of P870 which recovered rapidly was closely related to the mole ratio of cytochrome/P870. Complete recovery with a halftime of 25 μsec occurred when the cytochrome/P870 ratio was above approx. 10. At cytochrome/P870 ratios lower than 10, only the fraction of the reaction-center complex which have cytochromes bound at the active site can recover with the rapid decay time. Ultrafiltration measurements showed that each particle of the reaction-center complex can bind approx. 24 cytochrome molecules.

4. An electro static interaction is expected simply from the large difference between the isoelectric points of cytochrome c ( 10) and that of the reaction-center complex (4.1 measured by electro-focusing). The electro static interaction was further evidenced by the effects of pH, ionic strength, and by polylysine displacement of binding sites on the coupled oxidation of ferrocytochrome c by P870. From the limiting polylysine concentration giving complete blocking of cytochrome coupling, it was calculated that each reaction-center complex with a particle weight of 6.5×10⁵ contained approx. 500 negative charges.

5. Arrhenius plot of the first-order rate constants vs. the reciprocal absolute temperature yielded an activation energy of 12 kcal/mole for the cytochrome/P870 reaction, which is presumably the energy needed for cytochrome to achieve the most favorable orientation for the rapid electron transfer. Below the freezing temperature of the sample, the cytochrome reaction appeared to be uncoupled. The temperature dependence is consistent with the effect of viscosity on the reaction rate.

6. Double flash excitations spaced 200 μsec apart showed that at a cytochrome/P870 ratio of 24, the first flash caused maximum oxidation, indicating that all the reaction-center particles have at least one cytochrome attached to the active site. However, only 60% of the particles have a second cytochrome closely attached and capable of undergoing the rapid electron transport.