Dual roles of ubiquinone as primary and secondary electron acceptors in light-induced electron transfer in chromatophores of Chromatium D.

The effects of isooctane-extraction on the quantum yield ofphotooxidation of cytochromes in chromatophores of Chromatiumvinosum, strain D, were investigated. The initial rate of photooxidation of cytochrome c-555 in theisooctane-extracted chromatophores was decreased by repeatedor prolonged preillumination in the presence of 30 mM ascorbate.The minimum number of light quanta absorbed during preilluminationto cause the maximum decrease in the photooxidation of cytochromec-555 was about 2% of the number of bacteriochlorophyll moleculespresent. In the absence of ascorbate no lowering of the initial rateof cytochrome photooxidation was observed after prolonged orrepeated illumination. No decrease in the initial rate due topreillumination was observed in lyophilized or ubiquinone-readdedchromatophores. The initial rate of photooxidation of both the cytochromes c-555and c-552 in partially isooctane-extracted chromatophores (50–90%extraction of ubiquinone) was also decreased by repeated orprolonged illumination in the presence of 30 mM ascorbate. Our previous and present studies indicate that about 10% ofthe total ubiquinone- 7 functions as the primary electron acceptorfor the photooxidation of cytochrome c-552, and that the majorpart of the ubiquinone functions as the common secondary electronacceptor for the photooxidation of cytochromes c-555 and c-552in Chromalium chromatophores. Therefore, ubiquinone probablyhas dual roles in the light-induced electron transfer of Chromatiumchromatophores. (Received July 23, 1975; )     

Light-induced blue shift of the carotenoid spectrum in chromatophores of Chromatium vinosum strain D

In Chromatium chromatophores, the response of part of the carotenoid complement to a light-induced membrane potential is a shift to the blue of its absorption spectrum, as indicated by the characteristics of the light-minus-dark difference spectrum. The spectrum in the dark of the population of carotenoid which responds to a light-induced membrane potential is located at least 1–2 nm to the red in comparison to the total carotenoid absorption. The results indicate that the proposed permanent electric field affecting the responding population has a polarity with respect to the chromatophore membrane opposite to that in Rhodopseudomonas sphaeroides chromatophores. The carotenoid absorption change interferes seriously with measurements of cytochrome c-555 redox changes at its α band.     

Evolution of photosystems of photosynthetic organisms

It is generally accepted that two photosystems function successively in photosynthetic electron transport chain of plants and algae. The interaction of these photosystems results in the enhancement of photosynthesis. It was suggested that only one photosystem is present in purple bacteria, the most primitive photosynthetic organisms. The functioning of this photosystem is accompanied by absorption changes at 890 nm. Recently new spectral changes were found inChromatium chromatophores under reductive conditions, more favorable for bacterial growth. Some of that spectral changes take place even atliquid nitrogen temperature. It is proposed these absorption changes could be related to other photosystem functioning in low potential region. Such a photosystem is necessary for reduction of NAD inChromatium, for which the reverse electron transport to NAD was not shown. In contrast to photosystems of plants, the bacterial photosystems appear to function independently because the enhancement of bacterial photosynthesis is not found. Apparently the evolution of photosystems involved interaction between independent photosystems, one of them functioning under more oxidative conditions.     

Photo-oxidation of membrane-bound and soluble cytochromec in the green sulfur bacteriumChlorobium tepidum

We studied the photosynthetic electron transfer system of membrane-bound and soluble cytochromec inChlorobium tepidum, a thermophilic green sulfur bacterium, using whole cells and membrane preparations. Sulfide and thiosulfate, physiological electron donors, enhanced flash-induced photo-oxidation ofc-type cytochromes in whole cells. In membranes,c-553 cytochromes with two (or three) heme groups served as immediate electron donors for photo-oxidized bacteriochlorophyll (P840) in the reaction center, and appeared to be closely associated with the reaction center complex. The membrane-bound cytochromec-553 had anE _m-value of 180 mV. When isolated soluble cytochromec-553, which has an apparent molecular weight of 10 kDa and seems to correspond to the cytochromec-555 inChlorobium limicola andChlorobium vibrioforme, was added to a membrane suspension, rapid photo-oxidation of both soluble and membrane-bound cytochromesc-553 was observed. The oxidation of soluble cytochromec-553 was inhibited by high salt concentrations. In whole cells, photo-oxidation was observed in the absence of exogenous electron donors and re-reduction was inhibited by stigmatellin, an inhibitor of the cytochromebc complex. These results suggest that the role of membrane-bound and soluble cytochromec inC. tepidum is similar to the role of cytochromec in the photosynthetic electron transfer system of purple bacteria.     

Some effects of o-phenanthroline on electron transport in chromatophores from photosynthetic bacteria

The midpoint potentials of the primary electron acceptors in chromatophores from Rhodopseudomonas spheroides and Chromatium have been studied by titrating the laser-induced P605 and cytochrome c oxidations, respectively. Both midpoint potentials are pH dependent (60 mV/pH unit).o-Phenanthroline shifts the midpoint potentials of the primary acceptors, by +40 mV in Rps spheroides and +135 mV in Chromatium. A similar though less extensive change in midpoint potential was observed in the presence of batho-phenanthroline, but not with 8-hydroxyquinoline. The shifted midpoints retain the same dependence on pH.Some of the effects of o-phenanthroline can be explained by assuming that it chelates the reduced form of the primary electron acceptor. This suggests the presence in the primary electron acceptor of a metal chelated by o- and batho-phenanthroline.In Rps spheroides chromatophores o-phenanthroline inhibits the laser- and flash-induced carotenoid shift at all redox potentials, stimulates the laser-induced P605 oxidation at redox potentials between +350 and +420 mV and slows the decay of the laser-induced cytochrome c oxidation below +180 mV. These effects show that o-phenanthroline may have more than one site of action.     

Effect of oxidation-reduction potential on light-induced cytochrome and bacteriochlorophyll reactions in chromatophores from the photosynthetic green bacterium Chlorobium

The reaction center bacteriochlorophyll of Chlorobium thiosulfatophilum has a midpoint oxidation-reduction potential (E_m) of +330 mV. Its photooxidation is unaffected by oxidation-reduction potentials in the range from +260 mV to ?70 mV but on further reduction is attenuated to zero in a one-electron transition with an E_m of ?130 mV.A c-type cytochrome with an E_m of +220 mV and absorption maxima at 551–552 nm (α-band) and 420 nm (γ-band) is present in Chlorobium chromatophores and undergoes photooxidation. Cytocrome c photooxidation is attenuated to zero in two 1-electron steps with E_m of +30 mV and ?130 mVPossible roles for +30 mV and ?130 mV components in photosynthetic electron transport in Chlorobium are discussed.     

Nature of photochemical reactions in chromatophores of Chromatium D. II. Quantum yield of photooxidation of cytochromes in Chromatium chromatophores

Initial rates of the light-induced absorption decrease in Chromatium chromatophores due to the oxidation of cytochromes were measured under various experimental conditions. The initial rate in the presence of 10 mM potassium ferrocyanide and 50 μM potassium ferricyanide was about one-half to two-thirds of that in the presence of 30 mM ascorbate or in a medium with a redox potential (E_h) of − 78 mV.

Light-minus-dark difference spectrum indicated that, in the presence of 10 mM ferrocyanide and 50 μM ferricyanide, only cytochrome c-555 was photooxidized. In the presence of 30 mM ascorbate or at E_h values lower than about 0 mV, both cytochrome c-555 and cytochrome c-552 were photooxidized. The quantum yield of cytochrome c-555 photooxidation was calculated to be about 0.4.

The results obtained in the present study are compared with other investigators' and the possibility of the presence of two types of associations between the cytochromes and reaction-center bacteriochlorophyll is discussed.     

A high-potential nonheme iron protein (HiPIP)-linked,thiosulfate-oxidizing enzyme derived fromChromatium vinosum

A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was inferred to be tetrathionate.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

Preparation of subunits of flavocytochromes c derived from Chlorobium limicola f. thiosulfatophilum and Chromatium vinosum.

The subunits of Chlorobium limicala f. thiosulfatophilum cytochrome c-553 and of Chromatium vinosum cytochrome c-552 have been obtained. Chlorobium cytochrome c-553 is split into the cytochrome and flavoprotein subunits by treatment with trichloroacetic acid; after the cytochrome is precipitated by 1–2% trichloroacetic acid, the cytochrome subunit is extractable with buffer, while the flavoprotein subunit is not dissolved. The subunits of Chromatium cytochrome c-552 can not be obtained by the trichloroacetic acid-treatment. The flavoprotein subunit of the Chromatium cytochrome is obtained by isoelectric focusing in the presence of 6 M urea and 1% mercaptoethanol, while the cytochrome subunit is prepared by gel filtration in the presence of 6 M urea with Sephacryl S-200. Molecular weights of the cytochrome and flavoprotein subunits from the Chlorobium cytochrome are 11,000 and 47,000, respectively, while those of the two subunits from the Chromatium cytochrome are 21,000 and 46,000, respectively. The molecule of each flavocytochrome c is composed of one molecule of each of the cytochrome and flavoprotein subunits.     

The cytochromec reductase/oxidase respiratory pathway ofParacoccus denitrificans: Genetic and functional studies

Data are presented on three components of the quinol oxidation branch of theParacoccus respiratory chain: cytochromec reductase, cytochromec ₅₅₂, and thea-type terminal oxidase. Deletion mutants in thebc ₁ and theaa ₃ complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochromec ₅₅₂ in electron transport between complex III and IV is presented.     

Cytochrome components of denitrifyingPseudomonas stutzeri

Cytochromesc-551,c-552,c-554,cd, ac type with low α/β peaks, and an acidicc-type cytochrome were detected in extracts ofPseudomonas stutzeri. The first four were purified and physically characterized. Light absorption spectra indicate a probably histidine-methionine liganding of heme iron inc-551 andc-554, but an absence of methionine in the ligand ofc-552 heme iron. In displaying two separately reduciblec-hemes, thec-552 appears homologous with that fromP. perfectomarinus.     

Complex formation between Chlorobium limicola f. thiosulfatophilumc-type cytochromes

It has been possible to demonstrate, using affinity chromatography, that Chlorobium flavocytochrome c-553 forms an electrostatically stabilized complex with Chlorobium cytochrome c-555. The binding site for cytochrome c-555 appears to be located on the heme-containing subunit of flavocytochrome c-553. This complex appears to be involved in the flavocytochrome c-553-catalyzed transfer of electrons from sulfide to cytochrome c-555. Complex formation has also been demonstrated between Chlorobium cytochromes c-555 and c-551, two components involved in the oxidation of thiosulfate by this green sulfur bacterium. Affinity chromatography data also suggest the possibility that the cytochrome binding sites on the Chlorobium flavocytochrome c-553 and on flavocytochrome c-552 from the purple sulfur bacterium Chromatium vinosum may be similar.     

Reconstitution of photosynthetic electron transport and photophosphorylation in cytochrome-c2-deficient membrane preparation of Rhodopseudomonas capsulata.

Cytochrome c₂ was removed by washing from heavy chromatophores prepared from Rhodopseudomonas capsulata cells. The easy removal of the cytochrome could indicate that it was attached on the outside of the membrane. Therefore, the membrane was probably oriented inside out in relation to the membrane of regular chromatophores, from which cytochrome c₂ could not be removed. Washing of the heavy chromatophores caused loss of photphosphorylation activity. The activity was restored to the resolved heavy chromatophores by the supernatant obtained during the washing or by the native cytochrome c₂, which was found to be the active component in this supernatant. The activity could not be restored by other c-type cytochromes. Ascorbate, which enhanced photophosphorylation activity in the heavy chromatophores at the optimal concentration of 8 mm, restored this activity to the washed heavy chromatophores, but at an optimum concentration of 50 mm. Cytochrome c₂ and dichlorophenol indophenol reduced the optimum of the ascorbate concentration to 7 mm. This might indicate that the effect of ascorbate is mediated through cytochrome c₂. Washing the heavy chromatophores caused 70% loss of the light-induced electron transport from ascorbate and from ascorbate-reduced dichlorophenol indophenol to O₂. However, this effect was only observed with the lower concentrations of ascorbate and the dye. The activity was restored either by the supernatant obtained from the washing or by various c-type cytochromes, reduced by ascorbate. Washing the heavy chromatophores did not affect succinate oxidation in the dark. It is suggested that cytochrome c₂ is one of the cytochromes catalyzing the photosynthetic cyclic electron transport, as has been seen from its high specificity in the reconstitution experiments. Light can induce oxidation of various c-type cytochromes and other redox reagents. However, reduction was specific for cytochrome c₂ from Rps. capuslata, since it was the only one which could be both reduced and oxidized as required from a component which is part of a cyclic electron transport chain. It is also suggested that cytochrome c₂ was not part of the succinate oxidase system.     

Observations on Light-Induced Oxidation Reactions in the Electron Transport System of Chromatium

Light-induced cytochrome oxidations in Chromatium subchromatophore particles were studied in detail. These reactions were found to be dependent not only on redox potential, but also on the efficiency of coupling of the redox buffer electrons to the cytochrome system. Light-induced oxidation of the high potential cytochrome (c-556) was dependent on (a) the availability of reduced cytochrome and (b) the rate of light-induced oxidation (as determined by light intensity) vs. rate of cytochrome rereduction. Chromatium high potential iron-sulfur protein (“HiPISP”) enhanced the rate of c-556 rereduction by mediating electron flow from artificial redox buffers to c-556. In these experiments, the light-induced oxidation of the low potential cytochrome (c-552.5) is dependent not only on the above parameters, but also on the rate of oxidation of the primary electron acceptor X. The interactions of purified Chromatium cytochromes with the light-induced cytochrome oxidation system are discussed.     

Evidence for a dual role of cytochrome c-553 and plastocyanin in photosynthesis and respiration of the cyanobacterium,Anabaena variabilis

The cytochrome oxidase activity (oxygen uptake in the dark) of a membrane preparation from Anabaena variabilis was found to be stimulated by cytochrome c-553 and plastocyanin obtained from this alga. Cytochrome c from horse heart was as active as cytochrome c-553, whereas little or no stimulation of oxygen uptake was obtained with cytochromes c ₂ from two Rhodospirillaceae, the plastidic cytochrome c-552 from Euglena, and plastocyanin from spinach. Cytochrome c-553 (A. variabilis) stimulated photosystem 1 activity in the same preparation much more than cytochrome c (horse heart). The results indicate that cytochrome c-553 and plastocyanin, besides their established function as electron donors of photosystem 1, participate in respiratory electron transport as reductants of a terminal oxidase. Photooxidation and dark oxidation show a different donor specificity.Abbreviations Chl chlorophyll a - TMPD N,N,N,N-tetramethyl-p-phenylenediamine     

Function and properties of a soluble c-type cytochrome c-551 in secondary photosynthetic electron transport in whole cells of Chromatium vinosum as studied with flash spectroscopy

Changes in the absorption spectrum induced by 10-μs flashes and continuous light of various intensities were studied in whole cells of Chromatium vinosum.This paper describes the role and function of a soluble c-type cytochrome, c-551, which was surprisingly found to act in many ways similar to the cytochrome c-420 in Rhodospirillum rubrum, described in a previous paper [1].After the photooxidation of the membrane bound high potential cytochrome c-555 by a 10-μs flash, (the low potential cytochrome c-552 was kept permanently in the oxidized state) the oxidation of c-551 is observed ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.3}\text{ms}$). From a careful analysis of the absorbance difference spectrum and the kinetics it is concluded that there is approximately 0.6–0.7 c-551 per reaction center and that essentially all the c⁺-555 is reduced via the cytochrome c-551. The oxidized-reduced difference spectrum of c-551 shows peaks at 551 and 421.5 nm. The reduction of c⁺-551 following the flash-induced oxidation is strongly inhibited by HOQNO, but only slightly by antimycin A.Cytochrome c-551 reduces only the oxidized high potential cytochrome c-555, which is probably located on the outside of the membrane, on the opposite side of the primary acceptor. The low potential cytochrome c-552 does not show any detectable interaction with cytochrome c-551. After the cells have been sonicated, no c-551 is photooxidized and at least part of the cytochrome occurs in the solution.Analysis of the reduction kinetics of c⁺-551 in the absence and presence of external donors suggests that c⁺-551 is partly reduced via a cyclic pathway, which is blocked by addition of o-phenanthroline, and partly via a non-cyclic pathway. The non-cyclic reduction rate of c⁺-551 (k = 6 s^?1) is increased approximately 5–10 times upon thiosulphate addition, suggesting a role for c-551 between the final donor pool and the oxidized membrane bound c-type cytochromes.     

Polarographic studies on ubiquinone-10 and rhodoquinone bound with chromatophores from Rhodospirillum rubrum.

Redox components bound with chromatophores of Rhodospirillum rubrum, and pure samples of ubiquinone-10 and rhodoquinone were studied polarographically at 24 degrees. In a mixture of ethanol and water (4 : 1, v/v) at pH 7, ubiquinone-10 and rhodoquinone had half-wave potentials (E1/2) OF +43 MV and -63 mV, respectively. For both quinones, values of the electron transfer number (n) were 2 , and plots of E1/2 versus pH formed straight lines with slopes of -30 mV/pH in the neutral pH range; thus, values of the proton transfer number (n-a) were estimated to be 1 for both quinones. When bound with chromatophores, ubiquinone-10 and rhodoquinone had E1/2 values of +50 mV (n=2) and -30 mV (n=2), respectively, at pH 7. Values of (n-a) were estimated to be 1 for ubiquinone-10 and 2 for rhodoquinone. A component (POC-170) thought to be one of the active center bacteriochlorophylls (Liac-890) was characterized; it has E1/2 value of -170 mV at pH 7 and its oxidation-reduction is possibly brought about by dehydrogenation-hydrogenation. Conceivably, the oxidation-reduction sites of ubiquinone-10, rhodoquinone and POC-170 partly, if not all, exist on the surface of chromatophore membrane or project outside the membrane, because of their accessibility to the polarographic electrode.     

Electron transport in the membrane of lutoids from the latex ofHevea brasiliensis

1. An antimycin-insensitive NADH-cytochromec oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex ofHevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH.2. Twob-type cytochromes are present in the membranes. Cytochromeb₅₆₃ is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 °K. A second cytochrome, cytochromeb₅₆₁, seems to be reducible by hydrosulfite only.3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochromeP-450 could not be demonstrated.4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

Temperature dependence of horse heart cytochromec oxidation by membrane preparations ofAnacystis nidulans: Characterization of two distinct arrhenius discontinuities

The oxidation of reduced horse heart cytochromec by membranes isolated from the cyanobacteriumAnacystis nidulans after growth at different temperatures was studied between 4°C and 41°C in the light and the dark using both spectrophotometric and polarographic techniques. Arrhenius plots of the temperature dependence of cytochromec photooxidation showed a single discontinuity at 25°C, 15°C, and 12°C in membranes derived from cells grown at 40°C, 30°C, and 25°C, respectively. By contrast. Arrhenius plots of the temperature dependence of dark respiratory cytochromec oxidation always displayed two distinct breaks at 25 and 18°C, 15 and 8.5°C, and 12 and 5.5°C in membranes isolated from cells grown at 40°C, 30°C, and 25°C, respectively. The results are discussed in terms of the thermotropic lipid-phase transitions known to take place in the membranes ofA. nidulans. Special reference will be made to possibly distinct localizations of the membrane-bound cytochromec oxidase complexes in respiration and photosynthesis.