Cytochrome b and photosynthetic sulfur bacteria

Chromatophores isolated from the purple sulfur bacterium Chromatium and the green sulfur bacterium Chlorobium exhibit absorbance changes in the cytochrome -band region consistent with the presence of a b-type cytochrome. Cytochrome content determined by reduced minus oxidized difference spectra and by heme analysis suggests that each bacterium contains one cytochrome b per molecule of photochemically active bacteriochlorophyll (reaction-center bacteriochlorophyll).

The b-type cytochrome in Chromatium has an -band maximum at 560 nm and a midpoint oxidation-reduction potential of −5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an -band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near −90 mV.

Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na₂S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria.     

The respiratory chain of plant mitochondria. XV. Equilibration of cytochromes c549, b553, b557 and ubiquinone in mung bean mitochondria: Placement of cytochrome b557 and estimation of the midpoint potential of ubiquinone

1. 1. Cycles of oxidation followed by reduction at pH 7.2 have been induced in uncoupled anaerobic mung bean mitochondria treated with succinate and malonate by addition of oxygen-saturated medium. Under the conditions used, cytochromes b₅₅₇, b₅₅₃, c₅₄₉ (corresponding to c₁ in mammalian mitochondria) and ubiquinone are completely oxidized in the aerobic state, but become completely reduced in anaerobiosis.

2. 2. The time course of the transition from fully oxidized to fully reduced in anaerobiosis was measured for cytochromes c₅₄₉, b₅₅₇, and b₅₅₃. The intramitochondrial redox potential (IMP_h) was calculated as a function of time for each of the three cytochromes from the time course of the oxidized-to-reduced transition and the known midpoint potentials of the cytochromes at pH 7.2. The three curves so obtained are superimposable, showing that the three cytochromes are in redox equilibrium under these conditions during the oxidized-to-reduced transition.

3. 3. This result shows that the slow reduction of cytochrome b₅₅₇ under these conditions, heretofore considered anomalous, is merely a consequence of its more negative midpoint potential of +42 mV at pH 7.2, compared to +75 mV for cytochrome b₅₅₃ and +235 mV for cytochrome c₅₄₉. Cytochrome b₅₅₇ is placed on the low potential side of coupling site II and transfers electrons to cytochrome c₅₄₉ via the coupling site.

4. 4. The time course of the transition from fully oxidized to fully reduced was also measured for ubiquinone. Using the change in intramitochondrial potential IMP_h with time obtained from the three cytochromes, the change in redox state of ubiquinone with IMP_h was calculated. When replotted as IMP_h versus the logarithm of the ratio (fraction oxidized)/(fraction reduced), two redox components with n = 2 were found. The major component is ubiquinone with a midpoint potential E_m7.2 = + 70 mV. The minor component has a midpoint potential E_m7.2 = − 12 mV; its nature is unknown.

Shifts of bacteriochlorophyll and carotenoid absorption bands linked to cytochrome c-555 photooxidation in Chromatium

Shifts in the absorption bands of bacteriochlorophyll and carotenoids in Chromatium vinosum chromatophores were measured after short actinic flashes, under various conditions. The amplitude of the bacteriochlorophyll band shift correlated well with the amount of cytochrome c-555 that was oxidized by P₈₇₀⁺ after a flash. No bacteriochlorophyll band shift appeared to accompany the photooxidation of P₈₇₀ itself, nor the oxidation of cytochrome c-552 by P₈₇₀⁺. The carotenoid band shift also correlated with cytochrome c-555 photooxidation, although a comparatively small carotenoid shift did occur at high redox potentials that permitted only P₈₇₀ oxidation.

The results explain earlier observations on infrared absorbance changes that had suggested the existence of two different photochemical systems in Chromatium. A single photochemical system accounts for all of the absorbance changes.

Previous work has shown that the photooxidations of P₈₇₀ and cytochrome c-555 cause similar changes in the electrical charge on the chromatophore membrane. The specific association of the band shifts with cytochrome c-555 photooxidation therefore argues against interpretations of the band shifts based on a light-induced membrane potential.     

Study of electrogenic electron transfer steps in chromatophore membrane of Chromatium vinosum by the response of merocyanin dye

1. Electrogenic steps in photosynthetic cyclic electron transport in chromatophore membrane of Chromatium vinosum were studied by measuring absorption changes of added merocyanin dye and of intrinsic carotenoid.

2. The change in dye absorbance was linear with the membrane potential change induced either by light excitation or by application of diffusion potential by adding valinomycin in the presence of K⁺ concentration gradient.

3. It was estimated that chromatophore membrane became 40–60 mV and 110–170 mV inside positive upon single and multiple excitations with single-turnover flashes, respectively, from the responses of the dye and the carotenoid.

4. Electron transfers between cytochrome c-555 or c-552 and reaction center bacteriochlorophyll dimer (BChl₂) and between BChl₂ and the primary electron acceptor were concluded to be electrogenic from the redox titration of the dye response.

5. No dye response which corresponded to the change of redox level of cytochrome b was observed in the titration curve. Addition of antimycin A slightly decreased the dye response.

6. The dye response was decreased under phosphorylating conditions.

7. From the results obtained localization of the electron transfer components in chromatophore membrane is discussed.     

Spectral and photochemical properties of subchromatophore fractions derived from carotenoid-deficient Chromatium by Triton treatment

Triton treatment of chromatophores of carotenoid-deficient Chromatium followed by density-gradient centrifugation led to a separation into three subchromatophore fractions. Unlike the case with chromatophores of regular Chromatium, Triton releases about 1/3 of the total bulk bacteriochlorophyll into one fraction (designated G, for green) whose major absorption-band maximum is at 780 nm. One fraction (H, for heavy) absorbs at 805 and 885 nm, with an absorbance ratio A₈₈₅ nm/A₈₀₅ nm between 1.5 and 2; another fraction (L, for light) absorbs at 805 nm and has a shoulder at 825 nm. The absorption and fluorescence emission spectra of the three fractions at room temperature and 77°K indicate that the different bacteriochlorophyll forms are efficiently separated by Triton treatment.

The reaction center P890 is concentrated exclusively in the H-fraction, at a level of 5–7% of the bulk bacteriochlorophyll. The solubilized bacteriochlorophyll absorbing at 780 nm can be totally and irreversibly bleached by 5 mM ferricyanide. The other bacteriochlorophyll forms in the H- and L-fractions are also irreversibly bleached by ferricyanide to variable extents. P890 is the only component that can be re-reduced by ascorbate after ferricyanide oxidation. The P890 content estimated by reversible chemical bleaching agrees well with that obtained by reversible light bleaching. The different bacteriochlorophyll forms, with the exception of the 780-nm absorbing form, are relatively stable toward light bleaching. Again, only P890 is reversibly bleached by light.

Cytochromes-555 and -553 are distributed in both the H-and L-fractions, but not in the solubilized-bacteriochlorophyll G-fraction. However, only cytochromes in the H-fraction which contains all of the P890 can undergo coupled oxidation. Excitation with 20-nsec ruby-laser pulses shows that cytochrome-555 can be oxidized in 2–3 μsec by photooxidized P890, indicating that necessary conformation for rapid electron transport is retained in the subchromatophore particles.

The data on fractionation and redox reactions obtained here, together with direct kinetic measurements recently reported in the literature lend further support to the view that oxidation of these two cytochromes is mediated by the same reaction center, P890.     

Properties of a cytochrome c-enriched light particulate fraction isolated from the photosynthetic bacterium Rhodopseudomonas spheroides.

Differential centrifugation of suspensions of French-press-disrupted Rhodopseudomonas spheroides yielded a light particulate fraction that was different in many properties from the bulk membrane fraction. It was enriched in cytochrome c and had a low cytochrome b content. When prepared from photosynthetically grown cells this fraction had a very low specific bacteriochlorophyll content. The cytochrome c of the light particles differed in absorption maxima at 77K from cytochrome c2 attached to membranes; there was pronounced splitting of the alpha-band, as is found in cytochrome c2 free in solution. Potentiometric titration at A552--A540 showed the presence of two components that fitted an n = 1 titration; one component had a midpoint redox potential of +345mV, like cytochrome c2 in solution, and the second had E0' at pH 7.0 of +110 mV, and they were present in a ratio of approx. 2:3. Difference spectroscopy at 77K showed that the spectra of the two components were very similar. More of a CO-binding component was present in particles from photosynthetically grown cells. Light membranes purified by centrifugation on gradients of 5--60% (w/w) sucrose retained the two c cytochromes; they contained no detectable succinate-cytochrome c reductase or bacteriochlorophyll and very little ubiquinone, but they contained NADH-cytochrome c reductase and some phosphate. Electrophoresis on sodium dodecyl sulphate/polyacrylamide gels showed that the light membranes of aerobically and photosynthetically grown cells were very similar and differed greatly from other membrane fractions of R. spheroides.     

The assignment of the 655 nm spectral band of cytochrome oxidase

The spectral characteristics of the ‘655 nm’ band of cytochrome oxidase were found to be affected by ligands of the binuclear centre, including formate and chloride, and by the resting/pulsed transition. The band titrated with near n=1 characteristics at a midpoint of about 400 mV, in contrast to haem a₃, which exhibits strong redox interaction and a titration range at significantly lower potential. Thus, although the total reduced-oxidised difference spectrum of haem a₃, shows a trough at about 655 nm, this characteristic is absent in the low potential region. The 655 nm feature may arise from a charge transfer band of ferric high-spin haem a₃, which is modulated by the redox state of Cu_B, as suggested by Beinert et al. [(1976) Biochim. Biophys. Acta 423, 339–355].     

Thermodynamic properties of the photochemical reaction center of Heliobacterium chlorum

The thermodynamic and spectral properties of the photochemical reaction center components of Heliobacterium chlorum have been examined. The primary electron donor bacteriochlorophyll has E_m,7 = +225 mV, and the ‘primary acceptor’ E_m,10 = −510 mV. The former has an EPR signal in its oxidised form near G = 2.0025, ΔH = 0.95 mT, reminiscent of the properties of the primary donor in bacteria containing bacteriochlorophyll a. The ‘primary acceptor’ has properties similar to those of the iron-sulfur cluster acceptors of green sulfur bacteria. H. chlorum contains a c-type cytochrome (E_m,7 = +170 mV) that donates electrons to the photooxidised primary donor with . The reaction center of H. chlorum is thus very similar to that found in representative green sulfur bacteria, but the cellular architecture and photopigments of this group are quite distinct from those of H. chlorum.     

Cytochrome oxidase and its derivatives. VIII. Formation and properties of the ‘oxygenated’ from of cytochrome oxidase and its reconversion to ferrous and ferric oxidase

1. The ‘oxygenated’ compound of cytochrome c oxidase used in our experiments is more stable than the compound of previous reports. It is quantitatively reversible to ferrous oxidase.

2. It is best formed with an excess of O₂ after reduction with a minimum amount of dithionite. It can also be formed at low O₂ tension, but then contains some ferric oxidase.

3. Its formation from ferrocyanide-reduced oxidase remains incomplete and subsequent reduction by dithionite is also incomplete.

4. Cyanide does not inhibit its formation from ferrous oxidase. If only ferricytochrome a but no ferricytochrome a₃ is reduced in the presence of cyanide by dithionite, there is no reaction with O₂.

5. The anaerobic reduction of ‘oxygenated’ oxidase by dithionite is monophasic and fast. In contrast, that of ferric oxidase is biphasic, with an initial fast reduction of ferricytochrome a followed by a much slower reduction of ferricytochrome a₃. The rate of cytochrome a, but not that of cytochrome a₃ reduction depends on dithionite concentration.

6. In the presence of dissolved O₂, the ferric oxidase reduction comes to a temporary standstill when one-third of the absorbance increase at 444 mμ has been reached.

7. Ethyl hydrogen peroxide reacting with ferrous oxidase forms a compound similar to the ‘oxygenated’ compound.

8. Hydrogen donors known to react with peroxidase-H₂O₂ complexes, particularly pyrogallol, accelerate the transformation of ‘oxygenated’ to ferric oxidase, though not at a rate comparable to that of cytochrome c.

9. These results strengthen the evidence for cytochromes a and a₃ but indicate that this difference has disappeared in ‘oxygenated’ oxidase.     

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.     

Function of three cytochromes in photosynthesis of whole cells of Rhodospirillum rubrum as studied by flash spectroscopy. Evidence for two types of reaction center.

1. Changes in the absorption spectrum induced by 10-mus flashes and continuous light of various intensities were studied in whole cells of Rhodospirillum rubrum in the presence and absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide(HOQNO) and antimycin A. 2. Three cytochromes, c-420 (cytochrome c2), c-560 (cytochrome b) and c-428 were photoactive and gamma and alpha peaks at 420 and 550, 428 and 560, and 428 and 551 nm, respectively; they were photooxidized following the flash with half times of 0.3, 0.6 and 7 ms in the approximate ratios of 1/100, 1/300 and 1/1000 (cytochrome oxidized/antenna chlorophyll) and became reduced with half times of 12 ms, 60 ms and 0.7 s, respectively. c-428 and c-560 have not been distinguished before. 3. From a detailed analysis of the kinetics of P+ (oxidized reaction center chlorophyll) and the cytochromes, we conclude that 5% of the P+ (P2+) oxidizes c-428, whereas the remaining 95% of P+ (P1+) oxidizes c-420. At actinic light intensities low enough to keep c-420 fully reduced, approx. 4-5% of P becomes oxidized, accompanied by all c-428. The P2+ -P2 difference spectrum induced by this weak light is, when corrected for a shift to longer wavelengths of the bacteriochlorophyll absorption band at 878 nm, identical to the difference spectrum caused by the photooxidation of the remaining P1. At low flash intensity, c-428 becomes preferentially photooxidized, which suggests that the reaction centers where c-428 functions as a secondary donor contain much more antenna pigments compared to the centers where c-420 serves this purpose. 4. c+-420 is reduced in a competitive way by reduced c-560 (t 1/2=7 ms), and by an electron donor pool, (t 1/2=15 ms). HOQNO inhibits both pathways; antimycin A only the first. In the presence of HOQNO, c-560 is in the oxidized state in the dark, and is reduced in a light flash (t 1/2=100 ms), indicating that c-560 acts in a cyclic electron transport chain connected to P1. 5. The ratio of numbers of molecules P1 and antenna bacteriochlorophyll, transferring excitation energy to P1, is P1/bacteriochlorophyll1=1/30 P2: bacteriochlorophyll2=1/300; c-420/P1=1:2; c-560/P1=1/6; C-428/P2=1/1; bacteriochlorophyll2=7:3. If P2 is oxidized, excitation energy is transferred from bacteriochlorophyll2 to bacteriochlorophyll1.     

Blue and red shifts of bacteriochlorophyll absorption band around 880 nm in Rhodospirillum rubrum

The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360–410 mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores.

The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C₃₄₀) which has an oxidation-reduction midpoint potential close to 340 mV (n = 1) at pH 6.0–7.6. The E_m of this component varies by 60 mV/pH unit between pH 7.6 and 9.2.

The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C₃₄₀.     

Membranes of Rhodopseudomonas sphaeroides. VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes.

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromotophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (Em = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

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.     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Qx absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect. 2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the alpha-band (lambda max at 551--551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c2 (lambda max at 549.5 +/- 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed. 3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b50 appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b-90 was observed. 4. Difference spectra attributed to (BChl)2, (Formula: see text), c type cytochrome and cytochrome b50 were resolved in the Soret region for Rhodopseudomonas capsulata. 5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.     

Rapid scan spectrometry: oxidation of substrates by coupled potato mitrochondria

By rapid scan spectrometry, spectral changes of redox components have been followed during the oxidation of substrates by coupled potato ( Solanum luberosum L. cv . Bintje) mitochondria under different redox conditions. The redox changes of the b cytochromes, the changes in the base line level and in the slope of the spectra during aerobiosis appear to be connected with changes of the membrane potential. As the latter does not collaps at anaerobiosis, cytochrome o₃ remains in an oxidized state. Collapse of the membrane potential by uncouplers induces partial oxidation of the b cytochromes and reduction of cytochrome a ₃, so that toe resulting redox states derive from thermodynamic equilibrium with the redox conditions of the substrates. However, in aerobiosis as well as in anaerobiosis, the amplitude changes of the b cytochromes vary with the substrate oxidized as if these components were not always completely connected with the cytochrome oxidase complex. The b cytochromes become fuliy reduced only after dithionite reduction.     

Energy transduction 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 (+355 mV); 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 CO-difference spectra and with an 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 “o” and considered the alternative CO-sensitive oxidase.     

Half reduction potentials and oxygen affinity of the cytochromes of Pseudomonas carboxydovorans

Abstract The midpoint redox potentials (E'₀) of the cytochromes of Pseudomonas carboxydovorans have been studied by means of coupled spectrum deconvolution and potentiometric analysis. Membranes of cells grown on different substrates (CO; H₂+ CO₂; or pyruvate) contained cytochromes with similar absorption peaks and redox potentials. The cytochromes of the CO-sensitive main electron pathway of the respiratory chain revealed redox potentials in the same range as mitochondrial cytochromes (cytochrome b -555, about −20 mV; cytochrome c and cytochrome a , about +220 mV). For the cytochromes of the CO-insensitive alternative electron pathway, which allows uninhibited growth and respiration in the presence of high concentrations of CO, redox potentials of approx. +50 mV (cytochrome b -558) and −11 to −215 mV (cytochrome b -561) were determined. Cytochrome [ib-561], earlier proposed as the alternative terminal oxidase o in this organism, was shown to possess the lowest half reduction potential of all the cytochromes present in the cells. Measurements of the apparent K _m value for oxygen revealed a low affinity of cytochrome a ( K _m/ 5 υ M O₂) and a very high affinity of the CO-insensitive oxidase ( K _m < 0.5 μ M O₂). The high affinity to oxygen might be responsible for the CO-insensitivity of this unusual cytochrome o .     

Photochemical activities and photosynthetic ATP formation in membrane preparation from a facultative methylotroph,Protaminobacter ruber strain NR-1

Membrane preparation from the bacteriochlorophyll-containing cells of a facultative methylotroph, Protaminobacter ruber strain NR-1, contained reaction center bacteriochlorophyll similar to those in many species of purple bacteria and contained a few cytochrome species. -Peak of the reduced-minus-oxidized difference spectrum of one of the cytochromes was at 554 nm. The midpoint potential of the cytochrome at pH 7 (E_m7) was 350 mV. Two other cytochromes had the same reduced-minus-oxidized difference spectra with a split -band at 557 and 566 nm, but had two different E_m7s' of 130 mV and 0 mV.On flash or continuous light the reaction center bacteriochlorophyll and the cytochrome with -peak at 554 nm were reversibly oxidized. Redox titration of the light-induced cytochrome oxidation gave an E_m7 value of 356 mV. Under continuous illumination the membrane preparation reversibly took up protons, and formed ATP in the presence of ADP and inorganic phosphate. The ATP formation activity on the bacteriochlorophyll basis was one-third to one-fifth that in chromatophores from Rhodospirillum rubrum under similar experimental conditions. These results clearly indicated that the membrane preparation from P. ruber which contained bacteriochlorophyll had a cyclic photosynthetic electron transfer system and coupled ATP formation activity.Abbreviations Bchl (only in figure legends) bacteriochlorophyll - CCCP carbonylcyanide-m-chlorophenylhydrazone - E_h the ambient redox potential - E_m7 the midpoint potential at pH 7 - PMS N-methylphenazonium methosulfate - MES morpholinoethanesulfonic acid - MOPS morpholinopropanesulfonic acid     

The pH dependence of the oxidation-reduction midpoint potential of cytochromes c2 in vivo.

A recent report by Pettigrew et al. [Biochim, Biophys. Acta 430, (1976), 197-208] has examined the pH dependence of the oxidation-reduction midpoint potential of cytochromes c2 in vitro. In media of low ionic strength, these workers identified several pKs on the oxidized forms of the cytochromes, and in some cases there were also pKs on the reduced species. In this work we examine the pH dependence of the midpoint potentials of the cytochromes in situ, attached to the chromatophore membrane. Under these conditions no pK values are detected, and we conclude that in vivo there is no net change in the protonation of cytochrome c2 during oxidation or reduction.