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.

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.     

Physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus

The nature and number of physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus have been probed by deleting the genes for cytochromes c₁ and b of the cytochrome bc₁ complex, alone or in combination with deletion of the gene for cytochrome c₂. Deletion of cytochrome c₁ renders the organism incapable of photosynthetic growth, regardless of the presence or absence of cytochrome c₂, because in the absence of the bc₁ complex there is no cyclic electron transfer, nor any alternative source of electrons to rereduce the photochemically oxidized reaction center. While cytochrome c₂ is capable of reducing the reaction center, there appears no alternative route for its rereduction other than the bc₁ complex. The deletion of cytochromes c₁ and c₂ reveals previously unrecognized membrane-bound and soluble high potential c-type cytochromes, with E_m7 = + 312 mV and E_m6.5 = +316 mV, respectively. These cytochromes do not donate electrons to the reaction center, and their roles are unknown.     

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.     

Oxidoreduction of cytochrome b in the presence of antimycin

1. The effect of oxidizing equivalents on the redox state of cytochrome b in the presence of antimycin has been studied in the presence and absence of various redox mediators.

2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site.

3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558.

4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant.

5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14.

6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c₁ (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH₂/QH^• couple and reduction of cytochrome b.     

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.     

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.     

Antimycin-insensitive mutants of Candida utilis. II. The effects of antimycin on cytochrome b

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutant. A similar reoxidation is observed in the wild type in the presence of low concentrations of antimycin.

2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steadystate reduction; reduction in the presence of substrate, cyanide and oxygen; the ‘red shift’ and lowering of E′₀ of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable.

3. The red shift in the mutant is more extensive than in the wild type.

4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes.

5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant.

6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH₂-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycinbinding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Cytochrome b-560, a new component of thylakoid membranes

During a survey of a series of electron transport mutants of Chlamydomonas reinhardtii, we have observed a hitherto unrecognised component, cytochrome b-560, in a strain (F18) which entirely lacks the cytochrome bf complex. This component is present at approx. 1 nmol/mg chlorophyll, has a midpoint potential of −125 mV (n = 1) at pH 7.2 and is slowly reduced by dithionite. The cytochrome was also observed in thylakoid membranes prepared from wild-type Chlamydomonas and from higher plants after extraction of the cytochrome bf complex by detergent treatment. It could not be observed in thylakoid membrane fragments prepared from the cyanobacterium Phormidium laminosum.     

Reducibility of cytochromes b in aerobic beef-heart mitochondria treated with antimycin

Under anaerobic conditions cytochrome b in beef-heart mitochondria is partially reduced in the presence of NADH, whereas other cytochromes are completely reduced. Addition of antimycin together with oxygen under these conditions causes an immediate reduction of cytochromes b-558, b-562 and b-566 and oxidation of cytochrome c. During the subsequent transient aerobic steady state cytochromes b-558 and b-566 are rapidly re-oxidized without changes in redox state of cytochrome c, but cytochrome b-562 remains reduced. When oxygen is consumed by the leak through or around the antimycin-inhibition site, cytochrome b-562 becomes oxidized with concomitant reduction of cytochrome c.

The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.

It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566.     

The b-type cytochromes of bovine heart mitochondria: Absorption spectra, enzymatic properties, and distribution in the electron transfer complexes

1. 1. Three b-type cytochromes (b_557.5, b₅₆₀, and b_562.5), plus a chromophore with an absorption peak at 558 nm at 77 °K, have been found to be associated with the electron transport system of bovine heart mitochondria. The reduced minus oxidized spectra of these components at 77 °K, as well as that of cytochrome c₁, have been recorded with a wavelength accuracy of ± 0.1 nm and presented to the nearest 0.5 nm. All the major and β absorption peaks of cytochromes b_557.5, b₅₆₀, b_562.5, c₁ and c have been shown by fourth derivative analysis to be present in the dithionite-reduced minus oxidized spectra of mitochondria and submitochondrial particles.

2. 2. The distribution of the above components has been studied in the four electron transfer complexes of the respiratory chain. Cytochromes b₅₆₀, b_562.5 and c₁, as well as chromophore-558, were found to fractionate into Complex III (reduced ubiquinone-cytochrome c reductase), whereas cytochrome b_557.5 was found in Complex II (succinate-ubiquinone reductase).

3. 3. Cytochrome b₅₆₀ was readily reduced by NADH or succinate, but b_562.5 was not reduced by substrates unless the preparation was treated with antimycin A. In antimycin-treated preparations pre-reduction of c₁ with ascorbate inhibited the subsequent reduction of b_562.5 by substrates. These results indicate that b₅₆₀ and b_562.5 correspond, respectively, to b_K and b_T previously described by Chance et al.¹⁴ (1970, Proc. Natl. Acad. Sci. U.S. 66, 1175–1182).

4. 4. Similar to b₅₆₀, chromophore-558 can be reduced by substrates in the absence or presence of antimycin A. However, in antimycin-treated preparations, pre-reduction of c₁ inhibits its subsequent reduction by substrates. This property is similar to that of b_562.5.

5. 5. Cytochrome b_557.5, which occurs in Complex II, appears to have a low mid-point potential. It can be reduced with dithionite and oxidized by fumarate or ubiquinone. CO treatment of dithionite-reduced b_557.5 neither modified the spectrum of this cytochrome nor diminished the extent of b_557.5 reoxidation by fumarate.

6. 6. Antimycin A treatment does not appear to alter the spectra of the above cytochromes. However, small amounts (< 4%) of ethanol or methanol, which are usually added to particles as solvent for antimycin A, have a pronounced effect on the peaks of cytochrome c₁. The spectrum of cytochrome c₁ at 77 °K as modified by 3% (v/v) ethanol is shown.

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.     

Characterization of a mutant of Schizosaccharomyces pombe lacking cytochrome b-566

1. A chromosomal respiration-deficient mutant of the petite-negative yeast Schizosaccharomyces pombe was isolated. Its mitochondria show respiration rates of about 7% of the wild-type respiration with NADH and succinate as substrate, and 45% with ascorbate in the presence of tetramethyl-p-phenylenediamine. Oxidation of NADH and succinate is insensitive to antimycin and cyanide and that of ascorbate is much less sensitive to cyanide than the wild type.

2. The amounts of cytochromes c₁ and aa₃ are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.

3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH₂-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.

4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.

5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.

6. Cytochromes aa₃, c and c₁ are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.

7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin.     

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.     

Redistribution of electric charge accompanying photo-synthetic electron transport in Chromatium

The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10⁴. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential (E_m) of that carrier should be a function of the solution ionic strength (I). of that carrier should be a function of the solution ionic strength (I).

The E_m values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The E_m of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases.

The E_m values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these E_m values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane.

The E_m of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction.     

Photochemical properties of a Photosystem II subchloroplast fragment

1. The Photosystem II fraction (D-10) obtained by incubation of spinach chloroplasts with digitonin was further purified by incubation with Triton X-100. The resulting Photosystem II subchloroplast fragment (DT-10) contained 1 mole of cytochrome b-559 per 170 moles of chlorophyll. It lacked cytochrome f and cytochrome b₆ and its content of P700 was low.

2. The DT-10 fragment showed only traces of photochemical activity with water as electron donor, but it was active in a Photosystem II reaction with 2,6-dichlorophenolindophenol as electron acceptor and diphenyl carbazide as donor. Photoreduction of NADP⁺ with diphenyl carbazide as donor was negligible. There was some photoreduction of NADP⁺ with ascorbate plus 2,6 dichlorophenolindophenol as donor but this activity could be accounted for by contamination with Photosystem I. These results are consistent with the Z-scheme of photosynthesis with Photosystems I and II operating in series for the reduction of NADP⁺ from water. DT-10 subchloroplast fragments showed a light-induced rise in fluorescence yield at 20 °C in the presence of diphenyl carbazide. A light-induced fluorescence increase also was observed at 77 °K.

3. During the preparation of the DT-10 fragment, the high potential form of cytochrome b-559 was largely converted to a form of lower potential and C-550 was converted to the reduced state. A photoreduction of C-550 was observed at liquidnitrogen temperature, provided the C-550 was oxidised with ferricyanide prior to cooling. Some photooxidation of cytochrome b-559 was obtained at 77 °K if the preparation was reduced prior to cooling, but the degree of photooxidation was variable with different preparations. C-550 does not appear to be identical with the primary fluorescence quencher, Q.

4. Photosystem I subchloroplast fragments (D-144) released by the action of digitonin were compared with Photosystem I fragments (DT-144) released from D-10 fragments by Triton X-100. There were no significant differences between D-144 and DT-144 fragments either in chlorophyll a/b ratio or in P700 content.     

Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature

1. Light-induced absorbance changes of cytochrome b-559 and cytochrome f in the -band region were examined in leaves and in isolated chloroplasts.

2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.

3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.

4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I.     

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.     

Coulometric and potentiometric evaluation of the redox components of cytochrome c oxidase in situ

A new coulometric-potentiometric titration cuvette is described which permits accurate measurements of oxidation-reduction components in membranous systems. This cuvette has been utilized to measure the properties of cytochrome c oxidase in intact membranes of pigeon breast muscle mitochondria. The reducing equivalents accepted and donated by the portion of the respiratory chain with half-reduction potentials greater than 200 mV are equal to those required for the known components (cytochrome a₃ and the high-potential copper plus cytochrome a, ‘visible copper’, cytochrome c₁, cytochrome c, and the Rieske iron-sulfur protein). Titrations in the presence of CO show that formation of the reduced cytochrome a₃-CO complex requires two reducing equivalents per cytochrome a₃ (coulometric titration). Potentiometric titrations indicate (Lindsay, J.G., Owen, C.S. and Wilson, D.F. (1975) Arch. Biochem. Biophys. 169, 492–505) that both cytochromes a₃ and the high-potential copper must be reduced in order to form the CO complex (n=2.0 with a CO concentration-dependent half-reduction potential, E_m). By contrast, titrations in the presence of azide show that the E_m value of the high-potential copper is unchanged by the presence of azide and thus azide binds with nearly equal affinity whether the copper is reduced or oxidized.     

Photochemical systems in mesophyll and bundle sheath chloroplasts of C4 plants

1. The agranal bundle sheath chloroplasts of Sorghum bicolor possess very low Photosystem II activity compared with the grana-containing mesophyll chloroplasts.

2. Sorghum mesophyll chloroplasts have a chlorophyll (chl) and carotenoid composition similar to that of spinach chloroplasts. In contrast, the sorghum bundle sheath chloroplasts have a higher chl a/chl b ratio; they are enriched in β-carotene and contain relatively less xanthophylls as compared to sorghum mesophyll or spinach chloroplasts.

3. Sorghum mesophyll chloroplasts with 1 cytochrome f, 2 cytochrome b₆ and 2 cytochrome b-559 per 430 chlorophylls have a cytochrome composition similar to spinach chloroplasts. Sorghum bundle sheath chloroplasts contain cytochrome f and cytochrome b₆ in the same molar ratios as for the mesophyll chloroplasts, but cytochrome b-559 is barely detectable.

4. The chl/P700 ratios of mesophyll chloroplasts of S. bicolor and mesophyll and bundle sheath chloroplasts of Atriplex spongiosa are similar to that of spinach chloroplasts suggesting that these chloroplasts possess an identical photosynthetic unit size to that of spinach. The agranal bundle sheath chloroplasts of S. bicolor possess a photosynthetic unit which contains only about half as many chlorophyll molecules per P700 as found in the grana-containing chloroplasts.

5. The similarity of the composition of the bundle sheath chloroplasts of S. bicolor with that of the Photosystem I subchloroplast fragments, together with their smaller photosynthetic unit and low Photosystem II activities suggests that these chloroplasts are highly deficient in the pigment assemblies of Photosystem II.