Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26

Cytochrome c₁ from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150 000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30 000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c₁, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c₁ is more exposed than is that of mammalian cytochrome c₁, but less exposed than that of cytochrome c₂. Ferricytochrome c₁ undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c₁, suggesting that the sulfhydryl groups of cytochrome c₁ are the electron donors for photoreduction. Purified cytochrome c₁ contains 3 ± 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c₁, the bacterial protein does not form a stable complex with cytochrome c₂ or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c₁ and bacterial ferricytochrome c₂, and between bacterial ferrocytochrome c₁ and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c₁ is calculated to be 228 mV, which is identical to that of mammalian cytochrome c₁.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

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

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

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.     

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.     

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.     

Photooxidative reactions of c-phycocyanin

1. Aging cultures of blue-green algae, as well as cultures kept under certain adverse physiological conditions, often show bleaching of photosynthetic pigments.

2. Crude extracts containing c-phycocyanin of the blue-green algae, Plectonema boryanum or Anacystis nidulans, bleach when exposed to short wavelengths of visible light. Purified preparations of c-phycocyanin are not light sensitive, but can be sensitized by addition of catalytic amounts of FMN. This reaction proceeds only in the presence of oxygen. FMN and light can be replaced by H₂O₂.

3. When phycocyaninless extracts of the algae are exposed to light, peroxides are rapidly built up. There is an inverse relation between concentration of peroxides and c-phycocyanin in algal cultures grown at various light intensities.

4. A protective function of c-phycocyanin in certain blue-green algae against photooxidative death is suggested.     

Mitochondrial cytochrome components of Paragonimus adult worms

The cytochrome components of adult Paragonimus miyazakii mitochondria were investigated by polyacrylamide gel electrophoresis. The mitochondria were found to contain cytochromes b, c₁, c and aa₃. Two types of mitochondria, lightweight mitochondria (LWMt) and heavyweight mitochondria (HWMt), were obtained by centrifugation from the mitochondrial fraction of the adult Paragonimus ohirai. The succinate-reduced and oxidized difference spectrum of LWMt and HWMt at −196°C revealed that both mitochondria contained at least functional levels of cytochromes b, c₁, c and a low value of aa₃. Although succinate-reduced cytochromes of LWMt reoxidized in the presence of air, those of HWMt did so only minimally.     

Iron protein content of Thiocapsa pfennigii, a purple sulfur bacterium of atypical chlorophyll composition

Four out of five soluble electron transport iron proteins of Thiocapsa pfennigii plus the particulate cytochromes have been found to be analogous to those of Chromatium vinosum strain D. In addition to ferredoxin, high potential iron-sulfur protein. cytochrome c′, and cytochrome c-552(550), T. pfennigii contains a cytochrome c-552(545) not previously isolated from photosynthetic bacteria. It is concluded that T. pfennigii is more closely related to C. vinosum than to Rhodopseudomonas viridis, the only other known bacterial species having bacteriochlorophyll b.     

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

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

The P-700-chlorophyl a-protein complex and two major light-harvesting complexes of Acrocarpia paniculata and other brown seaweeds

Acrocarpia paniculata thylakoids were fragmented with Triton X-100 and the pigment-protein complexes so released were isolated by sucrose density gradient centrifugation. Three main chlorophyll-carotenoid-protein complexes with distinct pigment compositions were isolated.

1. (1) A P-700-chlorophyll a-protein complex, with a ratio of 1 P-700: 38 chlorophyll a: 4 ta-carotene molecules, had similar absorption and fluorescence characteristics to the chlorophyll-protein complex 1 isolated with Triton X-100 from higher plants, green algae and Ecklonia radiata.

2. (2) An orange-brown complex had a chlorophyll a : c₂ : fucoxanthin molar ratio of 2 : 1 : 2. This complex had no chlorophyll c₁ and contained most of the fucoxanthin present in the chloroplasts. This pigment complex is postulated to be the main light-harvesting complex of brown seaweeds.

3. (3) A green complex had a chlorophyll a : c₁ : c₂ : violaxanthin molar ratio of 8 : 1 : 1 : 1. This also is a light-harvesting complex.

The absorption and fluorescence spectral characteristics and other physical properties were consistent with the pigments of these three major complexes being bound to protein. Differential extraction of brown algal thylakoids with Triton X-100 showed that a chlorophyll c₂-fucoxanthin-protein complex was a minor pigment complex of these thylakoids.     

Influence du pH sur la synthèse de l''oxidase (cytochrome a3) chez Bacillus coagulans, microorganisme thermophile, capable d'' “adaptation respiratoire”

In a new series of experiments on Bacillus coagulans (ATCC 11.369), it was demonstrated that this organism possesses a respiratory system with cytochromes b, c₁, c, (a+a₃) and also cytochrome o. A small decrease in the pH of the growth medium from 6.5 to 5.5 increases the respiratory activity by a factor of 4 and induces a variation of the absorption ratio [₆₀₃ (a+a₃)]/[₅₆₀ (b+c)] resulting in a preponderant increase in the ₆₀₃ absorption. The kinetic studies of the respiratory system synthesis during the phenomenon of “respiratory adaptation” have shown that lowering the pH of the adaption medium has the same effect. Spectral studies of membrane fractions (red dithionite) with or without carbon monoxide showed a preferential synthesis of oxidase a₃.     

The reaction of antimycin with a cytochrome b preparation active in reconstitution of the respiratory chain

1. Succinate-cytochrome c reductase activity was reconstituted by incubating a mixture of succinate dehydrogenase, cytochrome c₁, ubiquinone-10, phospholipid and a preparation of cytochrome b, made by the method of .

2. Preparations of cytochrome b active in reconstitution contained 5–28% native cytochrome b, as adjudged by reducibility with succinate in the reconstituted preparation and by lack of reaction with CO. Preparations of cytochrome b containing no native cytochrome b according to this criterion were inactive in reconstitution.

3. With a fixed amount of cytochrome b, the activity of the reconstituted preparation increased with increasing amounts of cytochrome c₁ until a ratio of about 2b (total): 1c₁ (allowing for the cytochrome c₁ present in the cytochrome b preparation) was reached.

4. The amount of antimycin necessary for maximal inhibition of the reconstituted enzyme is a function of the amount of the cytochrome b and is independent of the amount of cytochrome c₁. It is equal to about one half the amount of native cytochrome b.

5. Preparations of intact or reconstituted succinate-cytochrome c reductase or of cytochrome b completely quench the fluorescence of added antimycin, until an amount of antimycin equal to onehalf the amount of native cytochrome b present was added. Antimycin added in excess of this amount fluoresces with normal intensity. The quenching is only partial in the presence of Na₂S₂O₄. Denatured cytochrome b does not quench the fluorescence.

6. Since preparations of cytochrome b active in reconstitution contained cytochrome c₁ in an amount exceeding one half the amount of native cytochrome b present in the preparation, there is no evidence that native cytochrome b has been resolved from cytochrome c₁. The stimulatory action of cytochrome c₁ may be due to the restoration of a damaged membrane conformation.

7. Based on the assumption that the bc₁ segment of the respiratory chain contains 2b:1c₁:1 antimycin-binding sites, the specific quenching of antimycin fluorescence by binding to cytochrome b enables an accurate determination of the absorbance coefficients of cytochromes b and c₁. These are 25.6 and 20.1 mM⁻¹×cm⁻¹ for the wavelength pairs 563–577 nm and 553–539 nm, respectively, in the difference spectrum reduced minus oxidized.     

Rhodobacter capsulatus MT113: A single mutation results in the absence of c-type cytochromes and in the absence of the cytochrome bc1 complex

MT113, a nonphotosynthetic mutant of Rhodobacter capsulatus previously characterized as lacking cytochrome c₂ is shown to lack also cytochrome c₁, the Rieske iron-sulfur cluster and the antimycin sensitive semiquinone Q_c, all components of the cytochrome bc₁ complex. Although MT113 contained b-type cytochromes and other iron-sulfur clusters at nearly wild-type level, it lacks c-type cytochromes. Based on antibody detection, c₂ apoprotein was absent in MT113, however the apoproteins corresponding to the cytochromes b and c₁ and the Rieske iron-sulfur cluster were present in reduced amounts. Genetic analysis indicated that the lesion appears to be due to a single mutation which is not localized in the structural genes of cytochrome c₂ or the bc₁ complex. These data taken together suggest that the pleiotropic mutation in MT113 might be related to the biosynthesis of c-type cytochromes.     

Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+a₃ maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls.

The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent.

Unlike other soluble c-type cytochromes, the maximum of cytochrome c₅₅₅ was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane.

Using cytochrome c₅₅₅-depleted mitochondria, a cytochrome c₁ pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c₅₅₅-depleted preparations exhibited an maximum at 553 nm at room temperature.

The reduced rate of succinate oxidation by cytochrome c₅₅₅-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c₁, even in the presence of antimycin, indicated that cytochrome c₅₅₅-mediated electron transfer between cytochromes c₁ and a+a₃ in a manner analagous to that of cytochrome c in mammalian mitochondria.     

Photostimulation of nitrogen fixation in Anabaena cylindrica

Photostimulation of N₂ fixation in the blue-green alga Anabaena cylindrica was investigated using monochromatic light and by comparing action spectra of C₂H₂ reduction with that of photosynthetic O₂ evolution. Maximum nitrogenase activity per unit energy was measured at a wavelength which corresponds to the maximum light absorption of chlorophyll a. The action spectrum of C₂H₂ reduction indicates a primary involvement of Photosystem I in N₂ fixation by blue-green algae.     

Cyanide-resistant respiration and a branched cytochrome system in Kinetoplastidae

Two functional terminal oxidases, cytochrome aa₃ and cytochrome o, are present in the cytochrome system in cyanide-sensitive trypanosomatids. The organisms examined included Crithidia fasciculata, Leptomonas sp., Blastocrithidia culicis, Herpetomonas muscarum, Leishmania tarentolae, Trypanosoma lewisi, Trypanosoma conorhini and Trypanosoma cruzi. A CO-binding protein with the spectral properties of cytochrome o has been solubilized and partially purified from C. fasciculata. In L. tarentolae, T. conorhini and C. fasciculata, 10–20% of the respiration is cyanide-resistant but is inhibited by salicylhydroxamic acid. The various possibilities of a branched electron transport system are examined and discussed.     

Activation by reduction of the resting form of cytochrome c oxidase: Tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2 · 10⁴ M⁻¹ · s⁻¹. In the absence of cyanide, ferrocytochrome a₃ appears at a rate (k_obs) of 0.016 s⁻¹. Ferricytochrome a₃ maintains its 418 nm Soret maximum until reduced. The rate of a₃ reduction is independent of dithionite concentration over a range 0.9 mM–131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a₃/cyanide complex occurs at the same rate as a₃ reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a₃ with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a₃ shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a₃. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (k_obs ≈ 0.02 s) entry of a third electron. Above +200 mV, the number of electrons taken up in the initial fast phase drops as a redox center (presumably Cu_A) titrates with an apparent mid-point potential of +240 mV. The slow phase of reduction remains at the more positive redox values. (4) The results are interpreted in terms of an initial fast reduction of cytochrome a (and Cu_A at redox values more negative than +240 mV) followed by a slow reduction of Cu_B. Cu_B reduction is proposed to spin-uncouple cytochrome a₃ to form a cyanide sensitive center, and trigger a conformational change to an activated form of the enzyme with faster intramolecular electron transfer.     

Further studies on the NADH oxidase of the cytoplasmic membrane of Mycobacterium tuberculosis

The cytoplasmic membrane of the H₃₇Ra strain of Mycobacterium tuberculosis has been isolated free of cell wall.

These membrane preparations contain very small quantities of cytochromes c, b and cytochrome oxidase. The cytochrome c is not extracted by any method attempted. The cytochrome b is reducible only by dithionite and is believed not to be involved in the direct transfer of electrons during the oxidation of NADH by these preparations. The NADH oxidase activity of the membrane is inhibited by high concentrations of cyanide and also by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide (HQNO). The cytochrome oxidase of the membrane contains both cytochromes a and a₃ and is present in low concentrations relative to cytochrome c. The cytochrome a₃ component was identified by characteristic complexes with both CO and cyanide and shows a γ-band absorption maximum at a slightly lower wavelength than the cytochrome oxidase of mammalian mitochondria (442 nm vs. 445 nm). The functional activity of the cytochrome oxidase is indicated by the inhibition of reoxidation of reduced cytochromes c and a in the presence of cyanide.     

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.