Oxidation-reduction potentials of respiratory chain components in ThiobacillusA2

(1) Cells of ThiobacillusA₂ grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a₃-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (E_m,7.0) and spectral maxima of c-, b-, a- and a₃-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the ‘invisible’, or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a₃ complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.     

Detection of two further b-type cytochromes in Rhodopseudomonas spheroides

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

Spectral and functional characterization of membrane fragments from the facultative photosynthetic bacterium Rhodopseudomonas blastica

The cytochromes of photosynthetically grown Rhodopseudomonas blastica have been thermodynamically characterized using the technique of redox titrations. Six cytochromes were present; two cytochromes c, E _m7= +295mV, E _m7=+345mV; and four cytochromes b, E _m7=+290mV, E _m7=+130mV, E _m7=+60mV, E _m7=-4mV. These cytochromes were tightly bound except for cytochrome c with E _m7 of+345mV which was mostly present in the soluble cell extracts.The effects of cyanide on both the cytochrome c oxidase activity and the NADH-dependent respiration, revealed the presence of a branched respiratory chain, one branch leading to a cyanide-resistant oxidase containing pathway and the other including the cyanide-sensitive cytochrome c-oxidase.The effects of antimycin A, myxothiazol and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) on the steadystate NADH-dependent respiration were also studied. Antimycin A and myxothiazol appeared to act at the level of the ubiquinol-cytochrome c oxidoreductase while UHDBT drastically affected both respiratory branches.Absorption spectra of chromatophore photopigments resulted to be similar to those reported in many species of facultative photosynthetic bacteria although carotenoid absorption maxima were blue-shifted by 5 nm.The light-induced oxygen reduction performed by chromatophores from R. blastica suggested a strict interaction between photosynthetic and respiratory apparatuses.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum. 1. A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells

Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined. Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with E _m,7 of +180, +72 and –5 mV (561–575 nm), and +244 and +27 mV (551–540 nm), respectively. Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with E _m,7 of +60 and –45 mV and +290, +250, +135, –20 and –105 mV, respectively. In addition to haems of the b- and c-type, two haems of the a-type (E _m,7 of +325 and +175 mV) were present only in cells grown in the dark. Four soluble cytochromes of the c type, but not cytochrome c ₂, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically. Inhibitory studies showed that 85–90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively). These results taken together were interpreted to show that the oxidative electron transport chain of Rsp. salinarum is linear, leading to a membrane-bound oxidase of the aa ₃ type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes. These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c ₂ or a branched respiratory chain. Received: 25 February 1997 / Accepted: 20 May 1997     

Spectroscopic and potentiometric characterization of cytochromes in two Sporomusa species and their expression during growth on selected substrates

The homoacetogenic bacteria Sporomusa ovata and Sporomusa sphaeroides were grown on betaine, betaine + formate, and acetoin in the absence of carbon dioxide, and the formation of membrane-bound cytochromes was determined. In S. sphaeroides, the growth substrate had little influence on the expression of cytochromes. In contrast, membranes from betaine-or acetoin-grown S. ovata cells had an 11-or 3-fold higher cytochrome b content than cells grown on betaine + formate. The cytochrome c content was reduced below the detection level after growth on the latter two substrates. The cytochromes in the membranes of S. sphaeroides and S. ovata were characterized by low-temperature difference spectroscopy, hemochrome difference spectroscopy, and redox potentiometry. Membranes of S. ovata were shown to contain two b-type cytochromes with E_m,7=-153±10 mV and E_m,7=-226±14 mV and two c-type cytochromes with E_m,7=-86±6 mV and E_m,7=-265±10 mV. In S. sphaeroides also two b-type cytochromes with E_m,7=-165±7 mV and E_m,7=-241±2 mV and two c-type cytochromes with E_m,7=-101±4 mV and E_{m, 8.5}=-338±9 mV could be distinguished. Cell extracts of S. sphaeroides were shown to contain all the enzymes of the acetyl-CoA (Wood) pathway. The degradation pathways of the substrates tested and the possible role of the cytochromes are discussed.Abbreviations E_m,7 midpoint potential at pH 7 and 25°C - H₄F tetrahydrofolate     

The electron transport system of Alcaligenes eutrophus H16

The electron transport system of autotrophically grown Alcaligenes eutrophus H16 has been investigated by spectroscopic and thermodynamic approaches. The results have been interpreted as evidence that isolated membranes contain a branched respiratory chain composed of three c-type haems (E _m,7=+160 mV, + 170 mV, and + 335 mV), five b-type haems (E _m,7=+ 5 mV, + 75 mV, + 205 mV, + 300 mV, and + 405 mV), two (possibly three) a-type haems [E _m,7= + 255 mV, + 350 mV, (+ 420 mV)], and nne d-type haem. EPR-analysis of the signals at g=1.93, g=2.02, and g=1.90 revealed the presence of iron-sulphur centres diagnostic of complexes I (NADH dehydrogenase), II (succinate dehydrogenase), and III (ubiquinol/cytochrome c oxidoreductase). The low potential b haems (+ 5 mV and + 75 mV) plus the Rieske protein (g=1.90, E _m,7=+ 280 mV), thought to be part of an orthodox bc ₁ complex, were present in low amounts as compared to their counterparts in membranes from Paracoccus denitrificans.CO-difference spectra in the presence of either succinate, NADH, hydrogen, ascorbate/TMPD, and/or dithionite as reductants, suggested the existance of four different oxidases composed by bo-, cb-, a-, and d-type haems.It is concluded that in contrast to other chemolithotrophes, e.g. P. denitrificans, autotrophic growth of Alcaligenes eutrophus utilizes a respiratory system in which the bc ₁ complex containing pathway is only partially involved in electron transport.Abbreviations Cytochrome c-551, number wavelength in nm - Cytochrome c ₂₇₀, number mid-point potential in mV - E _m,7 mid-point potential of an oxidation-reduction couple at pH 7.0 - KP buffer, potassium phosphate-buffer - OD optical density at 436 nm, 1 cm light path - TMPD N,N,N,N-tetramethyl-p-phenylenediamine     

The Cytochromes of Prototheca zopfii

The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a₃. Cytochromes a and a₃ could be resolved spectrally in the α-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a₃ complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a₃, and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).     

The cytochromes of a marine Beggiatoa

Naturally grown Beggiatoa filaments, occurring in massive near-mono-cultures at a “black smoker” wall of the Guaymas Basin hydrothermal vent site, were harvested and used for the analysis of their cytochromes. The cytochromes have been characterized by gel permeation chromatography, optical spectroscopy and redox potentiometry. Only c-type cytochromes were detected; a small, high potential cytochrome c that seems typical of its class, and a large complex (M_r 210,000) containing at least four thermodynamically distinct c-type hemes, which was partially dissociated by chromatography on DEAE-Sepharose. The hemes of the large complex have appropriate oxidation-reduction midpoint potentials (E_m7 +240 mV, +15 mV,-160 mV,-340 mV) to be involved in the metabolism of sulfide, which is presumed to be the source of reductant for this organism.     

A thermodynamic characterisation of the cytochromes of chromatophores from Rhodopseudomonas capsulata

1. The cytochromes of chromatophores from photosynthetically grown Rhodopseudomonas capsulata have been characterised both spectrally, using the carotenoid free mutant Ala Pho⁺, and thermodynamically, using the technique of redox titrations. Five cytochromes were present; two cytochromes b, E′₀ = 60 mV at pH 7.0; and three cytochromes c, E′₀ = 340 mV, $\text{E}\text{t}\text{?}{}_0\text{= 120}\text{mV}$, E′₀ = 0 mV at pH 7.0.2. Redox titrations at different values of pH indicated that the mid point potentials of all the cytochromes varied with pH over some parts of the range between pH 6 and 9, with the possible exception of cytochrome c₃₄₀.3. The effects of succinate and NADH on the steady state reduction of the cytochromes are reported. Succinate could reduce cytochromes c₃₄₀, c₁₂₀ and b₆₀; NADH could reduce cytochromes c₃₄₀, c₁₂₀, b₆₀ and b_?25. Cytochrome c₀ could be reduced by dithionite but not by the other substrates tested.     

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in Complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with α-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, ?75 and 187 mV, respectively. In addition, two very small contributions to the α-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c₁ (Λ_m(25°C) = 553.5 nm; E′₀ = 238 mV) and four cytochromes bΛ_m(25°C) = 558.6, 561.2, 562.1, 566.1 nm and E′₀ = ?83, 26, 85, ?60 mV).     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

Characteristics of b-type cytochromes in brain microsomes: Comparison with liver microsomes

Biochemical aspects of b-type cytochromes in swine cerebral microsomes were different from those of cytochrome b₅ in liver microsomes, as well as the difference in absorption spectra. First, the kinetic constants, K_m and V_max, in rotenone-insensitive NADH-cytochrome c reductase activity were different from those of liver microsomes, and the activity of cerebral microsomes was higher than that of liver microsomes. Second, midpoint potentials (E_m) of b-type cytochromes in cerebral microsomes were measured and compared with liver microsomal cytochrome b₅. In cerebral microsomes two components of b-type cytochromes were resolved, and showed E_m's of ?30 and +50 mV, respectively, in the presence of 2 mm KCN. On the other hand, the E_m of liver microsomal cytochrome b₅ was ?6 mV. The high-potential component of cerebral microsomal b-type cytochromes was identified as brain-b′₅ [S. Yoshida, T. Yubisui, and M. Takeshita (1983)Biochem. Int. 7, 291–298] and the low-potential component as brain-b₅. The significance of the difference between cerebral and liver microsomal b-type cytochromes was discussed.     

Oxidation-reduction potentials and spectral properties of some cytochromes from Thiobacillus versutus (A2)

Cytochromes c-550 (acidic), c-550 (basic), c-551 and c-552.5 from Thiobacillus versutus have been highly purified and characterized. Their spectral properties at 77 K are described. Oxidation-reduction titrations of cytochromes c-550 (acidic) and c-550 (basic) showed them to exhibit Nernst values of n = 1, with single redox centres in the cytochromes, and to have midpoint redox potentials at pH 7.0 (E_m,7) of 290 and 260 mV, respectively. Cytochrome c-551 contained two separately titratable redox components, each giving n = 1. The low potential centre (55% of titratable cytochrome) and the high potential centre (45%) had E_m,7 values of ?115 and +240 mV, espectively. Cytochrome c-552.5 also contained at least two redox centres. One (65% of titratable cytochrome) had n = 1 and E_m,7 = 220mV. The remaining 35% appeared to be a low potential component with an E_m,7 possibly as low as ?215 mV. the roles of these cytochromes in respiratory thiosulphate oxidation are discussed.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.     

Electron-transport chains of phototrophically and chemotrophically grown Chloroflexus aurantiacus

The membrane-bound electron-transfer chain components of both phototrophically and chemotrophically grown Chloroflexus aurantiacus have been characterized. Membranes isolated from chemotrophically grown Chloroflexus have been shown to contain at least three c-type cytochromes and at least three b-type cytochromes. In addition, these cells appear to lack a photochemical reaction center and the high potential (E_m = +260 mV) cytochrome c-554 that serves as the immediate donor to the reaction center in phototrophically grown Chloroflexus. Phototrophically grown cells contain a CO-binding c-type cytochrome, apparently absent in the chemotrophically grown cells. However, a different CO-binding component, which may function as the terminal oxidase, is present in chemotrophically grown cells.     

Resonance Raman resolution of a-, b- and c-type cytochromes in membrane vesicles of alkalophilic bateria

Resonance Raman spectroscopy has been used to obtain complete spectra of each individual cytochrome type — a, b and c — in the reduced state within membrane vesicle preparations from two species of obligately alkalophilic bacteria: Bacillus alcalophilus and Bacillus firmus RAB. The vibrational spectra, in the range 250–1700 cm^?1, were obtained with tunable dye laser excitation in the wavelength range 550–600 nm tuned to resonance with the appropriate reduced alpha band maximum for the cytochrome type of interest. The spectra reveal details which serve to characterize the specific type of cytochrome as well as to confirm the similarity of the heme prosthetic group to previously well-characterized cytochromes of the the a- b- or c-type. Preliminary evidence in support of heterogeneity of b-type, and possibly a-type cytochromes, or of heme-heme interaction within the membrane is presented.     

The respiratory electron transport system of heterotrophically-grown Rhodopseudomonas palustris

The enzymatic activities and the cytochrome components of the respiratory chain were investigated with membrane fractions from chemoheterotrophically grown Rhodopseudomonas palustris. Whereas the level of electron transfer carriers was not distinctly affected by a change of the culture conditions, the potential activities of the enzymes were clearly increased when the cells were grown aerobically. Reduced-minus oxidized difference spectra of the membrane fractions prepared from dark aerobically grown cells revealed the presence of three b-type cytochromes b ₅₆₁, b ₅₆₀ and b ₅₅₈, and at least two c-type cytochromes c ₅₅₆ and c ₂ as electron carriers in the electron transfer chain. Cytochrome of a-type could not be detected in these membranes. Reduced plus CO minus reduced difference spectra of the membrane fractions were indicative of cytochrome o, which may be equivalent to cytochrome b ₅₆₀, appearing in substrate-reduced minus oxidized difference spectra. Cytochrome o was found to be the functional terminal oxidase. CO difference spectra of the high speed supernatant fraction indicated the presence of cytochrome c′. Succinate and NADH reduced the same types of cytochromes. However, a considerable amount of cytochrome b ₅₆₁ with associated β and γ bands at 531 and 429 nm, respectively, was reducible by succinate, but not by NADH. A substantial fraction of the membrane-bound b-type cytochrome was non-substrate reducible and was found in dithionite-reduced minus substrate-reduced spectra. Cytochrome c ₂ may be localized in a branch of the electron transport system, with the branch-point at the level of ubiquinone. The separate pathways rejoined at a common terminal oxidase. Two terminal oxidases with different KCN sensitivity were present in the respiratory chain, one of which was sensitive to low concentrations of KCN and was connected with the cytochrome chain. The other terminal oxidase which was inhibited only by high concentrations of cyanide was located in a branched pathway, through which the electrons could flow from ubiquinone to oxygen bypassing the cytochrome chain.