Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

Purification and characterization of two constitutive cytochromes P-450 (F-1 and F-2) from adult female rats: identification of P-450F-1 as the phenobarbital-inducible cytochrome P-450 in male rat liver

Two hepatic microsomal cytochromes P-450, P-450F-1 and P-450F-2 were purified to electrophoretic homogeneity from untreated adult female rats by high-performance liquid chromatography (HPLC) with anion-exchange, cation-exchange, and hydroxyapatite columns. Cytochromes P-450F-1 and P-450F-2 were not adsorbed with the anion-exchange column, but were retained on a cation-exchange column and were separated poorly. These forms separated on hydroxyapatite HPLC. The molecular weights of cytochromes P-450F-1 and P-450F-2 were 50,000 and 49,000, respectively. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of cytochromes P-450F-1 and P-450F-2 were at 450 and 448 nm, respectively. Both forms catalyzed the N-demethylation of benzphetamine and had low catalytic activity for 7-ethoxycoumarin. Cytochrome P-450F-1 had low 2 alpha-hydroxylation activity toward testosterone. Cytochrome P-450F-2 had low 15 alpha-hydroxylation activity. On the basis of these results and those of NH2-terminal sequence analysis, cytochrome P-450F-2 seemed to be the typical female-specific cytochrome P-450. The NH2-terminal sequence of cytochrome P-450F-1 was identical to that of cytochrome P-450PB-2 purified from hepatic microsomes of male rats treated with phenobarbital. Cytochromes P-450F-1 and P-450PB-2 had identical chromatographic properties, minimum molecular weight, spectral properties, and peptide maps. Furthermore, the antibody to phenobarbital-inducible cytochrome P-450PB-2 gave a single immunoprecipitin band with cytochrome P-450F-1 by Ouchterlony double-diffusion analysis.     

Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats.

At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators.     

Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. I. Purification and spectral properties

A form of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation (tentatively called "P-450(14)DM") was purified from microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae to gel electrophoretic homogeneity. An apparent monomeric Mr = 58,000 was estimated for the purified cytochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both optical and EPR spectra of oxidized P-450(14)DM are characteristic of low spin ferric heme proteins, and its reduced CO complex showed a Soret absorption peak at 447 nm. As in the case of hepatic microsomal cytochromes P-450, the ethyl isocyanide complex of reduced P-450(14)DM was in a pH-dependent equilibrium between two states having Soret peaks at 429 and 453 nm, the equilibrium being considerably shifted toward the 453-nm state. Oxidized P-450(14)DM was peculiar in that in its CD spectrum there was a negative shoulder at 425 nm and the 350- and 414-nm troughs possessed larger and relatively smaller [theta] values, respectively, than those reported for other low spin ferric cytochromes P-450. Lanosterol was the only compound which caused a Type I spectral change in oxidized P-450(14)DM. The lanosterol-induced low to high spin state change was, however, only slight even at saturating concentrations of the sterol, indicating that the lanosterol-P-450(14)DM adduct was in a spin state equilibrium.     

Induction by Triacetyloleandomycin and partial purification of a LM3 form of cytochrome P-450 from rabbit liver microsomes

Treatment of rabbits with Triacetyloleandomycin (a currently used antibiotic in human therapy) at 1 mmol per kg of body weight daily for 5 days results in a significant induction of liver microsomal cytochrome P-450, (2.6 nmol/mg proteins). Electrophoresis in SDS polyacrylamide gels shows this increase in P-450 is associated to the appearance of a strong band in a zone located between the major bands of microsomes induced by phenobarbital and β-naphtoflavone (LM₃ forms in Coon's terminology). Partial purification of this P-450 LM₃ (TAO) was undertaken by chromatographic procedures (CM cellulose and hydroxylapatite). Its subunit molecular weight is 52 000; the absolute spectra in the oxidized, ferrous and CO-ferrous forms present maxima at 417, 536, and 570 nm; 415 and 548 nm; 450 and 555 nm respectively. Monooxygenase activity of LM₃ (TAO) was compared with that of LM₂ and LM₄ in a reconstituted system containing NADPH cytochrome P-450 reductase and phosphatidylcholine; the activity of P-450 LM₃ (TAO) was higher than that of LM₂ and LM₄ with chlorcyclizine as a substrate. According to these observations, LM₃ (TAO) resembles LM₃ (b), a constitutive form of untreated rabbit liver microsomes.     

Separation of two constitutive forms of cytochrome P-450 active in aminopyrine N-demethylation from rabbit intestinal mucosa microsomes

Two constitutive forms of cytochrome P-450, designated P-450ib and P-450ic, were purified from intestinal mucosa microsomes of untreated rabbits. P-450ib and P-450ic have minimal molecular weights of 56 000 and 49 000, respectively, as determined by calibrated sodium dodecyl sulphate polyacrylamide gel electrophoresis. The CO-reduced difference spectral maximum of cytochrome P-450ib is at 450 nm and P-450ic is at 451 nm. Both the cytochromes preferentially demethylate aminopyrine, benzphetamine and N,N-dimethylaniline in the presence of NADPH-cytochrome P-450 reductase. Cytochrome P-450ib has absorption maxima at 417, 535 and 573 nm in the oxidized form, indicating that this cytochrome is in a low-spin state. Ouchterlony double-diffusion studies show that cytochrome P-450ib does not cross-react with antisera against liver cytochrome P-450LM2 purified from phenobarbital-treated rabbits, but P-450ic cross-reacts with spur formation. Unlike cytochrome P-450ib, P-450ic is very similar, if not identical, to liver cytochrome P-450LM2 on the basis of its molecular weight, spectral properties, catalytic activities and immunochemical properties.     

A highly purified preparation of cytochrome P-450 from microsomes of anaerobically grown yeast.

Cytochrome P-450 was purified from microsomes of anaerobically grown yeast to a specific content of 12–15 nmoles per mg of protein with a yield of 10–30%. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis, the purified preparation yielded a major protein band having a molecular weight of about 51,000 together with a few faint bands. It was free from cytochrome b₅, NADH-cytochrome b₅ reductase, and NADPH-cytochrome c (P-450) reductase. In the oxidized state it exhibited a low-spin type absorption spectrum, and its reduced CO complex showed a Soret peak at 447–448 nm. It was reducible by NADPH in the presence of an NADPH-cytochrome c reductase preparation purified from yeast microsomes. Its conversion to the cytochrome P-420 form was much slower than that of hepatic cytochrome P-450.     

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.     

Cytochrome P-450 in human liver microsomes: high-performance liquid chromatographic isolation of three forms and their characterization

Three forms of cytochrome P-450, designated as P-450-HM1, P-450-HM2, and P-450-HM3, were isolated from human liver microsomes using high-performance liquid chromatography (HPLC) techniques. Each purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the results of SDS-PAGE, the molecular weights of P-450-HM1, P-450-HM2, and P-450-HM3 were estimated to be 51,000, 54,000, and 52,000, respectively. The oxidized absolute spectra of these three forms of cytochrome P-450 showed Soret absorption peaks at around 417 nm, indicating that these forms were in the low spin state. In a reconstituted system, P-450-HM1 showed the highest catalytic activities of nifedipine and (S)- or (R)-nilvadipine oxidases. The same form showed higher activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. P-450-HM2 showed high N-demethylase activities for benz-phetamine and aminopyrine, and also showed the highest activity of testosterone 16 beta-hydroxylase among the three forms, while it did not show detectable activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. Anti-P-450-HM1 immunoglobulin G (IgG), but not anti-P-450-HM2 IgG, inhibited the activities of testosterone 6 beta-hydroxylase and nifedipine and nilvadipine oxidases in human liver microsomes. Anti-P-450-HM1 IgG was also inhibitory against progesterone 6 beta- and 16 alpha-hydroxylases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low temperature spectral properties of subchloroplast fractions purified from spinach

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F_I, F_II, and F_III, corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at −196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F_II (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at −196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F_I, F_II, and F_III at −196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F_I showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F_II and F_III in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F_I and F_II caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at −196 C with an increase in the yield of fluorescence especially in the case of F_I.     

Selective loss of pulmonary cytochrome P-450I in rabbits pretreated with polychlorinated biphenyls

The polychlorinated biphenyls mixture, Aroclor 1254, generally considered a powerful inducer of rat hepatic and pulmonary microsomal monooxygenases, caused a 70% decrease in ethylmorphine N-demethylase activity, a 31% decrease in benzo(a)pyrene hydroxylase activity, and a 42% decrease in cytochrome P-450 content in rabbit lung microsomes. When pulmonary cytochrome P-450 was solubilized and subjected to column chromatography, the elution profiles of the two forms of the hemeprotein showed a marked decrease in cytochrome P-450I in treated rabbits, with no significant alteration in cytochrome P-450II content. These data were confirmed by subjecting the two cytochromes to gel electrophoresis and staining the electrophoretic bands for protein and heme-associated peroxidase activity. Cytochromes P-450I and P-450II isolated from Aroclor 1254-treated rabbits showed differences in spectral properties as well as in their stabilities. The CO difference spectral determinations showed absorbance maxima at 452 and 450 nm for cytochromes P-450I and P-450II, respectively. At room temperature, cytochrome P-450II was much more stable than P-450I. The present studies provide evidence not only for species differences in the biological actions of the polychlorinated biphenyls but also demonstrate differential effects of the environmental pollutant on the two major forms of cytochrome P-450 and associated enzymic activities in rabbit lungs.     

Purification and properties of cytochrome P-450 generally acting as a catalyst on benzo[a]pyrene hydroxylation from liver microsomes of untreated rats

A form of cytochrome P-450 generally catalyzing benzo[a]pyrene (B[a]P) hydroxylation was purified from liver microsomes of untreated rats on the basis of the catalytic activity. The purification procedures consisted of cholate solubilization and chromatography in 3 steps, on DEAE-Toyopearl (at room temperature), hydroxylapatite, and CM-Toyopearl columns. Cytochrome P-450 purified in this way (named P-450/B[a]P) was homogeneous on SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated to be 51,000. The absorption spectra of the oxidized form of P-450/B[a]P showed a Soret peak at 417 nm, characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 451 nm. Immunochemical analysis of P-450/B[a]P indicated that P-450/B[a]P is immunologically distinct from P-450b (a major phenobarbital-inducible form of P-450) and P-450c (a major 3-methylcholanthrene-inducible form of P-450, which highly catalyzes the hydroxylation of B[a]P). B[a]P hydroxylase activity in liver microsomes of untreated rats was inhibited to about 20% by the P-450/B[a]P antibody. These results demonstrate that P-450/B[a]P is a different form of P-450 from P-450b and P-450c, and generally catalyzes B[a]P hydroxylation in liver microsomes of untreated rats.     

Complexes of trivalent oxygenated phosphorus compounds with cytochrome P-450 and cytochrome P-420: the origin of double Soret spectra.

Trivalent oxygenated phosphorus ligands include alkyl and aryl phosphites, (RO)3P, phosphonites, (RO)2PR, and phosphinites, ROPR2. All such compounds tested, with the exception of triphenyl phosphite, interact with ferrous cytochrome P-450 and its denatured form, cytochrome P-420, to produce complexes having two peaks in the Soret region of their optical difference spectra. Careful evaluation of these spectra indicate that they arise for different reasons for each of the two cytochromes. Clear evidence shows that cytochrome P-450 is not denatured by these ligands. The high affinity of these ligands for heme iron is indicated by small Ks values. The experimental results are used to substantiate a theory of the origin of microsomal double Soret spectra and the nature of the environments available for microsomal cytochromes P-450 and P-420.     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

The interaction of aliphatic analogs of methylene-dioxyphenyl compounds with cytochromes P-450 and P-420.

The spectra resulting from the interaction of a series of substituted dioxolanes with microsomal cytochromes P-450 or P-420, as well as purified cytochrome P-450, were measured. With the exception of dioxolane, 4-methyldioxolane and 4-ethyldioxolane, these compounds interacted with ferric cytochrome P-450 to give complexes exhibiting type I optical difference spectra, and, after incubation with NADPH, spectra with peaks at about 430 nm. These complexes, as well as those formed from dioxolanes in the presence of cumene hydroperoxide, inhibit the binding of CO to the cytochrome. Consideration of the known chemistry of dioxolanes, together with recent advances in the understanding of double Soret spectra, lead to a possible explanation for the differences between the spectra of dioxolanes and their aromatic analogs, the methylenedioxyphenyl compounds.     

Physiology and metabolism of pathogenic Neisseria: partial characterization of the respiratory chain of Neisseria gonorrhoeae.

The cell membrane-associated respiratory electron transport chain of Neisseria gonorrhoeae was examined using electron paramagnetic spectroscopy (EPR) at liquid helium temperatures and optical spectroscopy at liquid nitrogen and room temperatures. EPR spectra of dithionite-reduced particles indicated the presence of centers N-1 and N-3 in the site I region of the respiratory chain, whereas reduction with succinate revealed the existence of center S-1 from the succinate cytochrome c reductase segment. Free radical(s) resembling that due to falvin semiquinone were observed with both reductants. Low temperature (77 K) optical difference spectra indicated the presence of cytochromes with alpha band maxima at 549, 557, and 562. Bands at 567, 535, and 417 nm, characteristic of the CO compound of cytochrome o, were also identified. Cytochromes a1 and a3 were not detected; however, a broad but weak absorbance with an alpha band maximun at 600 nm and a Soret shoulder at 440 nm was observed. Hence the respiratory chain of N. gonorrhoeae appears to contain several nonheme iron centers, cytochrome c, two b cytochromes, with cytochrome o which probably serves as the terminal oxidase.     

Induction of multiple cytochrome P-450 species in housefly microsomes--SDS-gel electrophoresis studies.

1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains.     

Induction of both cytochromes P-450 and P-448 by 2,3',4,4',5-pentabromobiphenyl, a component of fireMaster

The synthesis and purification of a component of fireMaster BP-6 and fireMaster FF-1, 2,3′,4,4′,5-pentabromobiphenyl, is described. The compound was found to be a potent inducer of liver microsomal drug-metabolizing enzymes in the rat, enhancing those enzymic activities induced by both phenobarbitone and 3-methylcholanthrene (i.e. cytochromes P-450 and P-448). The pentabromobiphenyl enhanced the activities of benzo[a]pyrene hydroxylase, dimethylamino-antipyrine N-demethylase and NADPH-cytochrome c reductase. The hepatic cytochromes b₅ and P-450 were increased and the Soret peak maximum of the latter was shifted to 448.5 nm. The relative peak intensities and spectral shifts for the ethylisocyanide-binding difference spectra confirmed the mixed induction characteristics of 2,3′,4,4′,5-pentabromobiphenyl.     

A -glucuronidase deficiency mucopolysaccharidosis: studies in cultured fibroblasts

Cytochrome P-450 cannot be detected spectrophotometrically in testis mitochondria of untreated rats because of the high cytochrome a₃ to Cytochrome P-450 ratio. Injection of human chorionic gonadotrophin (HCG) causes a large increase in mitochondrial cytochrome P-450. After 14 days injection, mitochondrial cytochrome P-450 levels are increased 15- to 30-fold (from 0.007 to 0.134 nmoles/mg protein) over control levels. Levels of cytochrome a + a₃ are not altered by this treatment. Mitochondrial cytochrome P-450 can also be demonstrated by injection of HCG into rats which were hypophysectomized 24 days previously. During hypophysectorny the mitochondrial cytochromes c + c_i, a + a₃ and mitochondrial protein decay with halflives of 14, 16, and 15.5 days, respectively. HCG treatment for 8 days increases mitochondrial cytochrome P-450 (from < 0.003 to 0.24 nmoles/mg protein) without altering the levels of the other mitochondrial cytochromes. The control of cytochrome P-450 levels in the mitochondria by HCG suggests that the level of this key component of cholesterol side-chain cleavage enzyme may be of importance in the regulation of steroidogenesis in the testis.     

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.