Cytochrome extinction coefficients

Cytochrome Determinations. Microsomal suspensions (1-5 mg protein/ ml) were assayed for cytochromes bs and P-A50 (12) using a Cary 15 spectrophotometer operated at room temperature (20-23°C). Suspensions in 0.05M phosphate buffer, pH 7.A, were contained in 3 ml cuvettes with a 1 cm path length. Sodium dithlo-nite was the reductant. The extinction coefficient of 171 mM 1 cm 1 was applied to the A28-A90 nm absorbance increment. 

A band of this type has been observed for an enzyme-substrate complex ES where the enzyme was represented by the oxidized form of peroxidase cytochrome c, cyt(Fe(III)) and the substrate was the reduced form of cytochrome c, cytj (Fe(II)) [298]. Indeed, on mixing the solution of cyt(Fe(I I)) and cytj (Fe(II)) there appeared a new absorption band with the absorption maximum at Emax = 1.4 eV, the extinction coefficient e = 0.35 M-1 cm-1, and the width a = 0.2 eV. This band was referred [298] to charge transfer via electron tunneling, [cyt(Fe(III))/ cyt, (Fe(II))] -> [cyt(Fe(II))/cytl(Fe(III))]. From a comparison of the data on the intensity of this band with the results of fluorescence measurements, the distance between the iron atoms Fe(III) and Fe (II) in the [cyt(Fe(III))/cyt1(Fe(II))] complex has been estimated to be R 15-20 A and the edge-to-edge tunneling distance Rt = 7 A. 

Chromophores free in solution and bound to macromolecules do not display identical s values and absorption peaks. For example, free hemin absorbs at 390 nm. However, in the cytochrome b2 core extracted from the yeast Hansenula anomala, the absorption maximum of heme is located at 412 nm with a molar extinction coefficient equal to 120 mM-1 cm-1 (Albani 1985). In the same way, protoporphyrin IX dissolved in 0.1 N NaOH absorbs at 510 nm, whereas when it is bound to apohemoglobin, it absorbs in the Soret band at around 400 nm. 

Two other features of these cytochromes are of interest. The Soret maxima are of high energy and low extinction coefficient and the Fe(III) forms will not form any complexes except under drastic conditions. It would appear that the haem unit is so buried in the protein as to be inaccessible and that the protein exerts a gross influence upon the haem geometry. 

Cytochrome 62 is found as a soluble protein in the autolysates of Sac-charomyces cerevisiae. The crystalline preparations of Appleby and Morton 278) were shown to sediment as a single peak in the ultracentrifuge. Minimum molecular weight based on amino acid analysis and a heme extinction coefficient of 232 mM cm was calculated to be 53,000 283). The heme extinction coefficient was then corrected to 183 mM- cm-, and the minimum molecular weight per mole of heme recalculated to be 58,600 284). It was concluded that cytochrome 6 is a tetrameric structure. This conclusion agreed with the results of X-ray diffraction studies on type I and type II crystals, which indicated molecular weights of 235,000 10,000 and 234,000 8,000, respectively, for these two preparations of cytochrome 2 285). The oxidized and reduced spectral bands of cytochrome ba are given in Table XIV. 

Fig. 46. Comparison of the absorption spectra of wild-type and mutant (cys G-439 and cys 1-68) sulfite reductases from Salmonella typhimurium. Spectra of S. typhi-murium sulfite reductase, cys G-439 NADPH-cytochrome e reductase, and cys 1-68 NADPH-oytoohrome c reductase, each dissolved in 0D5 M potassium phosphate buffer, pH 7.7, containing 0.1 mM EDTA, were read against a blank containing only buffer. The spectrum of each enzyme is presented in terms of its millimolar extinction coefficients, assuming 8 moles of flavin per mole of enzyme. Light broken line, calculated difference spectrum between those of wild-type and cys G enzymes when both enzyme solutions contain equal concentrations of flavin. From Siegel et al. (394).

Dutton, P. L., Petty, K. M., Bonner, H. S., and Morse, S. D., 1975, Cytochrome c and reaction center of Rhodopseudomonas sphaeroides Ga membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field altertions. Biochim. Biophys. Acta, 387 5369556. 

The data given is collected from Refs. 74-79. Cytochrome aa, contains two haems and two coppers per monomer. Considerable variations in the literature are due in part to differences in protein determination, and in part to use of erroneous extinction coefficients. The ratio of Complex 1 Complex 111 Cytochrome c Complex IV Ubiquinone is about 1 4 8 8 64 in most mitochondria. The content of cytochrome c is somewhat variable, however, and is lowered by extensive washing of mitochondria is salt solutions. 

Fig. 8. Absorption changes during photooxidation and dark re-reduction of the primary electron donor P (A) and oxidation of cytochrome c (B) following a brief, intense flash. Figure source Parson and Clayton (Straley, Parson, Mauzerall and Clayton) (1973) Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas sphaeroides. Biochim Biophys Acta. 305 606.

By flash excitation of this reaction system, it is not only possible to measure the absorbance change at 870 nm produced by photooxidation of the primary electron donor but also that at 550 nm produced by the oxidation of cytochrome c, as shown in Fig. 12, lower right trace. Since the differential extinction coefficient of cytochrome c was precisely known, the authors were able to use the differential extinction coefficient of P870 commonly accepted at the time, and the measured amplitudes of absorbance changes at 870 and 550 nm, to calculate a stoichiometry of 1 P870 1 cyt c for this reaction. Parson and Clayton " later utilized this model system to obtain conversely a more accurate differential extinction coefficient of 128 mM -cm" forP870 (see Section II. of Chapterd for further details). 

Hematin a was prepared from purified cytochrome oxidase as described (29). The hematin solution was made by dissolving the dried samples in phosphate buffer containing 1% Emasol 1130. Its concentration was determined by the pyridine hemochromogen method in a mixture of 20% pyridine and 0.05N NaOH an extinction coefficient of 27.4 mM cm." at 589 m/x was used (29). 

P.L. Dutton, K.M. Petty, H.S. Bonner, and S.D. Morse, Cytochrome C2 and Reaction Center of Rhodopseudomonas spheroides Ga. Membranes. Extinction Coefficients, Content, Half-Reduction Potentials, Kinetics and Electric Field Alterations, Biochim. Biophys. Acta 387 536 (1975). 

Compounds resembling cytochrome c of heart muscle have been isolated from many sources. The cytochrome c of skeletal muscle of the king penguin has been purified by the same methods used for ox heart cytochrome c, and has been crystallized from almost saturated ammonium sulfate. A precipitate of the oxidized cytochrome was obtained that exhibited birefringence but was not crystalline addition of reducing agent to the ammonium sulfate solution permitted crystals of reduced cytochrome c to form. Measurements of the catalytic activity and extinction coefficients gave values equivalent to those of the ox heart preparation within the limits of error. In a preliminary report, the... 

The apparent extinction coefficient of cytochrome P-450 is increased after 3-methylcholanthrene treatment and is slightly greater in female than in male rats. 

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