Absorption spectra of cytochrome

FIGURE 21.9 Typical visible absorption spectra of cytochromes, (a) Cytochrome c, reduced spectrum (b) cytochrome c, oxidized spectrum (c) the difference spectrum (a) minus (b) (d) beef heart mitochondrial particles room temperature difference (reduced minus oxidized) spectrum (e) beef heart submitochondrial particles same as (d) but at 77 K. a- and /3- bauds are labeled, and in (d) and (e) the bauds for cytochromes a, h and c are indicated. 

FIGURE 19-4 Absorption spectra of cytochrome c (cyt c) in its oxidized (red) and reduced (blue) forms. Also labeled are the characteristic a, fi, and y bands of the reduced form. 

Wray S, Cope M, Delpy DT, Wyatt JS, Reynolds EOR. Characterization of the near-infrared absorption-spectra of cytochrome-Aa3 and hemoglobin for the non-invasive monitoring of cerebral oxygenation. Biochimica et Biophysica Acta 1988, 933, 184—192. 

Several observations made in whole membranes or in the isolated complexes are in line with these concepts the shifts induced by antimycin A [110,137] and myxothiazol on the absorption spectra of cytochromes b and the alterations of the ESR spectrum of the FeS protein by UHDBT or DBMIB [131]. Moreover, the oxidant-induced reduction of cytochromes b, the key observation for accepting these electron transfer schemes, has been demonstrated in all h/c, complexes isolated so far from mitochondria [134], chloroplasts [111], cyanobacteria [112] and photosynthetic bacteria [110]. In the chloroplast b /f complex this reaction has been demonstrated also in the absence of any exogenously added quinol, indicating that possibly a structurally bound quinone (quinone is always present in the isolated complexes with a stoicheiometry of about 0.5-0.7 mol/mol of cyt. c, [110,111]) is sufficient to drive the reduction of cytochromes [138]. Since a detailed treatment of the genera] mechanism, as well as of the more specific problems of the mitochondrial respiratory chain, are reported in Chapter 3 of this volume, the following discussion will deal only with the specific features of the electron transfer chains in photosynthetic membranes. 

Fig. 2. Absolute and difference light absorption spectra of cytochrome c peroxidase and Compound ES at pH 7 and 20° (-----------------) enzymes and (—) + CjHiOOH.

K. Ruckpaul, and L.A. Blyumenfel d (1978). Absorption spectra of cytochrome P-450 nonequilibrium states formed during the low-temperature reduction of protein. Dokl. Acad. Nauk SSSR 241, 707-709. 

Absorption spectra of cytochrome P450CAM in the reaction with peroxy acids. FEBS Lett. 156, 244-248. 

Figure 1.23. Absorption spectra of cytochrome hi core in the oxidized (a) and in the reduced forms (b). Reduction occurs with dithionite which absorbs below 400 nm. Cytochrome hi core...

If the various forms of cytochrome P450 have different specificities for substrates, they must also differ in amino acid composition. It is now established that in all forms so far characterised, differences in molecular weight, amino acid composition and terminal amino acids exist. Figure 3 shows the optical absorption spectra of cytochrome P450 from Ps. putida. 

Fig. 4. Absorption spectra of cytochrome c in a thin-layer cell with 2,6-dichlorophenolin-dophenol mediator in 0.5 M phosphate buffer, pH 7.0, for a series of applied potentials. "...

Optical studies on Rps. viridis RCs provided evidence that all four hemes of cytochrome c have different absorption spectra in the a-band domain as well as midpoint potentials [1-4].In this work we used the individual spectra of each heme and linear least-squares method to elucidate the quantity of every heme from full absorption spectra of cytochrome c in the -band region as function of redox potential. For all four hemes the values of midpoint potentials established by the method of decomposition are in good agreement with those determined earlier [1]. 

Fig. 4.15 Absorption spectra of cytochrome c with the bound heme in its reduced (solid curve) and oxidized (dotted curve) forms (A), and of bacteriochlorophyll a in methanol (B)...

Fig. 13. Absorption spectra of cytochrome b. (-----) Air-oxidized form, (-----) dithionite-...

Figure 16-7 Absorption spectra of oxidized and reduced horse heart cytochrome c at pH 6.8. From data of Margoliash and Frohwirt.98...

The Fe atoms of the cytochromes undergo oxidation and reduction during respiration, cycling between the ferrous (Fe2+) and ferric (Fe3+) oxidation states. The absorption spectra of the oxidized and reduced forms differ (fig. 14.4). In the 1930s, David Keilin used this property to measure the oxidation-reduction states of cytochromes in living cells. Under anaerobic conditions, the cytochromes rapidly became reduced in the presence of 02, they became oxidized. Certain molecules that inhibited respiration (CO, N3, or CN ) blocked the oxidation other inhibitors (amy-tal, rotenone, and malonate) blocked the reduction. Keilin found that the transfer of electrons from cytochrome c to 02... 

Optical absorption spectra of a 10 /m solution of cytochrome c in the reduced (blue) and oxidized (red) states. 

The traces shown here are measurements of optical absorbance changes at 870 and 550 nm when a suspension of membrane vesicles from photosynthetic bacteria was excited with a short flash of light. Downward deflection of the traces represent absorbance decreases. Explain the observations. (Absorption spectra of a c-type cytochrome in its reduced and oxidized forms are described in the previous chapter.)... 

Fig. 7. The absorption spectra of oxidised rhodospirillum rubrum cytochrome cc (oxidised) with change of pli...

Fig. 8. The absorption spectra of reduced rhodospirillum rubrum cytochrome cc with change of pH. This figure and figure 7 are reproduced with permission (67)...

Electron transport in cytochromes occurs by direct electron transfer between Fe2+ and Cu+ in cytochromes a and a3. These changes in metal-ion oxidation state lead to changes in the visible absorption spectra of the cytochromes spectrophotometric measurement of these changes allows quantification of the electron flow. 

The absorption spectra of the model complexes of haem a, made by Lemberg and his collaborators (134), have been taken to indicate that the high-spin Fe(III) haem a complexes have absorption bands at 660 mp while the low-spin Fe(III) haem complexes have bands at 595 mp (135). These data allow an analysis of the spin-states of the cytochromes a. The analysis has been carried out by Williams (135), Vanneste (136), Williams, Lemberg and Cutler (137). 

It is well known that the O2 reduction site of bovine heart cytochrome c oxidase in the fuUy oxidized state exhibits variable reactivity to cyanide and ferrocytochrome c, which is dependent on the method of purihcation (Moody, 1996). Some preparations react with cyanide extremely slowly at an almost immeasurable rate and are known as the slow form. Other preparations, which react at a half-Ufe of about 30 s, are known as the fast form (Brandt et al., 1989). Electronic absorption spectra of the slow-and fast-form preparations exhibit Soret bands at 418 and 424 nm, respectively. The two forms often coexist in a single preparation (Baker et al., 1987). Both forms exhibit an identical visible-Soret spectrum in the fully reduced state. The slow-form preparation can be converted to the fast form by dithionite reduction followed by reoxidation with O2. The fast form thus obtained returns to the slow form spontaneously at a rate much slower than the enzymatic turnover rate. Thus, the slow form is unlikely to be involved in the enzymatic turnover (Antoniniei a/., 1977). It should be noted that no clear experimental evidence has been reported for direct involvement of the fast form in the enzyme turnover, although its direct involvement has been widely accepted. The third species of the fully oxidized O2 reduction site, which appears in the partially reduced enzyme, reacts with cyanide 10 —10 times more rapidly than the fast form (Jones et al., 1984). In the absence of a reducing system, no interconversion is detectable between the slow and the fast forms (Brandt et al., 1989). Thus, the heterogeneity is expected to inhibit the crystallization of this enzyme. In fact, the enzyme preparations providing crystals showing X-ray diffraction at atomic resolution are the fast form preparation. 

Fio. 38. The absorption spectra of crj stalline Pseudomonas cytochrome oxidase. 

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).

On reaction with a stoichiometric amount of hydroperoxide, catalase and horseradish peroxidase are converted to a green colored intermediate. Compound I (5). The chemical nature of Compound I has been extensively debated since its discovery by Theorell 59). Recently, Dolphin et al. 60) have demonstrated that upon one-equivalent oxidation several metalloporphyrins are converted to stable porphyrin jr-cation radicals, the absorption spectra of which possess the spectral characteristics of Compound I, namely, a decreased Soret w-n transition and an appearance of the 620-670-nm absorption bands. Since Moss et al. 61) proposed the presence of Fe(IV) in Compound I of horseradish peroxidase from Mossbauer spectroscopic measurements, it is attractive to describe Compound I as Fe(IV)-P, where P is a porphyrin w-cation radical. Then, Compound I and Compound ES become isoelectronic. Both contain Fe(IV) and a radical the former as a porphyrin radical (P ) and the latter as a protein radical (R ). Then the reaction cycles of horseradish and cytochrome c peroxidases may be compared as shown in Fig. 4. 

Vanneste, W. H., 1966, The stoichiometry and absorption spectra of components a and aj in cytochrome c oxidase. Biochemistry 5 838n848. 

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