Cytochromes absorption bands

Organism and cytochrome Absorption bands Reduced Oxidized MW pi Eo (mV) Hemes Ref. 

Figure 12 Graph allowing calculation of the error In measured peak height for a given monochromator spectral bandwidth and natural bandwidth of an absorption band. Spectral bandwidth can be obtained from manufacturers Information or by knowing the physical slit width of the monochromator and the reciprocal dispersion (nm/mm). Since natural bandwidths of, for example, cytochrome absorption bands are about 10 nm at room temperature a spectral bandwidth of 2.5 nm (ratio on the abscissa of 0.25) will Introduce no more than a 3% error in measured peak height. However, for low temperature spectra, spectral bandwidths of about 0.5 nm are required.

Table IV summarizes our present knowledge about the position of cytochrome absorption bands at 77° K in S. pombe. It must be pointed out that the positions of the 0 and 7 bands are very tentative and also that additional pigments might well be present.

Cytochrome P450 monooxygenases are characterized through the presence of the heme (protoporphyrin IX) prosthetic group (Scheme 10.1) that is coordinated to the enzyme through a conserved cysteine ligand. They have obtained their name from the signature absorption band with a maximum near 450 nm in the difference spectrum when incubated with CO. The absorption arises from the Soret Jilt transition of the ferrous protoporphyrin IX-CO complex. 

Electronic spectra of metalloproteins find their origins in (i) internal ligand absorption bands, such as n->n electronic transitions in porphyrins (ii) transitions associated entirely with metal orbitals (d-d transitions) (iii) charge-transfer bands between the ligand and the metal, such as the S ->Fe(II) and S ->Cu(II) charge-transfer bands seen in the optical spectra of Fe-S proteins and blue copper proteins, respectively. Figure 6.3a presents the characteristic spectrum of cytochrome c, one of the electron-transport haemoproteins of the mitochondrial... 

Porphyrins 21 are the backbone of major players in life cycles—cytochromes (Scheme 8). There are three types of cytochromes, classified by their color, or more precisely by their long-wavelength absorption band, as a (600 mn), b (563 nm), and c (550 nm). They are protein conjugates of a porphyrin complex with iron(II), which is a coenzyme called heme (22). In plants, porphyrins form a complex with magnesium-(II) chlorophylls a and b (23), vital in photosynthesis. Porphyrin derivatives are used in photodynamic therapy for dermatological diseases such as psoriasis, and for skin or subcutaneous cancer.5c-e... 

The use of the reversion spectroscope enabled the position of the absorption bands to be determined accurately and to be conclusively distinguished from hemoglobin and myoglobin. It became clear that there were three different intracellular respiratory catalysts— cytochromes a,b,c—common to animals, bacteria, yeast and higher plants. In 1925 a preliminary scheme for the passage of O2 from blood to tissue was proposed ... 

The above observations provide a clear demonstration that cosolvents in selected ranges of concentration create reversible perturbations of protein similar to those induced by other modifiers. The reversibility of the cosolvent effect is a prerequisite to cosolvent use and will depend on the concentration of cosolvent, which in turn will vary markedly with the type of solvent used. For instance, polyols can be used at concentrations up to 8 Af while methanol at 3 M causes the appearance of a new absorption band (410 nm) and, after further increases in concentration, an irreversible conversion of cytochrome P-450 into P-420. Other aliphatic alcohols cause denaturation at much lower concentrations. 

Keilin soon realized that three of the absorption bands, those at 604,564, and 550 nm (a, b, and c), represented different pigments, while the one at 521 nm was common to all three. Keilin proposed the names cytochromes a, b, and c. The idea of an electron transport or respiratory chain followed6 quickly as the flavin and pyridine nucleotide coenzymes were recognized to play their role at the dehydrogenase level. Hydrogen removed from substrates by these carriers could be used to oxidize reduced cytochromes. The latter would be oxidized by oxygen under the influence of cytochrome oxidase. 

The cytochromes were first observed by MacMunn as early as 1886. He described their spectral absorption bands in a large variety of organisms and tissues. His discovery was, however, forgotten after a controversy with Huppc-Scyler had raised doubts as to the validity of some of his conclusions, and it was not until 1925 that Kcilin independently rediscovered the remarkable cytochrome spectrum in the flight muscles of a living insect. 

The appearance of similar absorption bands has also been observed upon the formation of a complex between the reduced form of cytochrome c and the simple inorganic acceptor Fe(III)(CN)6[106]. The tunneling distance evaluated from the intensity of this band amounts to 7—10 A. However, more recent experiments have failed to detect such a band [107]. The situation is more favourable in the system [cytochrome c/P870] of the Chromatium reaction centre, where the intensity of the charge transfer band centred at 200 nm could be correlated with the data obtained in kinetic experiments [108]. 

Heme coenzymes, iron-sulfur clusters, flavin coenzymes, and nicotinamide coenzymes cooperate in multienzyme systems to catalyze the chemically remarkable hy-droxylations of hydrocarbons such as steroids (chapter 20). In these hydroxylation systems, the heme proteins constitute a family of proteins known as cytochrome P450, named for the wavelength corresponding to the most intense absorption band of the carbon monoxide-liganded heme, an inhib-... 

The reduction of UQ can be measured by the disappearance of an absorption band at 275 nm. Using this technique, it was shown that adding a substrate such as succinate caused a rapid reduction of essentially all the UQ present in the inner membrane the resulting UQH2 could be reoxidized by the cytochrome system in the presence of 02. To determine whether UQ is a necessary participant in electron transport from succinate to 02, the quinone was removed from mitochondria by selective extraction with an organic solvent. The depleted mitochondria were incapable of respiration but recovered this activity when UQ was added back. 

The reduction of ring IV in chlorophylls a or b changes the optical absorption spectrum of the molecule dramatically. Whereas the long-wavelength absorption band of a cytochrome is relatively weak (see fig. 14.4), chlorophyll a has an intense absorption band at 676 nm (fig. 14.5). Chlorophyll b has a similar band at 642 nm. Bacteriochlorophylls a and b have strong absorption bands in the region of 770 nm (see fig. 15.5). The chlorophylls thus absorb red or near-infrared light very well. 

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. 

When a protein possesses a prosthetic group such as heme, its concentration is usually determined at the absorption wavelengths of the heme. The most important absorption band of heme is called the Soret band and is localized around 408-425 nm. The peak position of the Soret band depends on the heme structure, and in cytochromes, this will depend on whether cytochrome is oxidized or reduced. 

The EPR-nondetectable ions in laccase function as a cooperative 2-e" unit (5). With cytochrome oxidase redox titrations based on the heme absorption bands (2) indicate the presence of a high and a low potential site (380 and 220 mV, respectively). On the other hand, the quasi equilibrium established in the rapid initial transfer of electrons from reduced cytochrome c to the primary electron acceptor in the oxidase, cytochrome a, indicates a potential of 285 mV for this site (18). 

Table 14-1 Absorption Bands (Amax) of Cytochromes (position of bands in nanometers, cytochromes in reduced state)...

There are now good theoretical descriptions of the electronic structures contributing to the optical absorption bands in spectra of porphyrin radicals and ferryl species [160,167] most charge-transfer bands in the latter are due to a transition from a porphyrin p orbital to an Fe-0 tt orbital [167], However, in the absence of a prior knowledge of the structure around the Felv site (and/or spectra of a variety of synthetic model compounds) it is not straightforward to assign an optical spectrum to a ferryl species. Thus the intermediate assumed to be the ferryl species in the binuclear haem c /Cub centre of cytochrome c oxidase [168] has a spectrum at 580 nm essentially identical [169] to that of low-spin ferric haem a3 compounds (e.g. cyanide). 

The cytochromes c (and / ) have an additional absorption band at 695 m/t which has been discussed earlier. 

These cytochromes contain haem a which differs from the haem of other haem-proteins in that it has an unsaturated substituent, —CHO. In accord with theoretical expectation such a substituent shifts all the absorption bands to lower energy, and increases the intensity of the a/J (especially the a) bands relative to the Soret band. Thus in this series both Fe(II) and Fe(III) haem a complexes have well-pronounced a-bands. The introduction of an aldehyde substituent is also likely to stabilise low-spin as opposed to high-spin states. Thus it is not surprising that magnetic susceptibility data on the cytochromes a show that neither the Fe(II) nor the Fe(III) forms are more than 75% high-spin (133). 

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). 

---