Cytochrome Soret band

The third class of haemoproteins, with hexa-coordinate low-spin iron, are the cytochromes. First discovered by McMunn in 1884, they were rediscovered in 1925 by David Keilin. Using a hand spectroscope he observed the characteristic absorption (Soret) bands of the three cytochromes a, b and c in respiring yeast cells, which disappeared upon oxygenation. 

In that protein, rhodospirillum x ubrum cytochrome cc1, which shows the most remarkable shift of the Soret band to short wavelengths for the Fe(III) state at low pH there is an opposite shift to long wavelengths in the Fe(II) state, Fig. 8 (67). In fact this cytochrome behaves remarkably like P-450 in its isonitrile complexes. 

Cytochrome P-450 enzymes have been isolated from a variety of mammalian tissues, insects, plants, yeasts and bacteria. The P-450 cytochromes (Gunter and Turner, 1991) are membrane bound mono-oxygenase enzymes which catalyse oxygen atom transfer to entrapped non-polar substrates. The binding of carbon monoxide to the enzyme produces a split in the 420 nm Soret band to give bands at 364 and 450 nm. The absorption at 450 nm distinguishes the hemoprotein from all others and hence provides... 

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. 

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. 

Some anomalous features appear occasionally, such as the weak 14 kK band of cytochrome-c (5, 52, 53), the split Soret band of cyto-chrome-cc (54), and the high energy of the Soret band in haem dimers and in some proteins (55). 

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). 

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. 

It has been shown recently that the mitochondrial electron transport system contains at least three different fe-type cytochromes 178). Two of these cytochromes are found in complex III, and under appropriate conditions are reducible with substrates. The third 6-type cytochrome was discovered by Davis et al. 178), and shown to fractionate exclusively into complex II. At 77°K, the cytochrome 6 of complex II exhibits a double a band at 557.5 and 550 nm, a prominent band at 531 nm, and a Soret band at 422 nm (Fig. 29). Cytochrome 6557.5 appears to have a low reduction potential. It is not detectably reduced by succinate in either complex II or respiratory particles, but its dithionite reduced form is rapidly oxidized by either fumarate or ubiquinone. The role of this cytochrome in mammalian mitochondria is not known. Davis et al. 178) have suggested that it might be an electron entry point for an unknown ancillary tributary of the respiratory chain. Further, Bruni and Racker 179) have shown that a preparation of cytochrome 6 is required for reconstitution of succinate-ubiquinone reductase activity (see below). 

Split-Soret Cytochrome. The split-Soret cytochrome (Ssc) and NapB proteins have no relationship except the fact that both contain a single stacked parallel diheme motif Ssc is a dimer of two identical 26.3 kDa subunits, which has so far been isolated only from the D. desulfuricans ATCC 27774, a species that can use sulfate or nitrate as terminal electron acceptors. Each monomer contains two c-type hemes with bis-histidinyl coordination and redox potentials of —168 and —330 mV. The name of this cytochrome derives from the fact that the reduced form displays a spht-Soret band with maxima at 420 mn and a shoulder at415mn. 

S. acidocaldarius (strain 7) contains a cyanide-sensitive cytochrome oxidase [24], The purified cytochrome (M, 150000) is composed of three subunits (M, 37000, 23 000, and 14000). Difference spectra following reduction with dithionite show a Soret band at 441 nm and a maximum at 603 nm characteristic of aa3-type cytochromes. In addition, there is a band at 558 nm whose connection to the oxidase is not clear. This oxidase is stimulated by cholate, but unlike the oxidase from the DSM 639 strain it is inhibited by low concentrations of cyanide (pM as opposed to mM) and oxidizes horse-heart cytochrome c, TMPD-ascorbate, and caldariella quinol. The rates of oxidation (pmol/min/mg protein) for cytochrome c, TMPD-ascorbate, and quinol are 63, 6.1, and 0.2, respectively. Another cytochrome oxidase that has an absorption maximum at 602 nm, oxidizes caldariella quinol, but does not oxidize cytochrome c, is also present in strain 7 so that the terminal portion of the electron transport system in S. acidocaldarius consists of at least three oxidases. It is suggested [8] that the presence of three oxidases in 5. acidocaldarius is unlikely and that the cyanide-sensitive oxidase was isolated from a different species, namely S. solfataricus. There is little taxonomic information in this assertion to judge whether strain 7 and DSM 639 are indeed different species. However, based on growth conditions reported by the investigators [12,28], which are unique for S. acidocaldarius and S. solfataricus [ 22, there is no reason to suspect that these organisms are different species. 

Cytochrome P450 monooxygenases, which are protoheme proteins, present an unusually red-shifted Soret band for the reduced CO complex at 450 nm [167]. Upon denaturation by various treatments, the cytochromes are always able to bind CO reversibly by losing their unique spectral properties as well as their catalytic activities. The spectral properties of the reduced carbonylated complexes of the denaturated form called P420 are identical to those of the dioxygen-carrying hemoproteins (Amax = 420 nm). These facts indicate that the unusual spectral properties of cytochrome P450 closely relate to its function. 

---