Cytochrome a-band

Fig. 10. Absorption spectrum of purified tubular membrane of Rb. sphaeroides at 77 K. The inset shows the dithionite-minus-ferricyanide difference spectrum of the same sample in the cytochrome a-band region. The right side shows results of gel-electrophoresis measurements. See text for discussion. Figure and data source Jungas, Ranck, Rigaud, Joliot and Vermdglio (1999) Supramoiecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides. EMBO J 18 536.

FIGURE 2. Spectra of absorbance changes in the cytochrome a-band region at selected time-intervals after the flash. The extents of absorbance changes, relative to the dark level, are plotted against wavelength of measurement. 

FIGURE 1. Flash-induced absorbance changes in PSI vesicles at 518 nm (A) and in the cytochrome a-band region (B). 

Absorbance changes elicited by 4 ]is flashes of red light were measured as previously described (Crowther, Hind, 1980). The Flash frequency was 1 Hz and traces were the average of 64 (P700 turnover) or 128 (cytochrome a-band region) records. Traces in the cytochrome a-band region were corrected for contributions of P700. 

Figure 6.3 (a) Visible absorption spectrum of cytochrome c in its reduced and oxidized states, (b) The three separate a bands in the visible spectrum of beef heart mitochondria (below) indicating the presence of cytochromes a, b and c, with the spectrum of cytochrome c (above) as reference. (From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc.)... 

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. 

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 ... 

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 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]. 

The position of the a-band lies in the ranges 580-590 (a), 555-560 (b), 548-552 (c) and 600-620 nm (d). The literature without doubt contains some cytochromes that are classified incorrectly. In addition there are cytochromes not covered by this classification. 

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. 

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). 

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). 

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). 

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