Cytochrome bg 567 



on adding lactate to the preparation. The difference in the behaviour of intact, non- 

 fluorescent, crystalline cytochrome b.,, and denatured cytochrome 6, indeed recall the 

 differences in the reactions of cytochrome h in the intact mitochondrial system as com- 

 pared with the reaction in the modified, non-phosphorylating fragments of sarcosomes. 

 Hence, at the present time, 1 feel that the conclusions of Chance (p. 565), that the 

 reaction of the flavin with the haematin of cytochrome ^^ is intermolecular rather than 

 intramolecular, must be treated with caution. The opposite conclusion reached in our 

 studies (Morton et ai, this volume, p. 519) may possibly be attributed to the difference 

 in the relative intactness of the preparations of cytochrome b^, used, and to the con- 

 ditions of the experiments. 



Chance: The turnover number of the preparation was 220 sec"^ at 26°C. 



The Contribution of the Prosthetic Groups to the Absorption 

 Spectrum of Cytochrome h^ 



Morton: The extinction coefficients (expressed on the basis of millimolar concentration 

 of protein-bound haem) are appreciably greater for the absorption bands of both 

 reduced and oxidized cytochrome 63 than for any other B group cytochrome as yet 

 described (see Table 5, Morton, Rev. pure appl. Chem. 8, 161, 1958). This is not 

 merely due to the occurrence of narrow, high absorption bands. The same picture 

 would emerge if areas under the bands were considered or extinctions were plotted 

 against frequency rather than wavelength. In the reduced cytochrome b.^,, the positions 

 of the a-, P- and y-bands (at room temperature) are very close to those of pyridine 

 protohaemochrome. 



As shown by Table 4 of our paper (Morton, Armstrong and Appleby, this volume, 

 p. 509), nucleotide-free cytochrome 6, has similar absorption bands in the visible 

 portion of the spectrum, but removal of the polydeoxyribonucleotide causes a shift 

 of the U.V. absorption band from 265 m/< to about 268 m//, and a decline of f^M from 

 about 199 at 265 m/< to about 135 at 268 m/<. In the flavin-free haemoprotein deriva- 

 tive, the U.V. maximum shifts to about 278 m/t and there is a further decline in CmM- 

 Hence the flavin makes a significant and readily-detectable contribution to the absorp- 

 tion spectrum of cytochrome b^, in the U.V. region. In the visible region, however, 

 the eflfects of the flavin group on the absorption spectrum are not so apparent. The 

 influence of the flavin group can be seen in the region of the cJ-band and at about 

 450-470 m// by comparison of reduced and oxidized cytochrome b^, (see Figs. 2 and 3 

 of Morton et at., this volume, p. 501 ; also Appleby and Morton, 1954, 1959, loc. cit.). 

 In the fully oxidized material (Fig. 2) and in the reduced material at pH 10 (Fig. 3), 

 however, much of the flavin would have dissociated from the enzyme. Hasegawa and 

 Ogura (this volume, p. 534) have provided direct evidence of the contribution of ribo- 

 flavin phosphate to the absorption spectrum since the difference spectrum (oxidized 

 minus reduced) of their dye-treated enzyme is that of oxidized flavin. 



The transient shift in the absorption bands of the lactate-reduced cytochrome b^ 

 when oxidized by air from approximately 557 and 528 m/< to 567 and 533 m// respec- 

 tively provides evidence of the formation of a 'lactate' — cytochrome b^ complex, since 

 the fully-reduced and fully oxidized compounds show no such unusual absorption bands, 

 (see Appleby and Morton, 1959, loc. cit., and Morton et at., this volume, p. 510). 



In order that such a complex may be formed and the absorption bands of the haemo- 

 protein be aff"ected, there must be a close juxtaposition of the haem and the flavin 

 groups on the protein so that direct interaction may occur between these groups in the 

 presence of 'lactate', thus accounting for the shift of the a- and /^-bands of the fully- 

 reduced cytochrome b.^. The considerable elevation of the extinction coefficients of 

 the absorption bands of cytochrome b.. as compared with other B group cytochromes 

 may also indicate some type of interaction of the flavin group with the haem group. 

 Another possible explanation, as suggested by Kamen, is that the enzyme exists as a 

 polymer of the monomeric structure of weight 80,000 g/mole, and that the elevation 

 of the absorption bands is due to the effects of polymerization. 



