510 



R. K. Morton, J, McD. Armstrong and C. A. Appleby 



Z?2 might be expected to shift as compared with those of pyridine proto- 

 haemochrome. Table 4 shows that this is not so. 



However, when a freshly-prepared pellet of crystalline cytochrome bo is 

 washed and dissolved in 0-5 m NaCl, containing 0-1 niM EDTA at pH 6-8 

 and at 0°, and then gently shaken in air, the intense absorption bands of the 



250 300 350 400 450 500 550 600 

 Wovelength, m/^ 



Fig. 3. Lactate-reduced crystalline (deoxyribonucleoprotein) material at 



pH 6-5 {A), 4-7 {B) and 100 (C). Curve D is of lactate-reduced nucleotide-free 



flavohaemoprotein at pH 6-8. 



reduced enzyme at 556-5 and 527 m/^ slowly fade and are replaced by some 

 what weaker bands at 567 and 533 m,a (Appleby, 1957 ; Appleby and Morton, 

 1959b). These bands fade completely on further oxidation. Addition of 

 further lactate, or of NaaSgOj, causes immediate re-appearance of the bands 

 of the reduced enzyme, at 556-5 and 527 m/^. The oxidized cytochrome b^ 

 has a typical ferrihaemochrome-type spectrum, with a weak, broad band 

 between 530 and 560 m/t (Fig. 2). Appleby and Morton (1959b) therefore 

 considered that the bands at 567 and 533 m/« were those of the enzyme 

 at an intermediate state of oxidation. If there was formed a stabilized 

 (lavohaemoprotein-lactate complex, in which the resonance pathway is 

 lengthened by interaction of the 77-electron systems of the flavin and haem 

 prosthetic groups, it would be expected that the absorption bands would be 

 at longer wavelengths as compared with those of the fully-reduced flavo- 

 haemoprotein. 



Our interpretation of the transient appearance of absorption bands at 567 

 and 533 m/i is that they are indeed the bands of an intermediate flavohaemo- 

 protein-lactate complex, and that they are due to the influence of lactate and 



