568 Discussion 



Drabkin: 1 would like to speak to Morton's question concerning possible evidence in 

 favour of his most interesting model of cytochrome b.2, structure in the bonding of the 

 protein with the protohaemin nucleus, namely his proposal that the isoalloxazine 

 nucleus is sufficiently close to the haemin so as to influence it. What I say applies 

 only to the spectrum, not to the role of the substance as an enzyme. 



As Chance knows, I have been anxious to re-examine cytochrome b^ spectro- 

 scopically. Morton's published spectrum of the reduced form was most provocative 

 from the viewpoint of additional light it shed on my analytical procedure (this 

 volume, p. 142). There were four main points of interest: 



(1) There was the band at 260 m/f, certainly due to the riboflavin phosphate, which 

 did not belong to my postulated main series. (2) There was nearly complete masking 

 of the so-called protein peak at 280 m/i (my No. 8 band), although there was a very 

 slight inflection in this region. (3) There was an anomalous band at about 475 m/j. 

 (4) Above all, all the maxima, a, /3, y (or No. 6) and d (or No. 7) were appreciably 

 elevated in comparison with corresponding maxima in the more usual chromoproteins. 



I have analysed the ultra-violet portion of the spectrum by my procedure. The 

 presence of two definite bands at 280 and 260 m//, which closely overlap, explains the 

 high extinction at 260 m/<, but does not fully account for the marked elevation of the 

 y-band, nor the more moderate elevation of the a- and /5-bands. 



It is perhaps possible to account for the maximum at 475 m// by the absorption 

 due to the flavin phosphate. I was hence left with two possible explanations. There 

 was either some systematic error in the spectral measurements (probably unlikely), or 

 indeed some modifying influence upon the haemin itself. Morton's proposal that the 

 flavin nucleus is in the proximity of the haemin is appealing from the viewpoint of the 

 modifying influence such a structure might have upon the absorption spectra of the 

 iron-porphyrin complex. 

 O'Hagan: I should like to draw attention to the spectra of a haemoprotein with the 

 opposite type of spectra to that described by Morton, namely, one with a very low 

 Soret peak. This may be seen in the curves for aetiomyoglobin and its CO- and ferri- 

 derivatives described by O'Hagan and George {Biochem. J. 74, 424, 1960). Meso- 

 haemin and aetiohaemin in chloroform have essentially the same absorption spectrum, 

 but when linked to apomyoglobin the meso spectrum is much more pronounced. It 

 may be that in cytochrome b^ a very strong linkage of the haem propionate groups to 

 the apoprotein may contribute to the absorption. 

 George: Is it possible that direct co-ordination between flavin and haem iron (presumably 

 through one of the nitrogen atoms) could conceivably play any part in cytochrome b^ 

 reactions? 

 Morton: We have given some consideration to this type of compound. In such a complex, 

 one might expect that the extinction coefficients of the principal absorption bands of 

 the haemochrome would be elevated as compared with haemochromes formed with 

 the flavin-free system. 



As pointed out in our paper (Morton, Armstrong and Appleby, this volume, 

 p. 510), we have observed a temporary shift of the a- and /^-bands of ferrocytochrome 

 ^2 by about 10 m/t towards longer wavelengths to occur under certain conditions. 

 This suggests that there is some type of flavin-haem interaction when the iactate'- 

 cytochrome b^ complex has lost one electron, or: viz. a free radical is formed. This 

 would support the idea that a nitrogen atom of the iso-alloxazine ring may be co- 

 ordinated to the iron atom of the protohaem in cytochrome b^. 



Of course, as suggested by Falk there may be steric restrictions. 



It is apparent that there is a need for study of possible complexes of riboflavin and 

 of related compounds with protohaem. 



The Oxidation-reduction Changes in the Reaction of Lactate with Cytochrome b^ 



By E. BoERi AND E. Cutolo (Ferrara) 

 BoERi : One experiment which we performed seems to me important and I want to refer 

 to it. We have titrated flavocytochrome b^ with the substrate. The titrations were 



