Cytochromes Cooled in Liquid Nitrogen 459 



soy bean root nodules, by simple extraction with ethyl acetate-acetic acid, of a haem 

 similar to haem c. 



It appears unlikely that the prosthetic group of yeast cytochrome c differs from that 

 of heart-muscle cytochrome c, yet these observations suggest that there may be only 

 one thio-ether link to the polypeptide chain in yeast cytochrome c, or possibly true 

 ether bonds instead of thio-ether bonds. J. Keilin {Biochem. J. 64, 663, 1956) has 

 pointed out a somewhat related position in respect of cytochrome It. Is there any 

 information as to the influence of the protein structure itself on the splitting of the 

 prosthetic group of cytochrome c by Paul's silver sulphate method? 

 Margouash: We have repeatedly observed that Paul's silver sulphate method for the 

 splitting off of the prosthetic group of cytochrome c works very readily with native 

 horse-heart cytochrome c and that the haem is entirely liberated within about one 

 hour at 60'C. However, when the peptic 'core' of cytochrome c, containing only an 

 1 1 -amino acid peptide is used, the reaction proceeds much more slowly and takes from 

 18-24 hr to go to completion. This is contrary to what may be expected, since if 

 anything, the presence of the entire intact protein should interfere with and not 

 facilitate an attack on a bond presumably holding the haem in a crevice. 

 Lemberg: While it appears to me unlikely that yeast cytochrome c contains mesohaem (it 

 could not then combine by thioether linkages with the protein) I should like to make a 

 plea to chemists interested in cytochromes c to investigate the compound obtained by 

 the silver sulphate method splitting in acetic acid more carefully. It should e.g. be 

 possible to differentiate between haematoporphyrin, vinyl-a-hydroxyethyl deutero- 

 porphyrin and ethyl-a-hydroxyethyl deuteroporphyrin. Unfortunately we have 

 obtained some evidence that the silver sulphate method may yield artifacts and requires 

 further study to exclude secondary alterations. 



Origin of tiie Fine Structure in the Absorption Spectra of Cytochromes at — 190°C 



Orgel: According to Piatt's {Radiation Biology 3, 101, 1956) theory of porphyrin spectra 

 the lowest excited state of a metal porphyrin with four-fold symmetry is doubly 

 degenerate and it is the splitting of this degeneracy which produces the more com- 

 plicated spectra of the free porphyrins since the latter have only two-fold symmetry. 

 I wonder if the splitting which Estabrook observes at low temperatures is a similar 

 but smaller splitting of the degeneracy of the excited state caused : 



1 . by the different side chains ; 



2. by the unsymmetrical environment of the protein. 



One would anticipate that such splittings would be very small and might only be 

 revealed when the absorption bands become very narrow as they do at sufficiently low 

 temperatures. It should be noted that splittings due to different types of asymmetry 

 might either add up or cancel out depending on the exact geometry of the situation. 



As suggested by Margoliash, the asymmetry might be envisaged as due to different 

 groups bound to co-ordination positions 5 and 6 of the iron, above and below the 

 plane of the haem. 



Estabrook: These possibilities certainly exist. One difficulty in interpretation which 

 must be considered is that one does not see a split of the absorption bands of haemo- 

 globin, myoglobin, catalase, or cytochrome a at the temperature of liquid nitrogen. 

 The inability to see a split with these compounds may be resolved when we complete 

 adapting the instrument for spectra at the temperature of liquid helium. 



Williams: The resolution of the fine structure of the a- and /?-bands of cytochromes on 

 cooling to low temperature is to be contrasted with the absence of resolution in the 

 Soret band region. If we accept that the resolution is due to coupling of vibrational 

 with electronic transitions then there are at least two explanations. The first is that 

 given by Orgel in which the vibrational structure is due to a breakdown of the four-fold 

 symmetry by substituents ; the other arises from the different natures of the electronic 

 transitions involved and would occur for a totally symmetrical porphyrin. The 

 position can be understood by reference to the spectrum of benzene which has a long 

 wave-length band with vibrational structure and a shorter wave-length band without 



