Electromctric and Other Studies of Cytochrome c 389 



ndicates that the modified cytochrome c has a more unfolded protein-structure than 

 native cytochrome c. With the same enzyme, native cytochrome c crystallized from 

 baker's yeast is much more rapidly digested than those from ox- and horse-heart. 



Using native cytochrome c, the reduced form is much more resistant to digestion 

 by the proteolytic enzyme than the oxidized form. In fact, if the rapid autoxidation of 

 the reduced cytochrome c, (which is catalyzed by proteolytic products obtained by 

 digestion of oxidized cytochrome c) is prevented by using anaerobic conditions, the 

 reduced form is not attacked by the proteolytic enzyme at all. 



On the other hand, if one uses crystalline enzymes, for example, bacterial amylase, 

 yeast alcohol dehydrogenase, animal triose-phosphate dehydrogenase, animal lactate 

 dehydrogenase, the enzymes in their enzymically-active state are not digested by the 

 proteolytic enzyme, while the denatured enzymes (regardless of whether the denatura- 

 tion is reversible or irreversible), are rapidly digested. Kunitz and co-workers have 

 reported that crystalline yeast hexokinase is digested by trypsin and that the enzyme 

 becomes more resistant against the digestion in the presence of the substrates than in 

 the absence (glucose > fructose > mannose). 



These considerations indicate that the reduced form of cytochrome c has a more 

 rigidly folded protein-configuration than the oxidized form. 



As mentioned by Smith in connexion with estimation of cytochrome c oxidase 

 activity (p. 260), using cytochrome c modified in its monomer state, one may not find 

 serious differences in enzymic reduction and oxidation of native and modified cyto- 

 chrome c. But generally speaking, enzyme proteins lose their enzymic activity at the 

 same time as they lose the folded (coiled) protein-configuration. This concept does 

 not fit with the enzymic reduction and oxidation of modified cytochrome c. From this 

 point of view, I am just wondering what function is coupled with the folded con- 

 figuration of the native cytochrome c, and with the alteration in the folded protein- 

 configuration which may result from the oxidation-reduction reaction of cytochrome c. 



Reactivity of Native Cytochrome c in Oxidative Phosphorylation 



Morrison: As Henderson and Rawlinson (p. 369) have pointed out, the modified heart- 

 muscle cytochrome c (fraction 2) obtained by the chromatographic method of Margo- 

 liash does not appear to have markedly decreased biological activity as judged by its 

 ability to transport electrons. This is not true when the role of cytochrome c in 

 oxidative phosphorylation is assayed (Morrison, HoUocher and Stotz, Arch. Biochem. 

 Biophys. 92, 33S, 1961. 



When this modified cytochrome c fraction was added to tightly-coupled rat liver 

 mitochrondria, there was no increase in this rate of phosphorylation with added 

 cytochrome c. This is in marked contrast with the results obtained with the so-called 

 'native' fraction of cytochrome c (fraction 1). 



These results point to the direct participation of the cytochrome c molecule in the 

 oxidative phosphorylative mechanism. 



Structure of Bacterial Cytochromes of c- type 



Postgate: My question is addressed to Margoliash. Several of the bacterial cytochromes 

 of the c-type are neutral or acid (see Morton, Rev. pure appl. Chem. 8, 161, 1958). 

 If electrostatic links between polar groups in the protein are responsible for the very 

 great thermal stability of this group of proteins, would not this thermostability be pH- 

 dependent? If so, would not a study of the pH-dependence of the thermostability 

 of a basic and a neutral cytochrome c show differences bearing on the nature of the 

 linkage in folding of the peptide chain ? 



Margoliash: My idea of hydrophobically shielded electrostatic bonds as the bonds 

 conferring on cytochrome c its native folded configuration and relative resistance to 

 some types of denaturation procedures, such as heating, was advanced for the common 

 mammalian types of cytochrome c which are strongly basic proteins. The thermo- 

 stability of such cytochromes c is certainly pH-dependent. 



