VOL. 12 (1953) COMPOUNDS OF FERRICYTOCHROME C 279 



the theoretical maximum for five odd electrons, 5.92. Considering the uncertainty of the 

 calculations, this difference is insignificant. Our conclusion is that in C3^t-2H+ the iron is 

 held by essentially ionic bonds. 



DISCUSSION 



On the basis of these results we arrive at the following picture of the compounds of 

 ferricytochrome c in acid solution. The links between the iron atom and the hemichrome- 

 forming group in Cyt can be broken by two protons. The two dissociation steps are very 

 strongly linked, [n = 2), the entrance of one proton greatly facilitating the entrance of 

 the second one. Two chloride ions can enter into Cyt-2H+. The interaction between the 

 two steps is again so strong that w = 2. We may thus represent the relations between 

 the three compounds by the formula: 



As seen in the formula, we presume that the chloride ions are interposed between posi- 

 tive nitrogen and iron atoms, forming a dipole chain system. Neither a ferric ion, nor a 

 positively charged nitrogen atom alone would be able to form compounds of low dissocia- 

 tion constants with chloride ions. But in this case the nitrogen atoms probably are held 

 by the protein part in essentially intact positions relative to the iron after the entrance 

 of the two protons. We may recall that in cytochrome c the prosthetic group and the 

 protein part are firmly joined by two thioether linkages^*-^^. It is, therefore, easy to 

 imagine that anions must be strongly attracted into the gaps between =^ N+H and Fe+. 

 There is an interesting sort of interaction between protons and anions; they cooperate 

 synergistically in the formation of the dipole chain. The distance between =^ N+H and 

 Fe+ must be of decisive importance for the association of the anions. The larger Kq_ at 

 pH I, compared with that of pH 2, can be explained if we assume that a deformation 

 of the protein part the molecule takes place at low pH, increasing the distance between 

 = N+H and Fe+ 



The results from 1941I''* suggested that the iron-linked nitrogen atoms were split 

 off one after the other at widely different pH values, giving three types (HI, H, I) of 

 ferricytochrome c with the transitions occurring at pH 2.5 and 0.5 respectively. "Type 

 HI" corresponds to "Cyt" in this paper, while "Type H" must have included both 

 Cyt-2H+ and Cyt-2H+-2Cl~.The older results were based on spectrophotometric deter- 

 minations in visible light (470 and 650 m/x) In connection with the present investiga- 

 tion a few experiments were made at 620 myu. They indicated a chloride-sensitive dis- 

 sociation constant pA' 2.5, in agreement with the old value. However, we now observed 

 a slow drift of the optical density with time amounting to a few per cent. This observa- 

 tion remains to be interpreted. Such complications did not seem to appear in the Soret 

 band region. 



References p. 282. 



