VOL. 12 (1953) COMPOUNDS OF FERRICYTOCHROME C 281 



that the differences in the mode of hnkage between hemin and protein in HRP and 

 cytochrome c are smaller than previously believed. 



Lewis, working in this institute^^, has found that the splitting of ferrihemoglobin 

 or ferrimyoglobin into hemin + colourless protein by means of HCl plus acetone occurred 

 at a considerably higher pH if NaCl was added. A similar but less pronounced effect was 

 observed for sulphate ions. 



The cooperation of anions, e.g. CI"-, with protons in the splitting of heme-protein 

 bonds thus appears to be a general phenomenon. Cytochrome c is the first case in which 

 it has been possible to give a structural explanation of this anion effect. 



ACKNOWLEDGEMENTS 



The authors' thanks are due to the Rockefeller Foundation for a fellowship to one 

 of us (E.B.) and to Knut och Alice Wallenbergs Stiftelse, Magn. Bergvalls Stiftelse and 

 the Rockefeller Foundation for financial support. 



SUMMARY 



1. The absorption spectra in the Soret band region and the magnetic properties of ferricyto- 

 chrome c at various chloride ion concentrations have been reinvestigated within the range pH 1-5. 



2. The form of ferricytochrome c, which exists at neutral pH ("Cyt", /Smax = 23.3 • 

 mole"^ at 40S m//) changes upon acidification to an acid form ("Cyt-2H+", /Jmax = 50' 

 mole~i at 395 m/i) with the simultaneous uptake of two protons (n = 2). Half of the total spectral 

 change is reached at pH 2.12. Simultaneously to the spectral change the paramagnetic susceptibility 

 increases from y,„ = 2120 (i odd electron) to 16,200- io~^ cgs emu (5 odd electrons). The values for 

 Cyt-2H+ have been obtained by extrapolation to [Cl~] = o. 



3. When increasing amounts of chloride ions are added to a solution, containing cytochrome c 

 mainly as Cyt-2H+, the cytochrome is gradually converted into another form, denoted Cyt-2H+-2C1~ 

 (j^max = 25.8- 10^ cm^ x mole"-'- at 402 m/n). Two chloride ions are taken up simultaneously {n = 2). 

 Kci- was found to be 7- lo^^ M- at pH i and about ten times lower at pH 2 and 3. Parallelling these 

 spectral changes the paramagnetic susceptibility decreases to Xm = 6000 at pH 3 and 7400- io~^ cgs 

 emu at pH 1.5 (3 odd electrons). The differences in Kc\- and y„t of Cyt-2H+-2C1"" at pH 1-1.5 are 

 tentatively explained as depending upon protein disconfiguration due to the acid milieu. 



4. The change from Cyt to Cyt-2H+ is interpreted as corresponding to a replacement of covalent 

 links between the iron atom and the two hemichrome-forming groups of the protein moiety by 

 ionic links. In Cyt-2H+-2C1~ a dipole chain arrangement is assumed. 



RfiSUMfi 



1. L'etude des spectres d'absorption dans la region de la bande de Soret et des proprietes 

 magnetiques du ferricytochrome c, en presence de diverses concentrations d'ions chlorures, a ^te 

 reprise entre pH i et pH 5. 



2. Le ferricytochrome c se presente a pH neutre sous une forme ("Cyt", jSmax = 23.3- 10' cni- 

 X mole"^ a 408 m/^) qui, paracidification, donne une forme acide ("Cyt"-2 H+, /^max = 50-10^ cm- 

 X mol~^ a 395 m/f), en iixant deux protons en meme temps (w = 2). La moitie du changement spectral 

 est atteinte a pH 2.12. En meme temps que le spectre se modifie, la susceptibilite paramagnetique 

 augmente de Xm = 2120 (i electron non aparie) a 16,200- lo"^ unites C.G.S. (5 electrons non aparies). 

 Ces valeurs pour Cyt-2H+ ont ete obtenues par extrapolation en supposant [Cl~] = o. 



3. Quand on augmente la quantite d'ions chlorure ajoutes a une solution contenant la plus 

 grande partie du cytochrome c sous sa forme Cyt-2H+, le cytochrome prend progressivement une 

 autre forme designee par Cyt-2H+-2 Cl~ (/Smax = 25.8- 10' cm^ X mol~^ a 402 mfi). Deux ions chlorures 

 sont fixes en meme temps {n = 2). Kci~ est de 7-10^^ M^ a pH i et environ dix fois plus faible a 

 pH 2 et 3. Parallelement a ces modifications du spectre, la susceptibilite paramagnetique decroit 

 jusqu'a x„i = 6000 a pH 3 et 7400- io~^ unites C.G.S. a pH 1.5 (3 electrons non aparies). II est possible 

 que les differences de Kqi- et y„i de Cyt-2H+-2 Cl~ a pH 1-1.5, s'expliquent par une modification 

 de la configuration de la proteine, due a I'acidite du milieu. 



References p. 282. 



