Electron transfer between ferro- and ferricytochrome c

BIOCHIMICA ET BIOPHYSICA ACTA

SHORT COMMUNICATIONS

sc 63178 Electron transfer between ferro- and ferricytochrome c The reduction of ferricytochrome c is known to be accompanied by a change in protein conformation. Thus, the ferri and ferro forms of this protein differ in susceptibility to tryptic digestion", crystal form 2, chromatographic behaviors and optical rotary dispersions. The necessity for this change of conformation might present a kinetic barrier to electron transfer between ferro- and ferricytochrome c. The large proportion of basic amino acid residues in cytochrome c5 and the consequent preponderance of cationic sites might also hinder electron transfer from the ferro to the ferri forms by virtue of the repulsion of like-charged molecules. Nevertheless, a very rapid electron transfer between the ferro and ferri forms of cytochrome c has been inferred from studies of the proton magnetic resonance of mixtures of these proteins", In the present report ferricytochrome c labeled by guanidination with [14C]O-methyl isourea is found to equilibrate rapidly with guanidinated ferrocytochrome c, O-methyl isourea and 14C-labeled O-methyl isourea as the neutral sulfates were prepared from cyanamide by the method of BELLo? [14CJcyanamide was obtained from the International Chemical and Nuclear Corporation. Radioactivity was measured by scintillation counting. Cytochrome c was dispersed through the use of hyamine or was dried onto slips of filter paper which were then placed directly in the phosphor-containing solvent. In either case corrections for quenching were made 0.8

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Fig. r A, Separation of ferro- and ferricytochrome c. LO zzrnole of ferro cytochrome cPlus LO fbtllole of ferricytochrome c in 2.0 ml of 0.05 M potassium phosphate buffer containing 10-' M ethylenediarninetetraacetare (pH 7.8) were placed on a I em X 23 em column of Amberlite CG-50 (200400 mesh) which had been thoroughly washed-! and equilibrated with 0.25 M ammonium acetate. The column was eluted with 0.25 M ammonium acetate at a flow rate of 0.2 mljmin and 6.0-ml fractions were collected. The absorbance of each fraction was measured at 550 m!l before (O-o) and after (0--0) the addition of a few crystals of sodium dithionite.

Biochim, Biopbys, Acta, lIS (1966) 419-42I

SHORT COM:\WNICATIONS

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through the use of internal standards. Horse-heart cytochrome c was obtained from the Sigma Chemical Company. Polymeric material was removed by the procedure of MARGOLIASH 8 . Monomeric cytochrome c was guanidinated for 72 h by the method of TAKAHASHI et al», It was then exhaustively dialyzed against cold distilled water. Dialysis membranes which had had their pore size reduced by heating"? were used to prevent excessive losses of cytochrome c during extended dialysis. Based on measurements of radioactivity it can be inferred that approximately sixteen guanidino groups were introduced per molecule of cytochrome c. Ferro- and ferricytochrome c were separated by column chromatography", The ion-exchange resin used was prepared according to MARGOLIASH AND LUSTGARTEN l l . Ferrocytochrome c was prepared by reduction of the corresponding ferri form with a minimal amount of dithionite followed by exhaustive dialysis against 0.05 M potassium phosphate, 10-3 M ethylenediaminetetraacetate at pH 7.8. All dialyses were performed at 0°. 0.32

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Fig. r B, Separation of ferro- and ferriguanidinated cytochrome G. 0.4 pmole of ferroguanidinated cytochrome G plus 0.4 pmole of ferriguanidinatecl cytochrome c in 4.0 ml of buffcr (pH 7.8) were placed on the column. For procedural details see Fig. IA.

As shown in Figs. IA and IE the oxidized and reduced forms of cytochrome c and of guanidinated cytochrome c were readily separated. In both cases the reduced form emerged from the column well before the oxidized form. The small amount of ferricytochrome c evident in the ferro cytochrome c peak must have been generated by air oxidation during the many hours which elapsed between elution from the column and the measurement of absorbances. Guanidinated ferrocytochrome c (2.0 ml of 2' 10-4 M) was mixed with an equal amount of 14C-labeled guanidinated. ferricytochrome c at 00. The solvent for both was 0.05 M potassium phosphate, ro- 3 M ethylenediarninetetraacetate at pH 7.8. This mixture was immediately placed on a colunm of Amberlite CG-50 and elution was carried out as described in Fig. IA. The ferro- and ferricytochrome c bands were visibly separated on the column within IS min. Guaniclinated ferro- and ferricytochrome c were recovered from the respective elution peaks by lyophilization Biochim; Biophys. Acta, II8 (1966) 419-421

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an d radioactivity was measured . Both were equ ally lab eled, emitting approximately IO 000 counts/min per ,umole. It follows th at guanidinat ed ferro- and ferricyto chrome c are capable of ra pid equilibrat ion by elect r on transfer. Since guanidination is re ported " not to change the obser vable properties of cytochrome c and since gu anidination does n ot change the charge of the amino groups derivatized , it may be inferred that ferro- and ferricytochrome c, p er se, wou ld very likel y be capab le of similar rapid electron exchange in dilute, aqueo us solutio ns. It has recently been re porte d v that adsorbed and soluble cy toc hrome c were red uced a t similar rates by washed heart-muscle particles. From this it was conclu ded t hat adso rption and desorption of cyt ochr ome c to these particles is a rapid pro cess. In view of the result report ed above, physi cal exchange between bound and soluble cytochrome c need not be in voked to explain such d ata since electron transfer between bound and soluble cyto chrome c could provide an adequate explanat ion. It has been reported'< that the cytochrome oxidase isolated from Pseudomonas aentginosa acts rapidly on th e cytochrome c isolated fr om this organism but not on yeast cytochrome c and that the presence of a small amount of the Pseudomonas cy t ochrome c then permits the oxidat ion of the yeast cyto chrome c. No ex planation was offered for this observati on but it is now clear that r apid electron transfer bet ween the cytochromes offers one . This work was supporte d in full by Grant GM-IOZ87-0 3 from the Nat ional Institu t e of Genera l Medica l Sciences, National I nstitutes of H ealth, Bethesda, Md . Dep artment of B iochem istry , D uke University Med ical Cent er,

IRWIN FRIDOVI CH

Durham , N .C. ( U.S .A.) I 2

3 4 5 6

7 8 9 10 .II

12

13

N OZA KI , M . MIZUSHIMA, T . HORro AN D K. OIWN U K I, ] . B iochem, Toky o, 45 (1 95 8) 81 5 . D. M. BLOW, G. BODO, M. G. ROS SMAN AND C. P o S. TAY LOR, J. Mo l. B ioI., 8 (19 6 4) 606. S . P ALEUS AN D ] . B. N IE LANDS , Acta Chern. S cand., 4 (195 0) 10 24. D. W . URRY AN D P . D OTY , ] . Am. Chern. So c. , 87 ( 1965) 2756. E . MA RGO L I AS H , J. B ioI. Chem., 237 (19 62) 2161. A. KO WALSK Y , B iochemistry , 4 (1965) 23 82 . ] . B E L L O, B iochim , B ioPhys. Ac ta, 18 (1955) 448. E. MARGOLIASH , B iochem , ]. , 56 (19 54) 535. K. TAKAHASHI, K. TITAN I , K . F ORUNO AND H. ISHIKUR A, j. B iochem- Tokyo, 45 (19 5 8) 37 5 · D. W. KUPKE, Comp t. Rend. T ra1J . Lab. Carlsberg, 32 (1960) 107 E. l\i[ARGOLIASH AND ] . LUSTGA RTEN, ]. BioI. Chem., 23 7 (!965) 3397· L. SMITH AND K. MINNAE RT , B iochi m , Biophys, Acta, 105 (1965) I . T . YAMANAKA AND K . O KU N U K I, B iochim. Biophys. .'lela, 6 7 (19 6 3 ) 379·

M.

Received January 25th, 1966 Biochim. B iop hy s. Acta, 118 (1966) 4 19-42 1