FOLDING zyxwvutsrqpon OF CYTOCHROME C Participation of the Protein Ligands in the Folding of Cytochrome zyxwvu c? Jorge Babul1 and Earle Stellwagen* ABSTRACT : Absorption spectral, circular dichroic spectral, and viscosity measurements indicate that the compact low- spin conformation characteristic of native cytochrome zyxwvuts c is quantitatively recovered from its extended high-spin confor- mation at pH 2 by titration to pH 4.0. This conformational transition has a midpoint of 2.5 and is very cooperative. Com- parison of the pH transitions of native and various carboxy- methylated derivatives of cytochrome c indicates that re- covery of the compact conformation of the protein is coinci- dent with coordination of histidyl-18 and does not require coordination of a second protein ligand. Extensive carboxy- methylation of cytochrome zyxw c including histidyl-18 stabilizes an unfolded high-spin conformation of the protein through- out the pH range 2-7. H orse heart ferricytochrome c is a relatively small glob- Materials and Methods ular protein containing 104 residues arranged in a known sequence along a single polypeptide chain (Margoliash zyxwvutsr et al., 1961). The protein contains no free sulfhydryl groups or di- sulfide bonds. A single heme moiety is covalently attached to the polypeptide by two thioether bridges. In the crystalline state, the polypeptide chain is folded about the heme moiety forming a nearly spherical molecule with a hydrophobic in- terior and a hydrophilic exterior (Dickerson et al., 1971). The heme iron is coordinated with two strong-field protein ligands, histidyl-1 8 and methionyl-80, and the heme moiety forms numerous noncovalent interactions with the polypeptide chain. The native conformation in solution as deduced by a variety of physical and chemical measurements is generally the same as that described for the crystalline state. The small size of cytochrome c, the absence of disulfide bonds, and the availability of a variety of measurements to characterize the conformation of the protein in solution makes cytochrome c an attractive model for studying the mechanism of folding of a polypeptide chain into its native conformation. The polypeptide chain of cytochrome c is extensively unfolded in the presence of concentrated urea solutions at neutral pH and is quantitatively refolded into the native conformation upon removal of the urea (Stellwagen, 1968b). However, spectral studies indicate that two strong- field protein ligands are coordinated with the heme iron in concentrated urea solution (Babul and Stellwagen, 1971). In order to study the entire folding process it is necessary to dis- sociate these protein ligands by acidification of the protein solution. This report examines the folding of the polypeptide chain of cytochrome c and various carboxymethylated de- rivatives upon neutralization of acidified protein solutions in the absence of urea and the roles of the two protein ligands for the heme iron in the refolding process. t From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52240. Receiwd June 18, 1971. This investigation was sup- ported by U. zyxwvutsrq S. Public Health Service Grant GM 13215 from the Institute of General Medical Sciences. $Taken in part from a dissertation submitted by J. B. in partial satisfaction of the requirements for the degree of Doctor of Philosophy, University of Iowa, 1971, Present address : Department of Biochemistry, University of Chile, Santiago, Chile. * U. S. Public Health Service Career Development awardee (Grant GM 08737) from the Institute of General Medical Sciences, Materials. Horse heart cytochrome c, type zyx I11 and VI, was obtained from the Sigma Chemical Co. The protein prepara- tions were quantitatively oxidized to ferricytochrome c which was used exclusively throughout this work. Bromoacetic acid was purchased from the Eastman Kodak Co. and recrystal- lized from petroleum ether (bp 30-60') prior to use. Methods. Amino acid compositions were determined using a Spinco 120C analyzer after hydrolysis of carboxymethylated derivatives of cytochrome c in 6 N HC1 for 24 hr zy in vacuo. The content of each amino acid was calculated relative to the sum of arginine, proline, leucine, and valine, assuming that each molecule of protein contains 13 of these residues. No corrections were made for destruction of amino acids during acid hydrolysis. The content of half-cystine and methionine was determined as cysteic acid and methionine sulfone, respec- tively, after oxidation with performic acid as described by Hirs (1967) prior to acid hydrolysis. Carboxymethylmethionine is not oxidized under these conditions (Neuman et al., 1962). All absorption spectra were obtained using a Cary Model 14 recording spectrophotometer. Extinction coefficients used to calculate the concentration of native ferricytochrome c at pH 7 and 410 nm, (CmMet)(CmHis) c1 at pH 7 and 408 nm, (CmMet)z c at pH 8 and 406 nm, (CmMet)2(CmHis)a c at pH 7 and 403 nm in 0.1 M imidazole, hemopeptide 14-21 at pH 7 and 406 nm in 0.1 M imidazole, and the carboxymeth- ylated hemopeptide at pH 7 and 403 nm in 0.1 M imidazole were 1.06 X 105, 1.06 X 105, 1.27 X lo5, 1.06 X lo5, 1.06 X lo5, and 1.05 X lo5 M-' cm-1, respectively. Circular dichroic spectra were obtained with a Cary Model 60 ORD-CD ap- paratus. Viscosity measurements, solvent perturbation direr- ence spectra, and spectrophotometric titrations of the phe- nolic ionization of tyrosyl residues at 243 nm were obtained as described previously (Stellwagen and Van Rooyan, 1967). Results Cytochrome c. At neutral pH, the visible absorption spec- 1 Abbreviations used are: (CmMet)(CmHis) c, cytochrome c having one methionyl and one histidyl residue carboxymethylated; (CmMet)z c, cytochrome c having both methionyl residues carboxymethylated; (CmMet)l(CmHis)s c, cytochrome c having two methionyl, three histidyl, and one-half of one lysyl residue carboxymethylated. BIOCHEMISTRY, VOL. 11, NO. 7, 1 9 7 2 1195