neurospora crassa cytochrome cneurospora rassa cytochrome c. ii a stoppered vial, were added 25 1l...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 241, No. 13, Issue of July 10, pp. 3165-3180, 1966Printed in U.S.A.

Neurospora crassa Cytochrome c

II. CHYMOTRYPTIC PEPTIDES, TRYPTIC PEPTIDES, CYANOGEN BROMIDE PEPTIDES, AND THECOMPLETE AMINO ACID SEQUENCE*

(Received for publication, February 17, 1966)

JORAM HELLER AND EMIL L. SMITH

From the Department of Biological Chemistry, School of Medicine, and the Molecular Biology Institute, Univer-sity of California at Los Angeles, Los Angeles, California 90024

SUMMARY

The complete amino acid sequence of Neurospora crassacytochrome c has been determined. The molecule consistsof a single polypeptide chain containing 107 residues. TheNH2-terminal residue is glycine, and the COOH-terminalresidue is alanine.

There are 39 amino acid differences between the cyto-chromes of N. crassa and those of the yeast Saccharomycescerevisiae, whereas there are 43 amino acid differencesbetween the cytochromes c of Neurospora and human heart.

For all of the presently known cytochromes c, the numberof amino acids which occupy the same position is reduced to39.

N. crassa cytochrome c has only 2 histidine residues inpositions 18 and 33, whereas tuna fish cytochrome has 2histidine residues in positions 18 and 26. It is suggestedthat the histidine residue in position 18 contributes the fifthligand to the heme iron, and that the sixth ligand in cyto-chrome c is not formed with histidine or with another ni-trogenous group, but with an unknown ligand.

The purification, some physical properties, the amino acidcomposition, and comparative chymotryptic and tryptic peptidemaps of cytochrome c from "wild type" and "poky" strains ofNeurospora crassa have been described in the preceding paper (1).The present communication is concerned with the elucidation ofthe complete amino acid sequence of Neurospora cytochrome c.The sequence was determined by three degradative procedures:chymotryptic and tryptic hydrolyses, and cyanogen bromidecleavage. The resulting peptides were isolated and purified, andthe individual sequences were determined. The three sets ofOverlapping peptides made it possible to deduce an unambiguousamino acid sequence for Neurospora cytochrome c.

As shown in the preceding paper (1), wild type and poky cyto-chrome c are identical. The cytochrome c used throughout thepresent work was obtained from the poky strain, which is pheno-

* This investigation was aided by a research grant from theNational Institute of General Medical Sciences of the NationalInstitutes of Health, United States Public Health Service.

typically characterized by a high content of cytochrome c, whichfacilitated obtaining the necessary amounts of pure material forsequence studies. A summary of the present work has been pub-lished (2).

EXPERIMENTAL PROCEDURE

The isolation and purification of N. crassa cytochrome c, ob-tained from the maternally inherited mutant mi-i, called poky,is described in the preceding paper (1).

Digestion with Chymotrypsin-Poky cytochrome c (23.3/moles) was denatured with alcohol as described by Margoliashet al. (3). The digestion mixture was maintained at pH 8.0 with0.0672 N NaOH in the Radiometer TTTlc pH-stat. Digestionwas performed for 30 hours at room temperature with 2% (w/w)a-chymotrypsin (Worthington, three times crystallized, Lot6007, in 0.001 N HC1, pH 3.0) added at zero time, 2% oa-chymo-trypsin added after 11 hours, and 3 % a-chymotrypsin added after20 hours. Hydrolysis was terminated by adjusting to pH 3.0with acetic acid.

Digestion with Trypsin-Denatured cytochrome c (15 moles)was hydrolyzed for 24 hours at pH 8.0 in the pH-stat at roomtemperature with 3% (w/w) trypsin (Worthington, twice crys-tallized, Lot TRL6257, in 0.001 N HCI, pH 3.0) added at zero time,2% trypsin added after 8 hours, and 2% trypsin added after 19hours. The digestion was terminated by adjusting the solutionto pH 3.2 with acetic acid.

Digestion with Carboxypeptidase A-Denatured cytochrome c(1.0 mole) was treated at room temperature with 0.01 /mole ofcarboxypeptidase A (Worthington, twice crystallized aqueoussuspension, Lot COA707-11, solubilized and treated with DFP'as described by Guidotti, Hill, and Konigsberg (4)) in 4.0 ml ofdeionized water adjusted to pH 7.5 with 10.0 #l in 1% ammonia.Aliquots of 1.0 ml were removed at zero time, 2, 18, and 40hours, and were treated with an equal volume of 20% trichlor-acetic acid. The precipitates were washed twice with 0.5 mlof 10% trichloracetic acid. The supernatants and washings ofeach sample were pooled and evaporated four times over solidNaOH in an evacuated desiccator. Samples were analyzed onthe amino acid analyzer.

Digestion of Peptides with Carboxypeptidase A or B-To ap-proximately 0.1 jtmole of peptide in 100 Il of deionized water, in

1 The abbreviations used are: DFP, diisopropyl fluorophos-phate; PTH-, phenylthiohydantoin; DNP-, dinitrophenyl-.

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a stoppered vial, were added 25 l1 of 0.4 M Tris buffer at pH 8.2and 10 1 of DFP-treated carboxypeptidase A (2.5 mAmoles, en-zyme to substrate ratio, 0.025) or DFP-treated carboxypeptidaseB, 0.05 mg (Worthington, Lot COB6069). Hydrolysis was per-formed at 40 ° and was terminated by adding 200 l of glacialacetic acid. Aliquots (10%) were examined by paper chromatog-raphy and, when necessary, were submitted to quantitative aminoacid analysis after they were first evaporated to dryness overNaOH.

Digestion of Peptides uith Aminopeptidase-To approximately0.1 mole of peptide in 100 #l of deionized water in a stopperedvial were added 25 l of 0.5 M Tris buffer at pH 8.5, 25 l of0.025 M MgCl 2, and 10 Al (0.05 mg) of DFP-treated leucine amino-peptidase (Worthington, Lot LAPDFP105). Digestion wasperformed at 40° and was terminated by addition of 200 l ofglacial acetic acid. Aliquots were examined and analyzed asnoted above for carboxypeptidase digests.

Digestion of Peptides with Trypsin-To approximately 2.0/moles of peptide dissolved in 2 ml of deionized water in a stop-pered vial were added 100 yl of 0.5 M Tris buffer at pH 8.0 and0.1 mg of trypsin (Worthington, Lot TRL6257). The digestion,performed at 400, was stopped by adding 100 ul1 of glacial aceticacid, and a 1% aliquot was taken for peptide mapping. The di-gestion products were separated on paper by chromatography orelectrophoresis.

Digestion of Peptides uith Papain-To 3.0 moles of peptide,dissolved in 1.0 ml of deionized water, were added 1.6 mg ofmercuripapain (a 70% ethanolic suspension prepared accordingto Kimmel and Smith (5)) and 0.6 ml of 0.1 M NaCN solution,freshly prepared and adjusted to pH 6.0. The digestion mixturewas incubated at 400 in a stoppered vial. The reaction wasterminated by freezing.

Peptide Maps-Peptide maps were prepared and developed asdescribed in the preceding paper (1).

Column Chromatographic Separation of Chymotryptic and Tryp-tic Peptides-The chymotryptic and tryptic digests were chro-matographed on a column (2 X 150 cm) of Dowex 50-X2 at 400with a flow rate of 60 ml per hour. The resin was washed, andthe column was packed by the procedures of Moore, Spackman,and Stein (6). The column was equilibrated with 0.2 M pyr-idinium-acetate buffer at pH 3.1. Fractions of 10.0 ml werecollected.

The chymotryptic digest was fractionated by eluting with 4.0liters of 0.2 M pyridinium-acetate buffer at pH 3.1. This wasfollowed with a linear gradient which was established with 2.2liters of 2.0 M pyridinium-acetate buffer at pH 5.0. The run wascompleted by passing 1.8 liters of 2.0 M pyridinium-acetate bufferat pH 5.0 and, finally, 1.0 liter of 4.0 AM pyridinium-acetate bufferat pH 5.6.

For the tryptic digest, after passing 4.0 liters of 0.2 M pyr-idinium-acetate buffer at pH 3.1, a linear gradient was estab-lished with 4.0 liters of 2.0 M pyridinium-acetate buffet at pH5.0. The run was completed by passing 2.0 liters of 2.0 M pyr-idinium-acetate buffer at pH 5.6 and, finally, 2.0 liters of 4.0 M

pyridinium-acetate buffer at pH 5.6.A sample of 250 pl was removed from every other tube of the

column fraction and was analyzed by the ninhydrin method, bothdirectly (7) and after alkaline hydrolysis (8). Appropriate frac-tions under each chromatographic peak were pooled, taken todryness in a rotary evaporator at 40°, transferred in 5.0 ml ofdeionized water to a vial, and stored frozen. Samples were taken

from each fraction for peptide maps and, when appropriate, foamino acid analysis after acid hydrolysis.

Purification of Peptides-Peptides (3.0 to 10.0 Amoles) fromthe chymotryptic and tryptic digests were purified on WhatmaNo. 3MM paper, either by electrophoresis at pH 4.7 in pyridinium-acetate buffer or by chromatography in 1-butanol.pyridine-acetic acid-water (150:100:30:120), or by a combine.tion of both methods. The purity of a peptide was judgedfrom the peptide map and ultimately from the amino acid anal.ysis. Yields were calculated from the amino acid analysis withno correction for losses in any of the separation and purificationsteps.

The chymotryptic heme peptide was prepared and purified bythe procedure of Hettinger and Harbury (9) from 10.0 moles ofpoky cytochrome c.

Amino Acid Composition of Peptides-Analyses were performedwith the Beckman model 120B automatic amino acid analyzer bythe procedures of Spackman, Stein, and Moore (10). Samplesof 0.03 to 0.1 Amole of peptide in deionized water were mixed withequal volumes of concentrated HC1 (analytical grade), and werehydrolyzed at 110° in sealed, evacuated (vacuum pump) Pyrextubes. Peptides were hydrolyzed for 24 hours unless specifiedotherwise. The hydrolysate was then evaporated twice oversolid NaOH in an evacuated desiccator

Hydrolysates of peptides were also examined qualitatively bychromatography on Whatman No. 3MM paper with 1-butanol-acetic acid-water (200:30:75) at room temperature for 24 hourswith a standard amino acid mixture as a control. Individualamino acids were identified from RF values and from the colorswith 0.1% ninhydrin-collidine spray.

Cleavage uith Cyanogen Bromide-Cleavage with cyanogenbromide was performed on 0.25 pmole of cytochrome c by themethod of Gross and Witkop (11), as modified by Steers et al(12). Denatured cytochrome c was dissolved in 1.0 ml of 70%formic acid in a stoppered test tube. Two milligrams of cyano-gen bromide were added (80-fold molar excess), and the mixturewas incubated at 300 in the dark for 40 hours. Two milliliters ofdeionized water were then added, and the mixture was evaporatedtwice over solid NaOH and then dissolved in 0.5 ml of deionizedwater, adjusted to pH 9.0 with ammonia. The mixture was ap-plied to a column (1 X 10 cm) of Sephadex G-50 (fine) equil-ibrated with 10% acetic acid; elution was performed with thesame solvent. The dark colored material (heme peptide, Frac-tion 1) was eluted first in a volume of approximately 2.0 miMore colorless material (20 ml) was collected (Fraction 2)After it was evaporated to dryness, Fraction 2 was resolved bJpaper chromatography in 1-butanol-pyridine-acetic acid-watefor 18 hours into two neutral peptides. The heme peptide anthe two colorless peptides were hydrolyzed with HCl for 40 hoursand were submitted to quantitative amino acid analysis.

NH2-terminal Residue of Cytochrome c-Reaction with fiuorodinitrobenzene was performed as described by Fraenkel-ConroiHarris, and Levy (13) on 1.0 mole of cytochrome c. The extraafter acid hydrolysis was dissolved in 0.2 ml of acetone, and saraples were chromatographed on Whatman No. 1 paper in the following systems: (a) two-dimensional chromatography, witItoluene-pyridine-ethylene monochlorohydrin-0.8 N ammonia(5:1:3:3) in one dimension and 1.5 M sodium phosphate buffs(pH 6.0) in the other; (b) 1-butanol-acetic acid-water (4:1:5); (O1.5 N sodium phosphate buffer (pH 6.0); and (d) tert-amyl alcoblsaturated with pH 6.0 phthalate buffer.

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Reaction with phenyl isothiocyanate (13) was performed on0.45 Cumole of cytochrome c. Samples were chromatographed onWhatman No. 1 paper in n-heptane-pyridine (7:3), with knownphenylthiohydantoin amino acids as markers and with theiodine-azide spray for detection as described by Sj6quist (14).

Edman Degradation-The Edman degradation with peptideswas performed as described previously (15, 16). Two chromato-graphic solvents were used in identifying the PTH derivatives:Solvent A was composed of n-heptane-pyridine (7:3), andSolvent F was composed of n-heptane-75% formic acid-ethylenechloride (1:2:2).

Removal of Heme From Heme Peptide-Performic acid oxidationof the heme peptide was performed by the method of Hirs (17).Approximately 3.0 moles of the papain heme peptide were evap-orated to dryness in a rotary evaporator at 40° , and were thendissolved in 1.0 ml of performic acid at 40 (made by mixing 9.5ml of 99 % formic acid with 0.5 ml of H202 and incubating at roomtemperature for 2 hours). After 3 hours at 40°, the mixture wasthen diluted with 10.0 ml of deionized water and was lyophilizedtwice. The papain heme peptide, initially dark red-brown, be-came light straw in color after about 1 hour. The oxidized hemepeptide was purified by paper electrophoresis at pH 1.9.

Nomenclature-The working nomenclature of peptides fromthe chymotryptic and tryptic digests was based on the elutionpatterns from the Dowex 50-X2 columns. When the sequence ofNeurospora cytochrome became known, the nomenclature waschanged, and each peptide was given a number correspondingwith its position in the sequence beginning at the NH 2 terminus.When a peptide was found in several fragments, the largest frag-ment was given the appropriate number, and the smaller peptideswere given a letter in addition to the same number, again startingat the amino-terminal end of the peptide. Free lysine, recoveredfrom the tryptic digest, was given four different numbers in orderto keep the nomenclature uniform. When chymotryptic pep-tides were further hydrolyzed with trypsin or papain, the result-ing fragments were designated T-1, T-2, or P-1, P-2, in additionto the peptide number, e.g. C-3-T-1. The cyanogen bromidepeptides were given roman numbers in accord with the sequence,starting from the NH2-terminal end of the protein.

RESULTS

Amino-terminal Residue of Neurospora Cytochrome c-The NH2 -terminal residue of poky cytochrome c was investigated by thefluorodinitrobenzene method of Sanger and the phenyl isothio-cyanate method of Edman.

When the dinitrophenyl-coupled protein was hydrolyzed for 4hours, a yellow material was recovered in low yields by ether ex-traction. On two-dimensional chromatography, with toluene-PYridine-ethylene monochlorohydrin-ammonia in one dimensionand 1.5 M sodium phosphate buffer at pH 6.0 in the other, and inone-dimensional chromatography with 1-butanol-acetic acid-water, the material appeared to be identical with a DNP-glycine.The yellow spot did not bleach on exposure to HCl fumes. Whenthe DNP-coupled protein was hydrolyzed for 20 hours, no DNP-amino acid could be found in the ether extract. Examination ofthe aqueous phase in the tert-amyl alcohol-phthalate systemYielded only e-DNP-lysine. Tests for DNP-arginine were nega-tive.

When the ether extract of the acid-hydrolyzed phenylthio-arbamyl derivative of cytochrome c was chromatographed in0divent A, only one spot was seen, which had the same R as

TABLE I

Digestion of poky N. cytochrome c with carboxypeptidase ADenatured cytochrome c (1.0 Amole), dissolved in 4.0 ml of

deionized water and adjusted to pH 7.5 with ammonia, was di-gested with 0.01 mole of DFP-treated carboxypeptidase A atroom temperature. Samples of 0.25 mole of cytochrome c weretaken at 0, 2, 18, and 40 hours and treated with an equal volumeof 20% trichloracetic acid, and the supernatant was taken foramino acid analysis of the neutral and acidic residues.

Amino acid

Alanine .......................Threonine ....................Glutamic acid.................Methionine sulfoxide ..........Isoleucine ......................Phenylalanine ..................

2 hrs 18 hrs 40 hrs

moles liberated/mole cytochrome c

0.07 0.37 0.480.04 0.15 0.130.01 0.07 0.070.02 0.14 0.160.01 0.08 0.100 0.04 0.04

PTH-glycine and gave an immediate and strong red color withthe iodine azide spray.

That glycine is the NH2-terminal residue was confirmed by thesequence studies reported below.

Carboxyl-terminal Residue-The identity of the carboxyl-termi-nal residue was established by time studies of the carboxypep-tidase A digestion of the denatured protein. From the resultsshown in Table I, it is evident that alanine is the carboxyl-termi-nal residue. It is not possible to deduce from these results theexact sequence, but it is evident that threonine, glutamic acid,methionine, isoleucine, and phenylalanine are present in theCOOH-terminal sequence. The fact that alanine is the carboxyl-terminal residue was independently confirmed by the studies ofthe tryptic digest and the products of cyanogen bromide cleavage.

Cleavage of Cytochrome c with Cyanogen Bromide-A peptidemap after cleavage with cyanogen bromide showed three pep-tides, as expected from the presence of 2 methionine residues, twoneutral peptides, well separated in the chromatographic dimen-sion, and a single heme peptide that remained near the origin.The heme peptide was separated on Sephadex G-50, and thesmaller, colorless peptides were purified by paper chromatog-raphy.

The composition of the resulting peptides is given in Table II.Cyanogen bromide cleaves methionine residues on the carboxylside, converting them to homoserine. As Peptide III does notcontain homoserine, and as Peptides I (heme) and II each containapproximately 1 residue of homoserine per peptide and no methi-onine, Peptide III is obviously derived from the carboxyl end ofthe cytochrome molecule. The three cyanogen bromide peptidesaccount, within experimental error, for the composition of thewhole molecule. The high figure for histidine (Peptide I) isprobably due to insufficient resolution from lysine or to the pres-ence of a breakdown product from the heme. The low figures forhalf-cystine and tyrosine in Peptide I are due to partial destruc-tion of these residues, as also noted by Black and Leaf (18).Otherwise, the results are in excellent agreement with cleavage tobe expected from the sequence deduced from the overlappingchymotryptic and tryptic peptides.

Hydrolysis u ith a-Chymotrypsin-From the known amino acidcomposition, 22 bonds in the cytochrome molecule are theoreti-cally susceptible to hydrolysis with a-chymotrypsin (Phe6, Tyr 4,Met 2, Leu7 , Trpl, His2). One histidine is, in all probability,

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Neurospora crassa Cytochrome c. II

adjacent to the heme, and is resistant to the action of the enzyme.After digestion for 30 hours, the total base consumed indicatedthat only 14.5 bonds were cleaved (assuming 50% ionization ofthe amino groups at pH 8.0). The digestion was terminated,despite the fact that only 69% of the theoretical 21 bonds were

TABLE II

Amino acid composition of peptides from cyanogen bromide cleavageof poky Neurospora cytochrome c

The composition of each peptide is given as the molar ratios.The theoretical number of amino acids is derived from the ex-pected peptides resulting from cyanogen bromide cleavage of thecytochrome structure as deduced from the complete sequence.

BrCN-I (beme) BrCN-II BrCN-IIIAmino acid

Found Theory Found Theory Found Theory

Lysine ............... 9.8 10 3.1 3 1.0 1Histidine ............ 2.9 2Arginine ............. 1.7 2 0.9 1Homoserine lactone... 1.2 0.9Homoserine .......... 0.3 1 0.4 1Aspartic acid......... 9.4 9 4.0 4 0.1Threonine ............ 6.9 7 1.0 1 0.9 1Serine ............... 2.8 3 0.1Glutamic acid........ 7.1 7 0.2 1.0 1Proline ............... 2.9 3Glycine .............. 13.2 13 2.2 2 0.1Alanine .............. 6.4 6 1.1 1 2.0 2Half-cystine .......... 1.0 2Valine ............... 1.1 1Isoleucine ............ 3.0 3 1.8 2Leucine ............. 6.0 6 1.0 1Tyrosine ............. 2.1 4Phenylalanine ........ 3.9 4 1.8 2

Total residues ....... 80" 83° 18 18 5 5

" The sum of the amino acids does not include the single residueof tryptophan which is destroyed upon acid hydrolysis.

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hydrolyzed, mainly to minimize the extent of minor cleavageIt is noteworthy that the actual number of major bonds hy.drolyzed was 15, and that complete hydrolysis of all susceptiblebonds would have yielded a number of free amino acids, th~complicating the sequence elucidation.

Hydrolysis with Trypsin-The composition indicates that 17bonds in the cytochrome molecule are susceptible to hydrolyss8with trypsin (Lys14, Arg3). Hydrolysis with trypsin resulted iathe cleavage of 18.8 bonds, as shown by the base uptake assume .ing 50% ionization of the amino groups at pH 8.0). The actualnumber of bonds cleaved by trypsin in poky cytochrome c was18, as revealed by the subsequent sequence studies. Two of thebonds hydrolyzed represent points of chymotryptic specificity.

Peptide Maps-The peptide map of the chymotryptic hydroly.sis products of poky cytochrome c (1) reveals 18 major peptides,as judged by the intensity of the ninhydrin color. Of these, twoare acidic, eight are neutral, and eight are basic at pH 4.7. Onlyone tryptophan-containing peptide (Ehrlich-positive) was de.tected.

The peptide map of the tryptic hydrolysis products (1) showsapproximately 18 to 19 major and five to six minor peptides.One was acidic, nine were neutral, and eight to nine were basisInterestingly, two heme peptides were repeatedly observed, onea little less basic than the other. Again, only one tryptophan.containing peptide was detected.

Column Chromatography of Digests-The elution patterns of thechymotryptic and tryptic digests are shown in Figs. 1 and 2, respectively. All pooled fractions reacted with ninhydrin bothdirectly and after alkaline hydrolysis. No heme peptides couldbe eluted from the columns, and the chymotryptic heme peptidewas isolated in a separate experiment.

Purification of Peptides

Chymotryptic Peptides-The following peptides required nofurther purification: C-I, C-2, C-8, C-9, C-10, C-12, C-13, C-14C-15, and C-16.

Basic Peptide C-4 was purified from two minor contaminantsby paper electrophoresis at pH 4.7. Neutral Peptides C-5 and

pH5.0

4.0

3.0

11

VOLUME- LITERS

FIG. 1. Elution pattern of peptides from chymotryptic hydrolysis of poky N. crassa cytochrome c. The digest (23.3 moles)chromatographed on a column (2 X 150 cm) of Dowex 50-X2 with pyridine-acetate buffers. The solid lines on the abscissa indicatefractions pooled. The numbers refer to the peptides found in each fraction (see "Nomenclature").

C 10

0.2M, GRADIENT C8pH3.1 TO 0.2M,

PYRIDINIUM- pH 5.0ACETATE C 14

Clio

1 2 3 4 5

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FIGo. 2. Elution pattern of peptides from tryptic hydrolysis of poky N. crassa cytochrome c. The digest (15 moles) was chromato-graphed on a column (2 X 150 cm) of Dowex 50-X2 with pyridine-acetate buffers. The solid lines on the abscissa indicate the fractionspooled. The numbers refer to the peptides found in each fraction (see "Nomenclature").

C-7 were present in the same fraction, and were separated bypaper chromatography with 1-butanol-pyridine-acetic acid-water.Peptide C-6 was a basic peptide that was separated from smallamounts of impurities by paper electrophoresis at pH 4.7. Pep-tide C-11, a neutral peptide, and Peptide C-llb, a basic peptide,were present in the same fraction, and were purified by chro-matography in 1-butanol-pyridine-acetic acid-water. PeptideC-1a, an acidic peptide, was separated from three accompanyingneutral peptides by electrophoresis at pH 4.7. Peptide C-llc, abasic peptide, was separated from minor neutral contaminants byelectrophoresis at pH 4.7.

Tryptic Peptides-Only Peptides T-13 and T-15 did not requirefurther purification.

Free lysine (T-2, T-7b, T-10, and T-13a) was separated fromneutral Peptide T-7c by electrophoresis at pH 4.7. Peptide T-1,a neutral peptide, was purified from minor contaminants bychromatography in 1-butanol-pyridine-acetic acid-water. Pep-tides T-3, T-6, and T-7a were separated by chromatography inl-butanol-pyridine-acetic acid-water. Peptides T-4 and T-14,basic peptides, were isolated by the same procedure. NeutralPeptides T-7 and T-13b, present in the same fraction, were sep-arated by chromatography in 1-butanol-pyridine-acetic acid-water. Neutral Peptide T-8 was purified by chromatography.Peptide T-9, a neutral peptide, was separated from basic PeptideT-12 by paper electrophoresis at pH 4.7. Peptide T-12 wasfurther purified by paper chromatography in 1-butanol-pyridine-acetic acid-water. Peptide T-11, a basic peptide, was purifiedfrom several minor basic contaminants by chromatography inl-butanol-pyridine-acetic acid-water.

Amino Acid Compositions of Chymotryptic and Tryptic Peptides-The compositions of the purified chymotryptic and tryptic pep-tides are given in Tables III and IV, respectively. The listedPeptides were used for sequence determinations.

Amino Acid Sequences of Chymotryptic Peptides

Peptide C-1 (Residues -4 to -3): Gly-Phe--Tbis neutral pep-tide gave a green color with the collidine-ninhydrin spray. One8ep of the Edman degradation yielded PTN-Gly,, and the res-'due was phenylalanine.

Peptide C-2 (Residues -2 to 10): Ser-Ala-Gly-Asp-Ser-Lys-Lys (Gly, Ala) Asn (Leu, Phe)-This basic peptide gave ablue color with the collidine-ninhydrin spray. The steps requiredto determine the sequence are given in Table V. The trypticpeptides were separated by chromatography in 1-butanol-pyri-dine-acetic acid-water, and the compositions were determined onpaper after 24 hours of acid hydrolysis. Four steps of the Edmandegradation established the sequence Ser-Ala-Gly-Asp-. Thecarboxypeptidase A digest established the sequence at the car-boxyl terminus as -(Gly, Ala) Asn (Leu, Phe), although not theorder of residues shown parenthetically. The fact that freelysine was obtained from the tryptic digest of C-2 shows that thesequence Lys-Lys is present. This, in combination with thecomposition of the tryptic peptides of C-2, established the se-quence as written above. The sequence Leu-Phe is evident fromthe absence of phenylalanine in Peptide C-2a, and the sequence-Gly-Ala- was determined from Peptide T-3 (see below).

Peptide C-2a (Residues -2 to 9): (Ser, Ala, Gly, Asp, Ser, Lys,Lys, Gly, Ala) Asn-Leu-This basic peptide gave a blue colorwith the collidine-ninhydrin spray. The composition clearlyindicates that it is derived from C-2 by loss of phenylalanine, thusestablishing the carboxyl-terminal sequence of C-2. Hydrolysisof C-2a with carboxypeptidase A for 3 min yielded only leucine(paper chromatography), while digestion for 6 hours yieldedleucine and asparagine.

Peptide C-S (Heme) (Residues 11 to 26): Lys-Thr-Arg-Cys-Ala-Glu-Cys-His-Gly-Glu-Gly-Gly-Asn-Leu- Thr-Gln-Thechymotryptic heme peptide was prepared and purified as de-scribed in "Experimental Procedure." The data establishing thesequence are summarized in Table VI. Carboxypeptidase Adigestion established glutamine as the COOH-terminal residue,and the next 3 residues as (Thr, Leu, Asn), without establishingthe order. Hydrolysis of Peptide C-3 with trypsin yielded aheme peptide (Cys 2, Ala, Glu3, His, Gly3, Thr, Leu, Asp) and abasic peptide (Lys, Thr, Arg). The latter peptide must bederived from the NH 2-terminal portion of the chymotryptic hemepeptide. That the sequence is Lys-Thr-Arg was establishedfrom the main tryptic digest, from which Peptide T-3, with aCOOH-terminal lysine, was isolated and, in addition, the unique

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peptide Thr-Arg. There are only 3 arginine residues in Neuro-spora cytochrome c; the others do not possess adjacent threonine.Consequently, Thr-Arg is derived from Peptide C-3.

Hydrolysis of the tryptic heme peptide (C-3-T-1) with papainat pH 6.0 and 40° for 20 hours yielded three main fragments: apapain heme peptide (Cys2, Ala, Glu, His, Gly), an acidic pep-

TABLE V

Amino acid sequence of Peptide C-2 (residues -2 to 10)

Sequence

Carboxypeptidase A

Tryptic peptidesT-1T-2T-3T-4T-5

Edman degradationStep 1

Step 2

Step 3

Step 4

Ser-Ala-Gly-Asp-Ser-Lys-Lys (Gly, Ala)Asn (Leu, Phe)

3 min: Phe, Leu, equal amounts (paperchromatography)

60 min: Phe, 0.94; Leu, 1.0; Asn, 0.68; Ala,0.63; Gly, 0.52

(Ser(2), Gly, Ala, Asp, Lys( 2))(Ser(2), Gly, Ala, Asp, Lys)Lys(Lys, Gly, Ala, Asp, Leu, Phe)(Gly, Ala, Asp, Leu, Phe)

PTH-Ser; residue: Lys, 1.70(2); Asp,2.05(2); Ser, 0.80(1); Gly, 2.00; Ala,1.95(2); Leu, 1.00; Phe, 1.00

PTH-Ala; residue: Lys, 1.56(2); Asp,2.10(2); Ser, 0.79(1); Gly, 1.93(2); Ala,1.20(1); Leu, 1.00; Phe, 1.00

PTH-Gly; residue: Lys, 1.75(2); Asp, 2.00;Ser, 0.84(1); Gly, 1.47(1); Ala, 1.20(1);Leu, 1.04(1); Phe, 0.92(1)

PTH-Asp; residue: Lys, 1.76(2); Asp,1.44(1); Ser, 0.65(1); Gly, 1.38(1); Ala,1.24(1); Leu, 1.03(1); Phe, 0.97(1)

tide (Glu, Gly2, Asp), and a neutral peptide (Leu, Thr, GluThree steps of the Edman degradation on the papain heme pqtide (C-3-T-1-P-1) after performic acid oxidation established tlsequence Cys-Ala-Glu-. One step of the Edman degradatifshowed that NH2-terminal glutamic acid and carboxypeptidasehydrolysis of the acidic peptide (C-5-T-1-P-2) liberated aspargine, establishing the sequence Glu-Gly-Gly-Asn. One step,the Edman degradation established the NH2-terminal residue Ineutral Peptide C-3-T-1-P-3 as leucine. It is obvious that Petide C-3-T-1-P-3 (Leu-Thr-Gln), containing the sole leucilresidue, is derived from the COOH-terminal portion of PeptidC-3. Carboxypeptidase A hydrolysis of the whole peptide haestablished glutamine as the COOH-terminal residue. This etablishes the sequence of Peptide C-3-T-1-P-3 as Leu-Thr-Gln.

The significant peptides isolated from the partial acid hydrolsis of the performic acid-oxidized heme peptide (C-3) were Glucysteic acid (PA-2) and Gly-Glu (PA-3). Peptide PA-2 coIfirms and extends the sequence Cys-Ala-Glu-Cys. PeptidPA-3 places a glycine residue in a position preceding a glutamiacid residue. As the sequence shows, the glutamic acid residucan only be the one at position 20.

Apart from these two peptides, it was possible to isolate froithe partial acid hydrolysate the following free amino acidcysteic acid, glutamic acid, aspartic acid, glycine, lysine, hitidine, and arginine. No histidine-containing peptide could bisolated from the partial acid hydrolysate of C-3. Neverthelessas the position of every other residue in Peptide C-3 has been ektablished, the only place remaining for the histidine residue is aposition 18, adjacent to the cysteine linked to the heme. Thisthe same position at which a histidine residue has been found Dthe cytochrome c of all species.

Peptide C-4 (Residues 27 to 33): Lys-lle-Gly-Pro-Ala-LeaHis-This basic peptide gave a blue color with the collidine-nin

TABLE VI

Amino acid sequence of Peptide C-3 (heme) (residues 11 to 26)

SequenceCarboxypeptidase A

Tryptic peptidesT-1 (heme)T-2

Papain peptides of C-3-T-1P-1 (heme)

Edman degradation (after performicacid oxidation)

P-2Edman degradationCarboxypeptidase A

P-3Edman degradation

Partial acid hydrolysis of Peptide C-3after performic acid oxidation (12N HCI, 370, 84 hrs)

PA-1PA-2

Edman degradationPA-3

Edman degradation

Lys-Thr-Arg-Cys-Ala-Glu-Cys-His-Gly-Glu-Gly-Gly-Asn-Leu-Thr-Gln6 hrs: Asp, 0.04; Thr, 0.16; (Gln + Asn), 0.44; Gly, 0.16; Leu, 0.16

12 hrs: Asp, 0.20; Thr, 0.48; (Gln + Asn), 0.96; Glu, 0.16; Gly, 0.24; Leu, 0.3624 hrs: Asp, 0.64; Thr, 0.68; (Gln + Asn), 1.16; Glu, 0.32; Gly, 0.58; Leu, 0.6040 hrs: Asp, 0.40; Thr, 1.03; (Gln + Asn), 1.63; Glu, 0.57; Gly, 1.63; Leu, 0.69

(Cys(2), Ala, Glu( 2 3), His, Gly( 2_3), Asp, Leu, Thr) (paper chromatography)(Lys, Thr, Arg) (paper chromatography)

(Cys2, Ala, Glu, His, Gly)Step 1: PTH-cysteic acid (separated by paper electrophoresis)Step 2: PTH-Ala; regeneration, Ala (amino acid analyzer)Step 3: PTH-Glu; regeneration, Glu (amino acid analyzer)Glu, 1.04(1); Gly, 2.50(2); Asp, 0.92(1)Step 1: PTH-Glu; residue: Asp, 0.80(1); Glu (0.31), Gly (2.20 (2))10 hrs: Asn,.0.74Leu, 1.00; Thr, 1.00; Glu, 1.00Step 1: PTH-LeuThe fragments were separated on a Dowex 50 column (0.9 X 90 cm)

Cysteic acid(Glu, cysteic acid)Step 1: PTH-Glu (?); residue: cysteic acid (paper chromatography)Gly-Glu (gray with the collidine-ninhydrin spray)Step 1: PTH-Gly (?); residue: glutamic acid (paper chromatography)

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J. Heller and E. L. SmithYae of July 10, 1966

ydrin spray. The sequence information is summarized inTable VII. Hydrolysis with carboxypeptidase A established thesequence Leu-His. Treatment with leucine aminopeptidase es-tablished (Lys, Ile) at the amino terminus. That the sequenceis Lys-Ile was established from the fact that free lysine was ob-tained from tryptic digestion of Peptide C-4 and from the Edmandegradation, which established the sequence Lys-Ile-Gly-Pro-.

Peptide C-5 (Residues 34 to 36): Gly-Leu-Phe-This neutralpeptide gave a gray color with the collidine-ninhydrin spray.The sequence was established by two steps of the Edman degrada-tion. Step 1: PTH-Gly; regeneration, Gly; residue, (Leu, Phe),by paper chromatography. Step 2: PTH-Leu; residue, Phe, bypaper chromatography.

peptide C-6 (Residues 37 to 46): Gly-Arg-Lys-Thr-Gly-Ser-Val-Asp- Gly-Tyr-This basic peptide gave a gray color with thecollidine-ninhydrin spray. The procedures used to determinethe sequence are given in Table VIII. Hydrolysis with car-boxypeptidase A gave only the expected tyrosine and a trace ofglycine. Four steps of the Edman degradation established the

TABLE VII

Amino acid sequence of Peptide C-4 (residues 27 to 33)

SequenceCarboxypeptidase AAminopeptidaseTryptic peptides

T-1T-2

Edman degradationStep 1Step 2Step 3

Step 4

Lys-Ile-Gly-Pro-Ala-Leu-His24 hrs: His, 1.00; Leu, 0.7124 hrs: Lys, 1.00; Ile, 0.97Purified by paper electrophoresisLys(Ile, Gly, Pro, Ala, Leu, His)

PTH-LysPTH-IlePTH-Gly; residue: His, 1.00; Pro, 0.94(1);

Lys, 0.04; Gly, 0.31; Ala, 1.02(1); Ile,0.07; Leu, 1.04(1)

PTH-Pro; residue: Lys, 0.02; His, 1.0;Pro, 0.30; Gly, 0.26; Ala, 1.00; Ile,0.04; Leu, 1.00

TABLE IX


Sequence Thr-Asp-Ala-Asn-Lys-GlnCarboxypeptidase A 60 min: Gin, 0.95Carboxypeptidases A + B 180 min: (Asn + Gin), 1.83; Lys,

0.87Aminopeptidase 18 hrs: Thr, 0.92Edman degradation

Step 1 PTH-Thr; residue: Lys, 0.88(1);Asp, 2.16(2); Thr, 0.09; Glu, 1.00;Ala, 1.10(1)

Step 2 PTH-Asp; residue: Lys, 0.90(1);Asp, 1.25(1); Thr, 0.06; Glu,0.98(1); Ala, 1.00

sequence at the amino-terminal end. The sequence of the trypticpeptide C-6-T-3 was determined by five steps of the Edmandegradation. The sequence of this part of Peptide C-6 was alsoconfirmed by the hydrolysis of Peptide T-7c with aminopeptidase(see below).

Peptide C-7 (Residues 47 and 48): Ala-Tyr-This neutral pep-tide gave a blue color with the collidine-ninhydrin spray. Onestep of the Edman degradation gave PTH-Ala, and the residuewas identified by paper chromatography as tyrosine.

Peptide C-8 (Residues 49 to 54): Thr-Asp-Ala-Asn-Lys-Gln-This neutral peptide gave a green-gray color with the collidine-ninhydrin spray. The sequence determination of this peptide issummarized in Table IX. Hydrolysis with carboxypeptidase Ayielded 1 residue of glutamine (identified by paper chromatog-raphy and quantitative amino acid analysis). Treatment withcarboxypeptidases A + B yielded, in addition to glutamine, 1residue of lysine and 1 of asparagine (identified as above); thisestablished the COOH-terminal sequence as Asn-Lys-Gln. Twosteps of the Edman degradation gave the sequence Thr-Asp-,and completed the sequence as written above.

Peptide C-9 (Residues 55 to 59): Lys-Gly-Ile-Thr-Trp--This

TABLE VIII


SequenceCarboxypeptidase AEdman degradation

Step 1

Step 2

Step 3

Step 4

Tryptic peptidesT-1T-2T-3

Edman degradation

Gly-Arg-Lys-Thr-Gly-Ser-Val-Asp-Gly-Tyr24 hrs: Tyr, 1.00; Gly, 0.12

PTH-Gly; residue: Lys, 1.10(1); Arg, 1.08(1); Asp, 1.05(1); Thr, 0.87(1); Ser, 0.80(1);Gly, 2.10(2); Val, 0.90(1); Tyr, 0.32(1)

PTH-Arg; residue: Lys, 0.70(1); Arg, 0.18; Asp, 1.15(1); Thr, 0.96(1); Ser, 0.85(1); Gly,2.05(2); Val, 1.03(1); Tyr, 0.33(1)

PTH-Lys; residue: Lys, 0.30; Arg, 0.20; Asp, 1.20(1); Thr, 0.95(1); Ser, 0.90(1); Gly, 2.25(2);Val, 1.00; Tyr, 0.05 (1 assumed)

PTH-Thr; residue: Lys, 0.28; Arg, 0.20; Asp, 1.10(1); Thr, 0.36; Ser, 0.70(1); Gly, 2.00;Val, 1.00; Tyr, 0.05 (1 assumed)

Lys (paper chromatography)(Gly, Arg) (paper chromatography)Asp, 1.03(1); Thr, 0.89(1); Ser, 0.83(1); Gly, 1.95(2); Val, 1.00; Tyr, 0.58(1)Step 1: PTH-ThrStep 2: PTH-Gly; residue: Asp, 1.02(1); Thr, 0.05; Ser, 0.57(1); Gly, 1.12(1); Val, 0.98(1);

Tyr, 0.25(1)Step 3: PTH-SerStep 4: PTH-Val; residue: Asp, 1.00; Ser, 0.18; Gly, 0.99(1); Val, 0.30; Tyr, 0.85(1)Step 5: PTH-Asp; residue: Asp, 0.49; Thr, 0.05; Ser, 0.17; Gly, 1.00; Val, 0.29; Tyr, 0.1

(1 assumed)

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basic peptide (Table X) gave a blue color with the collidine-ninhydrin reagent and a positive Ehrlich reaction. Carboxy-peptidase A liberated tryptophan as the carboxyl-terminalresidue. Three steps of the Edman degradation established thesequence Lys-Gly-Ile.

Peptide C-10 (Residues 60 to 65): Asp-Glu-Asn-Thr-Leu-Phe-This acidic peptide gave a blue color with the collidine-ninhy-drin spray. Time studies with carboxypeptidase A establishedthe sequence -Thr-Leu-Phe (Table XI). Three steps of theEdman degradation established the sequence Asp-Glu-Asn.

Peptide C-11 (Residues 66 to 74): (Glu, Tyr, Leu) (Glu, Asn,Pro, Lys) Lys-Tyr-This neutral peptide gave a blue color withthe collidine-ninhydrin spray. Digestion with carboxypeptidaseA for 10 hours yielded only tyrosine (identified by paper chro-matography), while digestion with carboxypeptidases A + B for40 hours yielded 1.0 residue of tyrosine and 1.05 residues oflysine. Hydrolysis with aminopeptidase for 40 hours yieldedGlu, 1.13; Tyr, 0.84; Leu, 1.00. The complete sequence wasestablished by studying Peptides C-lla, C-llb, and C-llc (seebelow).

Peptide C-1 a (Residues 66 and 67): Glu-Tyr--This acidic pep-tide gave a blue color with the collidine-ninhydrin spray. Onestep of the Edman degradation yielded PTH-Glu, and the residuewas identified as tyrosine by paper chromatography.

Peptide C-11b (Residues 68 to 74): (Leu, Glu, Asn, Pro, Lys,Lys, Tyr)-This basic peptide gave a blue color with collidine-ninhydrin spray. This peptide is obviously part of Peptide C-11,minus Glu-Tyr, and differs from Peptide C-llc by containing 1residue of leucine. No sequence determinations were done onthis peptide.

Peptide C-11c (Residues 69 to 74): Glu-Asn-Pro-Lys-Lys-Tyr

TABLE X



Aminopeptidase


Step 2Step 3

Lys Gly Ile-ThrTrp3 min: Trp (identified by paper chroma-

tography)10 min: Lys, 0.60; Gly, 0.37; Ile, 0.26; Thr,

0.26; Trp, 0.1960 min: Lys, 1.25(1); Gly, 0.98; Ile, 1.00;

Thr, 1.00; Trp, 0.92

PTH-Lys; residue: Lys, 0.14; Gly, 1.17(1);Ile, 0.95(1); Thr, 0.95(1)

PTH-GlyPTH-Ile; residue: Gly, 0.53; lie, 0.25;

Thr, 1.00



Step 2

Step 3

Asp-Glu-Asn-Thr-Leu-Phe3 min: Phe, 0.70; Leu, 0.28

60 min: Phe, 1.00; Leu, 1.0025 hrs: Phe, 1.00; Leu, 1.15(1); Thr, 1.00

PTH-Asp; residue: Asp, 1.15(1); Thr,0.92(1); Glu, 1.02(1); Leu, 1.00; Phe, 1.00

PTH-Glu; residue: Asp, 1.13(1); Thr,0.96(1); Glu, 0.20; Leu, 1.03(1); Phe, 1.00

PTH-Asn (identified by paper chroma-tography)

TABLE XII

Amino acid sequence of Peptide C-llc (residues 69 to 74)

Sequence Glu-Asn-Pro-Lys-Lys-TyrCarboxypeptidase A 60 min: Tyr (identified by paper chror

tography)Edman degradation

Step 1 PTH-Glu; residue: Lys, 1.78(2); Asp, 1.Glu, 0.17; Pro, 1.00

Step 2 PTH-Asn; residue: Lys, 1.83 (2); Asp, 0.Glu, 0.16; Pro, 1.00; Tyr, 0.25 (unerected)

Step 3 PTH-Pro; residue: Lys, 2.00; Asp, 0.Glu, 0.23; Pro, 0.51; Tyr, 0.27 (unereacted)

TABLE XIIIAmino acid sequence of Peptide C-12 (residues 75 to 80)

Sequence Ile-Pro-Gly-Thr-Lys-MetCarboxypeptidases A + B 3 min: Met, 0.97

60 min: Met, 1.03; Lys, 0.77; T0.16

40 hrs: Met, 1.00; Lys, 0.95; T1.00

Edman degradationStep 1 PTH Ile; residue: Lys, 0.71

Thr, 1.0; Pro, 1.0; Gly, 1.06Met, 0.40(1); Ile, 0.0

Step 2 PTH-Pro; residue: Lys, 0.94Thr, 1.00; Pro, 0.13; Gly, 1.06Met, 0.78(1); Ile, 0.0

-This basic peptide gave a gray color with the collidine-ninldrin spray. The sequence determinations are summarizedTable XII. Digestion with carboxypeptidase A yielded oityrosine. Three steps of the Edman degradation establishthe sequence Glu-Asn-Pro-, and completed the sequencewritten above.

Peptide C-12 (Residues 75 to 80): Ile-Pro-Gly-Thr-Lys-MeThis basic peptide gave a blue color with collidine-ninhydspray. Hydrolysis with carboxypeptidases A + B for differtimes established the COOH-terminal sequence -Thr-Lys-lI(Table XIII). Two steps of the Edman degradation establishthe sequence Ile-Pro- and completed the sequence. The Imethionine after the first step of the Edman is due presumato destruction on acid hydrolysis.

Peptide C-13 (Residues 81 and 82): Ala-Phe-This neutpeptide was blue with collidine-ninhydrin. One step ofEdman degradation gave PTH-Ala, and the residue was idtified as phenylalanine by paper chromatography.

Peptide C-14 (Residues 83 to 85): Gly-Gly-Leu-This neutpeptide gave a green-yellow color with collidine-ninhyd'Two steps of the Edman degradation gave in each case PTH-Gand the residue after the second Edman step was leucine (pachromatography).

Peptide C-15 (Residues 86 to 9): Lys-Lys (Asp, Lys, AArg) Asn-Asp-Ile-Ile-Thr-Phe-This basic peptide gave a bcolor with collidine-ninhydrin. Carboxypeptidase A positiophenylalanine as COOH-terminal (Table XIV), but didestablish the order of (Ile2, Thr) and (Asp, Asn). Aminoptidase established the order Lys-Lys at the NH2 terminus.

TABLE XI

Amino aid sequence of Peptide C-10 (residues 60 to 65)

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TABLE XIV


Sequence

Wsrboxypeptidase A

Aminopeptidase

byptic peptidesT-1T-2T-3T4

Edman degradation

Lys-Lys (Asp, Lys, Asp, Arg) Asn-Asp-Ile-Ile-Thr-Phe20 min: Phe, 1.00; Ile, 0.07; Thr, 0.0660 min: Phe, 1.00; Ile, 0.44; Thr, 0.24

6 hrs: Phe, 1.00; Ile, 1.68; Thr, 0.85; Asp, 0.28; Asn, 0.273 hrs: Lys, 1.20

10 hrs: Lys, 2.17

Lys (identified by paper chromatography)(Lys 2_3, Asp)(Lys, Arg, Asp2 )(acidic peptide, color gray): Asp, 2.20(2); Thr, 0.95(1); Ile, 1.58(2); Phe, 1.00Step 1: PTH-Asn; residue: Asp, 1.04; Thr, 1.00; Ile, 1.59(2); Phe, 1.0Step 2: PTH-Asp; residue: Asp, 0.31; Thr, 1.04(1); Ile, 1.70(2); Phe, 0.98(1)Step 3: PTH-Ile; residue: Asp, 0.25; Thr, 0.94(1); lie, 1.00; Phe, 1.00Step 4: PTH-Ile; residue: Asp, 0.30; Thr, 0.84(1); Ile, 0.38; Phe, 1.00

portion (Asp, Lys, Asp, Arg), a unique peptide by composition,was determined from Peptide T-13b (see below). Four stepsof the Edman degradation and the carboxypeptidase resultsestablished the sequence of Peptide C-15-T-4.

Peptide C-16 (Residues 98 to 103): Met-Lys-Glu-Ala-Thr-Ala-This neutral peptide gave a blue color with collidine-ninhydrin.Hydrolysis with carboxypeptidase A (Table XV) established thearboxyl-terminal residue as alanine and the next 2 residues as

(Ala, Thr), although the sequence was left ambiguous. Diges-tion with aminopeptidase established the presence of glutamicacid and the absence of glutamine, in confirmation of the neutralelectrophoretic behavior. Four steps of the Edman degradationyielded the sequence Met-Lys-Glu-Ala-, thus establishing thesequence as written above.

Amino Acid Sequences of Tryptic Peptides

Peptide T-1 (Residues -4 to 4): Gly-Phe-Ser (Ala, Gly, Asp,Se, Lys)-This neutral peptide gave a gray color with the colli-dine-ninhydrin spray. The sequence at the NH 2-terminal endwas determined by three steps of the Edman degradation. StepI: PTH-Gly; residue: Lys, 0.87(1); Asp, 1.25(1); Ser, 1.73(2);Gly, 1.20; Ala, 1.15(1); Phe, 0.92(1). Step 2: PTH-Phe; residue:Lys, 1.00; Asp, 1.30(1); Ser, 1.44(2); Gly, 1.13(1); Ala, 1.08(1);Phe, 0.09. Step 3: PTH-Ser; residue: Lys, 0.86(1); Asp, 1.37(1);Ser,0.90(l); Gly, 1.15(1); Ala, 1.00; Phe, none. This uniquepeptide, having 2 of the 3 serines in the cytochrome molecule,obviously overlaps Peptides C-1 and C-2, and the Edman deg-radation confirms the presence of an unacetylated, free NH 2groupof glycine at the amino terminus.

Peptide T-3 (Residues 6 to 11): Gly-Ala (Asn, Leu, Phe, Lys)-This basic peptide gave a gray color with the collidine-ninhydrinSpray. Two steps of the Edman degradation established thefollowing sequence. Step 1: PTH-Gly; residue: Lys, 0.45(1?);Asp, 0.96(1); Gly, 0.23; Ala, 0.96(1); Leu, 1.00; Phe, 1.00. Step2: PTH-Ala; residue: Lys, 0.91(1); Asp, 1.03(1); Gly, 0.23; Ala,0.13; Leu, 1.00; Phe, 1.00. This peptide is part of Peptide C-2,pius lysine from Peptide C-3 (heme). Studies of Peptide C-2established that asparagine is present, and this is confirmed byte basic properties of Peptide T-3. This peptide established

sequence -Gly-Ala-, which was in doubt from studies oftide C-2.Peptide T-4 (Residues 12 and 13): Thr-Arg--This basic pep-ie gave a gray color with the collidine-ninhydrin spray. One


Aminopeptidase

Edman degradationStep 1Step 2

Step 3

Step 4

Met-Lys-Glu-Ala-Thr-Ala3 min: Ala, 1.20; Thr, 0.547 min: Ala, 1.60; Thr, 0.823 min: Met (paper chromatography)

60 min: Thr, 1.03(1); Glu, 1.04(1); Ala,1.97 (2); Met, 0.97 (1); no analysis for Lys

PTH-MetPTH-Lys; residue: Lys, 0.21; Thr, 0.98(1);

Glu, 1.00; Ala, 2.10(2); Met, 0.06PTH-Glu; residue: Lys, 0.15; Thr, 1.00;

Glu, 0.30; Ala, 2.05(2); Met, nonePTH-Ala; residue: Lys, 0.15; Thr, 1.00;

Glu, 0.30; Ala, 1.60(1); Met, none

step of the Edman degradation yielded PTH-Thr, and the residuewas identified as arginine by paper chromatography.

Peptide T-6 (Residues 28 to 36): (Ile, Gly, Pro, Ala) Leu-His-Gly-Leu-Phe-This basic peptide gave a blue color with thecollidine-ninhydrin spray. Hydrolysis with carboxypeptidase Afor 3 min released phenylalanine and leucine in equal amounts asjudged by paper chromatography, while digestion for 90 minyielded phenylalanine, leucine, glycine, and histidine. Thissequence proves the relative placing of Peptides C-4 and C-5and shows once more the position of histidine as carboxyl-terminalin Peptide C-4. The absence of a basic residue indicates that thispeptide resulted from a chymotryptic cleavage.

Peptide T-7 (Residues 37 to 53): (Gly, Arg, Lys, Thr, Gly, Ser,Val, Asp, Gly, Tyr, Ala, Tyr, Thr, Asp, Ala, Asn, Lys)-Thisneutral 17-residue peptide gave a blue color with the collidine-ninhydrin spray. No sequence studies were performed with thispeptide, but its composition provided information for overlappingPeptides C-6, C-7, and C-8, since it contains the arginine andserine residues.

Peptide T-7a (Residues 37 and 38): (Gly, Arg)-This basic pep-tide gave a green color with collidine-ninhydrin. From the colorand the tryptic specificity, the sequence is Gly-Arg. This pep-tide is part of Peptides C-6 and T-7 (amino-terminal in both),and results from a chymotryptic cleavage in the tryptic digest atphenylalanine at position 36.

Peptide T-7c (Residues 40 to 53): Thr-Gly-Ser-Val (Asp, Gly,

TABLE XV


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Tyr, Ala, Tyr, Thr, Asp, Ala, Asn, Lys)-This neutral 14-residuepeptide gave a gray color with the collidine-ninhydrin spray.Hydrolysis with aminopeptidase for different lengths ofj timegave the following results. At 30 min: Thr, 0.59; Gly, 0.21;Ser, 0.13; Val, 0.06. At 60 min: Thr, 0.77; Gly, 0.32; Ser, 0.25;Val, 0.16. At 3 hours: Thr, 1.00; Gly, 0.74; Ser, 0.67; Val,0.51. At 10 hours: Thr, 1.00; Gly, 1.04; Ser, 1.03; Val, 0.85.These results established the sequence at the amino terminus asThr-Gly-Ser-Val-. This peptide belongs to Peptides C-6, C-7,and C-8, and supplies, together with Peptide T-7, the necessaryoverlaps for the three peptides.

Peptide T-8 (Residues 54 and 55): Gln-Lys-This neutral pep-tide gave a blue color with the collidine-ninhydrin spray. Fromthe composition and tryptic specificity, this peptide is the linkbetween Peptides C-8 and C-9.

Peptide T-9 (Residues 56 to 72): (Gly, Ile, Thr, Trp, Asp, Glu,Asn, Thr, Leu, Phe, Glu, Tyr, Leu, Glu, Asn, Pro, Lys)-Thisneutral 17-residue peptide gave a yellow-green color with thecollidine-ninhydrin spray and a positive Ehrlich reaction. Thispeptide provides uniquely the overlaps for Peptides C-9, C-10,and C-ll.

Peptide T-11 (Residues 74 to 79): Tyr (Ile, Pro, Gly, Thr, Lys)-This basic peptide gave a blue color with the collidine-ninhydrinspray. It provides the overlap between Peptide C-11 (fromwhich the tyrosine is derived) and Peptide C-12. Aminopep-tidase digestion liberated only tyrosine as the N112-terminalresidue.

Peptide T-12 (Residues 80 to 86): Met-Ala-Phe (Gly, Gly, Leu,Lys)-This basic peptide gave a blue color with collidine-ninhy-drin. Hydrolysis with aminopeptidase gave the followingresults. At 5 min: methionine and alanine (identified by paperchromatography). At 60 min: Met, 1.02; Ala, 1.03; Phe, 1.00;Gly, 0.05. This peptide establishes the order of the two smallpeptides, C-13 and C-14, and links them to C-15 (from whichthe lysine is derived).

Peptide T-13 (Residues 87 to 97): (Lys, Asp, Lys, Asp, Arg,Asn, Asp, Ile, Thr, Phe)-This neutral peptide, obtained inonly 2% yield, gave a blue color with the collidine-ninhydrinspray. This peptide represents a chymotryptic cleavage in thetryptic digest at phenylalanine residue 97.

Peptide T-13b (Residues 88 to 91): Asp-Lys-Asp-Arg-Thisneutral peptide gave a blue color with the collidine-ninhydrinspray. Since this peptide is obviously part of Peptide C-15, itwas used to establish this part of the sequence in the latter pep-tide. Three steps of the Edman degradation gave the followingresults. Step 1: PTH-Asp; residue: Lys, 0.88(1); Arg, 1.12(1);Asp, 1.13(1). Step 2: PTH-Lys; residue: Lys, 0.18; Arg, 1.00;Asp, 1.15. Step 3: PTH-Asp; residue: arginine (identified bypaper chromatography).

Peptide T-14 (Residues 98 and 99): (Met, Lys)-This basicpeptide gave a gray color with collidine-ninhydrin. This peptideresults from tryptic hydrolysis at lysine and a chymotrypticcleavage between -Phe-Met-, resulting in Peptide T-13.

Peptide T-15 (Residues 100 to 103): (Glu, Ala, Thr, Ala)-Thispeptide was the only acidic peptide isolated from the trypticdigest, and gave a blue color with the collidine-ninhydrin spray.This particular peptide has to be derived from the carboxylterminus of the cytochrome molecule because (a) it lacks a pointof tryptic cleavage; (b) its composition is in accord with theresults obtained with carboxypeptidase A digestion of the wholemolecule; and (c) its composition fits that of the cyanogen bro-

mide Fragment III, i.e. (Lys, Glu, Ala, Thr, Ala) minuslysine residue.

Complete Sequence of N. crassa Cytochrome c

The data used in establishing the complete sequence are shoin Fig. 3. At the initiation of the sequence studies, it was knothat unacetylated glycine is the amino-terminal residue and talanine is carboxyl-terminal. The sequences of each individ,chymotryptic peptide and the compositions (in some cases wparts of the sequence) of the tryptic peptides were establish(The composition of the three cyanogen bromide fragments athe sequence of the chymotryptic heme peptide were also estalished. With this information, it was possible to deduce tentire sequence.

1. Peptide C-16 has to be the terminal peptide at the carboend. It is the only peptide with COOH-terminal alanine, andclearly related to Peptides T-15 and BrCN-III, the latter beithe only fragment from the cyanogen bromide action which lahomoserine.

2. The cyanogen bromide fragments account for the compotion of the whole protein; Fragment BrCN-III, as discussabove, is COOH-terminal, and Fragment I is the heme fragmeThis places BrCN-II either between Fragments I and III oramino-terminal to BrCN-I. The latter possibility is incopatible with the sequence as a whole. Fragment BrCN-II repsents the sum of Peptides C-13, C-14, and C-15. That PeptiC-15 precedes Peptide C-16 was proved by showing that PeptiT-12, which incorporates Peptides C-13 and C-14, containsNH2-terminal methionine residue, and thus has to be the iibetween Peptides C-12 and C-13.

3. The order of Peptides C-13 and C-14 is establishedPeptide T-12, which has the sequence Met-Ala-Phe- at its Nterminals, thus showing the order to be C-13 and C-14.

4. Peptides C-1 to C-12 account for the composition of BrC(heme). This fragment has only 1 homoserine residue fromethionine, and it obviously has to be COOH-terminal. Apafrom Peptide C-16, the only other methionine residue is presentPeptide C-12, and in this peptide methionine is the COOterminal residue. This links C-12 to C-13 through T-12,explained in Point 2 above.

5. Peptide T-11 represents a tryptic cleavage of Peptide C-plus a tyrosine resulting from a tryptic cleavage of Peptide C-There are only 4 tyrosine residues in Neurospora cytochromebut only the one at the COOH terminus of C-11 is situatedproduce the NH 2-terminal tyrosine of Peptide T-11.

6. Peptide T-9 contains the unique tryptophan residue aobviously represents parts of Peptides C-11, C-9, and all of CThe fact that Peptide C-9 is amino-terminal in Peptide T-9indicated by the fact that the only glycine in T-9, which has toderived from C-9, is NH2-terminal.

7. Peptide T-8 represents uniquely the overlap between Pttides C-8 and C-9.

8. Peptide T-7 includes parts of Peptides C-6, C-7, andFrom the composition of Peptide T-7c, C-6 is amino-termin,T-7.

The bond between residues 36 and 37, -Phe-Gly-, was foto be hydrolyzed in both the chymotryptic and tryptic dig,and no overlapping peptide was found. It is possible, howeto continue with the sequence deduction, starting now fromamino terminus.

9. It was established independently that the amino-tern

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de of July 10, 1966 J. Heller and E. L. Smith

I- . -- T3 T4 T 5 ----- T6-

2-Gly-Phe-Ser-Ala-Gly-Asp-Ser-Lys-Lys-Gy-Ala-Asn-Leu-Phe-Lys-Th r-Arg-Cys-Ala-Glu-Cys-His-Gly-Glu-Gy-Gly-Asn-Leu-Thr-GIn-Lys- le-Gly-Pro-Ala--4 A, - I I 10A 20 , 30

<Ct ,, C2…----- --

( …------ C2a…-------

C3 C4-

r<______ _ _--- - - - - - - - - - - - - - - - - -BrCN I - -- ----

-T<-T7a -><_74 - -- T7c - - - - - - - - - - - )

- T6 -- - -- -- - T7- - - ---- -T8> -- T9-

leu-His-Gly-Leu-Phe-Gly-A rg-Lys-Th r-Gly-Ser-Va-Asp-Gly-Tyr-Ala-Tyr-Th r-Asp-Ala-Asn-Lys-GI n-Lys-Gly-I le-Thr-Trp-Asp-Glu-Asn-Th r-Leu -P he-GI u-Tyr-40

-C4 >4- C54- t,b

Tp~ 50 T~ c 60,->- C74- C8- )-- C9 -C10 CI-

CIIa-

…-…-…-…-…-…-…-…-…----- - ---- - - - -B- BrCN I-- - - - - - - - - - - - - - - - - - - - - - --

T----T9-- --- TI-I I10I-

Leu-Glu-Asn- P ro-Lys-Lys-Tyr- Ile-P ro-Gly-Thr-Lys-MetI-A la-P he-G ly-Gly-Leu-Lys-Lys -A sp-Lys-Asp-A rg-As n-A sp- I le- IIe-Thr-P he-Met-Lys-G I u -Aa-Th r-AIaCOOH

70 80 90 100 103

II- Cl- C12 13 C14 ------ C1t6

i I - ---- - - ---

'- - - - - -- BrCN I - - BrCN II ..... BrCNIr---

FIG. 3. The complete amino sequence of N. crassa cytochromet. The tryptic peptides (T-) are shown above the sequence, andthe chymotryptic peptides (C-) and cyanogen bromide fragmentsBrCN-) are shown below. A solid line shows that the detailed

sequence of the peptide was determined, and a dashed line indi-cates that only the composition of the peptide was ascertained.

ePidue in Neurospora cytochrome c is unacetylated glycine.naong the chymotryptic peptides remaining to complete the

a'puence (C-1, C-2, C-3, C-4, C-5), two possess NH2 -terminalycine, i.e. C-1 and C-5, whereas, among the remaining trypticTides (T-1, T-2, T-3, T-4, T-5, T-6), two have NH2-terminal

Ycine, T-1 and T-3. However, only C-1 (Gly, Phe) has afmon sequence with T-1 (Gly, Phe), and so these must be the'terminal peptides.0. Peptide T-1 thus provides the overlap between Peptide

The tryptic heme peptide (T-5) was not isolated. The glycine atresidue 1 is equivalent to the acetylated NH2-terminal glycine invertebrate cytochromes in order to match the position of thecysteine residues attached to the heme. The residues indicatedby negative numbers indicate additional residues not present in thevertebrate cytochromes.

C-1 and the part of C-2 that has a unique composition, having 2serine residues of the 3 in the entire molecule.

11. Peptide T-3 is the overlap between Peptides C-2 and C-3(heme), sharing a lysine residue of the heme peptide and linkingPeptides C-1 and C-2 with the amino end of the chymotrypticheme peptide.

12. Only Peptides C4 and C-5 remain to be considered. Theorder is established as C-4 followed by C-5 by Peptide T-6, whichhas the sequence -His-Gly-Leu-Phe at its COOH-terminal end.

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The sequence as presented accounts for the complete aminoacid composition of Neurospora cytochrome c (1), and placesuniquely all of the chymotryptic and tryptic peptides and thecyanogen bromide fragments in a single sequence.

DISCUSSION

Specificity of Proteolytic Enzymes-Chymotrypsin hydrolyzedcompletely 15 bonds, and three additional bonds were partiallyhydrolyzed. Complete hydrolysis occurred at all the aromaticamino acid residues, except the Tyr-Leu bond at residues 67 and68, which was only partially cleaved. Of the 7 leucine residuesin the molecule, only one bond was completely hydrolyzed (posi-tion 85), whereas two more were partially hydrolyzed (positions9 and 68) and four were left intact. It is interesting that twothat were not hydrolyzed occurred in the sequence -Leu-Phe-(positions 35 and 65) and one occurred in the sequence -Leu-His(position 32), and that the bonds at the phenylalanine andhistidine residues were completely hydrolyzed. The fourthleucine bond that was not affected by chymotrypsin occurred inthe sequence -Asn-Leu-Thr-Gln- (residues 23 to 26). Thereason for the resistance to chymotryptic action of this bond isnot apparent.

The bond between Met-Ala at residues 80 and 81 was completelyhydrolyzed, whereas the bond -Met-Lys at residues 98 and 99 wasnot hydrolyzed at all. However, this methionine residue ispreceded by phenylalanine, and this bond was completely hy-drolyzed, leaving methionine as the amino-terminal residue inthis particular peptide. The bond -His-Gly- at residues 33 and34 was hydrolyzed completely.

The only unusual cleavages occurred in the sequences Asn-Lys-Gln-Lys (residues 52 to 55) and Asn-Leu-Thr-Gln-Lys (res-idues 23 to 27), in which the Gln-Lys bonds were completelyhydrolyzed. Similar Asn-Lys bonds in horse heart (19) and hu-man heart (20) as well as in other cytochromes c have been foundto be hydrolyzed by chymotrypsin. Hydrolysis by chymotryp-sin at glutamine or asparagine residues has been observed withother proteins and it is noteworthy that the amide residue isfrequently followed by a lysine or arginine residue.

Tryptic hydrolysis of the denatured protein did not result incleavage of all susceptible arginine and lysine bonds. The bondLys-Asp (residues 89 and 90) was not hydrolyzed at all, and thebonds Gly-Arg-Lys-Thr (residues 37 to 40) and Lys-Asp (resi-dues 87 and 88) were only partially hydrolyzed.

In the tryptic digest, there were two points of hydrolysischaracteristic of chymotrypsin specificity: at Phe-Gly (residues36 and 37) and Phe-Met (residues 97 and 98). This type ofhydrolysis is presumably due to the inherent chymotrypticactivity of trypsin. Since such action is limited, these bonds maybe the most susceptible to chymotrypsin and among the first to behydrolyzed when chymotrypsin itself acts on the protein, butbecause of the much higher ratio of enzyme to substrate used inthe latter case, this selectivity is not observed in the completedigest.

Structural Features of N. crassa Cytochrome c-Our studiesindicate that N. crassa cytochrome c is clearly related to allother mammalian type cytochromes c. Like all other cyto-chromes c of this type, it has a basic isoelectric point, is easilypurified on the acidic resin Amberlite CG-50, possesses a typicalspectrum both in the oxidized and the reduced states, and has theexpected molecular weight of about 13,000. Moreover, it hasbeen demonstrated earlier that Neurospora cytochrome c reacted

with mammalian cytochrome oxidase (21). Nevertheless,amino acid composition of Neurospora cytochrome c indicathat the structure had to differ considerably from that ofclosest relative, baker's yeast cytochrome c (22). Inspectof the amino acid sequence of N. crassa cytochrome c rev(that it bears an over-all structural similarity to other knocytochromes c and is clearly homologous with such molecuNeurospora cytochrome c is a single chain polypeptide conting 107 residues. Like yeast (22) and moth (23) cytochronand differing from all vertebrate cytochromes, it is unacetylatcontaining instead 4 additional residues at the NH 2 terminIt has the 2 heme-linked cysteine residues, spaced by thequence -Ala-Glu-, preceded by an arginine residue, and followby a histidine residue. It is noteworthy that there is presin all known cytochromes c at position 13 a basic residue, arginor lysine, preceding the heme linkage, and a histidine at posit18.

There are only 2 histidine residues in Neurospora cytochrolone in position 18 and the other in position 33, and 1 tryptoplin position 59. The sequence -Lys-Thr-Arg- precedingcysteine residue in position 14 is common to Neurospora ayeast (22) cytochrome c, but is different from all others. sequence following the histidine residue in position 18 is totsdifferent in Neurospora cytochrome c from any previouslycorded. Indeed, Thr at 19, Val at 20, Glu at 21, and Gly atand 24, which were found to be constant in all other known cychromes (24, 25), are all replaced in Neurospora cytochrom(The histidine residue in position 26, previously assumed toconstant in all cytochromes, is replaced in Neurospora cytochroby glutamine. The second histidine residue in Neurospora cychrome is present at position 33. Other amino acid replacemewhich have been observed for the first time in N. crassa cychrome c are Ser at -2, Ser at 3, Asn at 8, Glu at 16, Ile atAla at 31, Asp at 44, Gln at 54, Asp at 88, Ile at 94, Phe atand Met at 98. In contrast to the variability shown bycytochrome c in other parts of the molecule, the sequence frpositions 70 to 80 inclusive is the same as in all other knocytochromes.

Comparison of the sequences of Neurospora and yeast cychromes c shows that there are 39 amino acid differences betwethem (plus an extra residue at the NH 2 terminus in yeast cychrome c, making a total of 40). There are 43 amino acidferences between Neurospora and human heart cytochrome c (2and 47 between Neurospora and moth cytochromes (23). Tbcomparisons are made to indicate that the cytochromes oftwo Ascomycetes differ almost as much from each other as tbmolecules differ from the very much more distantly related insand man.

With the elucidation of the amino acid sequence of Neurospcytochrome c, the number of residues found to be constant incytochromes of known sequence (2) has been reduced to 3938% of the 103 residues which are possessed in common.would appear that this value is approaching the minimal numof residues necessary to define the essential structural reqAments for a cytochrome c molecule. Although some ofconstant residues have an obvious function, e.g. the 2 belinked cysteine residues, the role of the majority of constresidues is not precisely known at the present time. Howesince many of these are glycine residues (2, 24), it is evident tsome of these residues play a major role in conformation rlthan in enzymic activity per se. Indeed, this is certainly

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efamong the variable residues, such as the large hydrophobic,dues, many of which are replaced conservatively (24, 25).Iost interesting is the sequence of 11 residues from positionsto 80 inclusive, constant in all known cytochromes c, whichids in a marked contrast to the second longest constantDence of only 3 residues. Yet no obvious conclusions can bewn about the function of the 11-residue sequence by inspectingamino acid sequence, other than the previous inferences (24,that it is clearly nonhelical because of the presence of 2

Line residues, and that it may represent a critical conforma-Mal area.Isoelectric Point-As noted in the preceding paper (1), N.tochrome c has an isoelectric point that is significantly loweran that of other mammalian-type cytochromes. The dif-rence in isoelectric point between horse heart (a typical repre-

entative) and Neurospora cytochromes can be explained withec knowledge of the amino acid composition and the amide

content. Horse heart cytochrome c contains 19 lysyl, 2 arginyl,aspartyl, and 2 glutamyl residues, plus the free a-COOH of theminus, for a net excess of 15 positive charges, neglecting theistidyl residues, which would presumably be uncharged atkaline pH values. Neurospora cytochrome contains 14 lysyl,arginyl, 7 aspartyl, and 6 glutamyl residues, for an excess of 4positive charges, again neglecting the histidyl residues andking into account the presence of both a-NH3+ and a-COO-roups, which compensate for one another. Thus, horse cyto-

chrome c contains an excess of 15 positive charges, as compared to4 for Neurospora cytochrome, and would be expected to have amuch higher isoelectric point (as already reported), approxi-mately pH 10 as compared to approximately pH 9.3 for Neuro-wora cytochrome c. Whether this change in isoionic point andin net charge at the pH of the mitochondrial complex reflects afhnctional requirement or is purely fortuitous is unknown.

Nature of Iron Ligands in Cytochrome c-On the basis ofphysicochemical studies on beef and horse heart cytochromes c,Theorell proposed in 1941 (26) that, in addition to the four ligandsto the pyrrol nitrogens, the fifth and sixth iron ligands involve 2of the 3 histidines in the molecule. The proposal (27, 28) thatone of the ligands is not histidine but lysine has been renderedhighly improbable by the work of Hettinger and Harbury (9),who demonstrated that completely guanidinated horse hearttochrome c has the same spectrum, oxidation-reduction po-

tential, and enzymic activity as the native molecule. Moreover,anger and Harbury (29) have shown that trifluoroacetylated

Vtochrome c has a normal spectrum. The histidine residue, No.18, immediately following the heme-binding cysteine residue hasgenerally been assumed to be one such ligand. This was basedO (a) its proximity to the heme, (b) the fact that steric modelsan be constructed that will place the imidazole in position 18 in

the right conformation to bind to the iron (30), and (c) the con-411ncy of histidine in this position in all cytochromes c examinedAfar (2, 24, 25), including bacterial cytochromes (31, 32). Most

mammalian type cytochromes c contain histidine residues atoidue positions 18, 26, and 33. Kreil (33) reported that tuna

ei cytochrome c contains only 2 residues of hisidine at posi-ns 18 and 26. The conclusion that the second histidineand is in position 26 is an obvious one, as pointed out byW (33). N. crassa cytochrome c also contains only 2 histi-.residues; these are, however, in positions 18 and 33.

There are several interpretations for the different positions ofsecond histidine residue. The suggestion that the heme iron

in cytochrome c has only five ligands seems impossible in thelight of all physicochemical properties of cytochrome c, e.g. thediamagnetism of the reduced molecule and the absence of ligandformation by the native protein. That either histidine 26 in tunafish or histidine 33 in Neurospora cytochrome could ligate withthe heme iron would imply that the conformation of these twocytochromes is quite different; this seems very unlikely in viewof the great similarity in properties of all the mammalian typecytochromes c. At present, the best hypothesis would seem tobe that some group other than histidine forms the sixth ligandwith the iron. It seems reasonable to assume that this groupought to be found among the constant amino acid residues inall of the presently studied cytochromes c. This view wouldalso aid in explaining the similarity of spectrum of all cytochromesc, including the cytochrome c of Pseudomonas, which containsonly a single histidine residue at the place in the sequence analo-gous to that of position 18 (31).

Recently, Harbury et al. (34) have studied the reactions of theheme octapeptide from horse heart cytochrome c with a numberof methionine derivatives, and have shown that mixed complexesare formed, presumably involving the methionine thioether sulfuras the sixth ligand. These investigators have considered thatsuch methionine-heme binding may exist in the cytochromes c.This interpretation would also be in accord with the results ofAndo, Matsubara, and Okunuki (35), that carboxymethylationof the methionine at residue 80 leads to loss of the electron trans-fer capacity of cytochrome c.

Obviously, knowledge of model compounds alone cannot solvethis problem and the possibility of a ligand with 1 of the constanttyrosine residues cannot yet be excluded. We feel that theconstant tyrosine residue at residue 74 should also be consideredas a possible site of heme binding. If the tyrosine at residue 74or the methionine residue at 80 is involved in the heme attach-ment, a convincing explanation would be at hand for the absoluteconstancy of residues 70 to 80 over the billions of years of theevolution of aerobic life during which mammalian type cyto-chrome c has existed.

Acknowledgment-We are greatly indebted to Dorothy McNallfor the quantitative amino acid analyses.

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Joram Heller and Emil L. SmithCYANOGEN BROMIDE PEPTIDES, AND THE COMPLETE AMINO ACID SEQUENCE

: II. CHYMOTRYPTIC PEPTIDES, TRYPTIC PEPTIDES,c Cytochrome Neurospora crassa

1966, 241:3165-3180.J. Biol. Chem.

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neurospora crassa cytochrome cneurospora rassa cytochrome c. ii a stoppered vial, were added 25 1l...

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