Ferricytochrome c - Journal of Biological Chemistry · THE JOUXNAL OF ~~IOLOGICAL CHEMISTRY Vol. 246, No. 5, hue of March 10, pp. 1511-1535, 1971 Printed in U.S.A. Ferricytochrome

THE JOUXNAL OF ~~IOLOGICAL CHEMISTRY Vol. 246, No. 5, hue of March 10, pp. 1511-1535, 1971

Printed in U.S.A.

Ferricytochrome c

I. GENERAL FEATURES OF THE HORSE AND BONITO PROTEINS AT 2.8 A RESOLUTION*

(Received for publication, August 7, 1970)

RICHARD E. DICKERSON, TSUNEHIRO TAKANO, DAVID EISENBERG,~ OLGA B. KALLAI, LALLI SAMSON, ANGELA COOPER, AND E. MARGOLIASH

From the Norman W. Church Laboratory of Chemical Biology, California Institute of Technology, Pasadena, California 91iO9, and the Department of Molecular Biology, Abbott Laboratories, North Chicago, Illinois 60064

SUMMARY

The structure of crystalline horse heart ferricytochrome c has been determined by x-ray methods to a resolution of 2.8 A, and the results have been extended to obtain the structure of bonito cytochrome c as well. There is no difference between the horse and bonito structures, other than the expected changes in side chains where the amino acid sequences differ. The tertiary folding of cytochrome c has been maintained constant since the ancestors of mammals and fish diverged 400 million years ago, and probably for much longer. The polypeptide chain of 104 amino acids is wrapped around the heme in two halves; residues 1 to 47 to the right and 48 to 91 to the left of the heme, which sits in the resultant heme crevice with one edge exposed to solvent. Residues 92 to 104 form an cx helical strap that rises over the top rear of the molecule and back across the right side again. Cysteines 14 and 17 and histidine 18 extend to the heme from the right wall of the heme crevice, and methionine 80 extends from the left wall. The heme is tightly enveloped in hydrophobic groups. Two “channels” filled with hydrophobic side chains lead to the right and left from the heme to the surface of the molecule. Each channel contains at least two aromatic rings in roughly parallel orientation, and each channel is surrounded by a cluster of positively charged lysines where it meets the surface. At the back rear of the molecule, between the two positive regions, is a cluster of nine negatively charged acidic groups. It is proposed that these surface features may be involved in binding to other macromolecular complexes, including the cytochrome oxidase system.

The principal folding influence on the molecule appears to be the heme group itself. Only residues 92 to 102 are genuinely (Y helical, although several other areas might have been were it not for interference from the heme. Methods currently proposed for predicting (Y helical regions

* This paper is Contribution 4085 from the Norman W. Church Laboratory~ of Chemical Biology, California Institute of Tech- nolog.y, Pasadena. California 91109. The work was SUDDOrk!d bv National Science Foundation Grant GB-6617, and National In- stitutes of Health Grant GM-12121.

$ Present address, Department of Chemistry, University of California at Los Angeles, Los Angeles, California 90024. This research was conducted in part during leave of absence from UCLA.

are promising but insufficiently discriminating. The abrupt 310 bend, occasionally observed in other globular proteins, occurs six times in cytochrome c, at locations where a sharp chain reversal is needed in wrapping around the heme.

The x-ray structure shows the reasons for many of the unchanging features apparent from comparison of amino acid sequences from over 30 different species. Hydrophobic groups are invariant because they play an essential role in providing the proper heme environment and causing the protein to fold around the heme. Acidic and basic chains are necessary for the preservation of segregated regions of charge on the surface. Glycines often occur where there is no room for a side chain, and serines and theronines play important hydrogen-bonding roles in maintaining the folding of the protein.

Cytochrome c is an electron-carrying protein found in mito- chondria of all aerobic organisms. It is part of the terminal oxidation chain, which completes the breakdown of foods to COZ and HzO, storing the liberated chemical energy in molecules of ATP. Like myoglobin, it is an iron porphyrin protein, made up of one heme group and one polypeptide chain. The iron atom alternates between the +2 and +3 oxidation state as the molecule interacts in turn with cytochrome reductase and cytochrome oxidase, each a large multimolecular complex (l-3). One of the goals of the present x-ray analysis is to understand how electron transfer occurs into and out of cytochrome c, which will ulti- mately require a knowledge of the molecular structure in both the ferric and ferrous states. This paper reports the structure of the ferric protein from horse and bonito hearts at a resolution of 2.8 A.

A second reason for the interest in the three-dimensional structure of cytochrome c is the vast amount of information accumu- lated about the amino acid sequences of cytochromes c from different species. Cytochrome c is found across the entire spectrum of animals, plants, and aerobic microorganisms except bacteria, making it an admirable tool for studying the process of molecular evolution (2, 4-20). The primary structures of the proteins of over 30 species are now known, including those of man, chimpanzee, rhesus monkey, horse, donkey, cow, pig, sheep,

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1512 Ferricytochrome c Xtructure at 2.8 A Resolution Vol. 246, No. 5

Horse and Bonito Cytochrome C

CH~-CO-NH-GLY-A~~-V~I-~~-L~S-GLY-L~S-L~S-~~-PHE-‘~

-Val-Gln-Lys-GYS-Ala-Gln-CYS-HIS-Thr-Val-Glu- kzz- ”

-Gly-Gly-Lys-His-LYS-~O~-GLY-PRO-Asn-LEU-T,~- His ‘.Ly-34

-Leu-Phe-Gly-ARG-Lys-Thr-GLY-Gln-Alo-PGI-46

-$F-TYR-Thr-Asp-ALA-ASN-Lys-&Lys-Gly-IIu- $~~-”

-TRP-~Y,S,-GIu-GA:U,-Thr-Leu-Met-Glu-TYR-LEU-Glu-ASN-70

-PRO-LYS-LYS-TYR-ILU-PRO-GLY-THR-LYS-MET-IIU-PHE-~”

-Ala-GLY-Ilu-Lys-LYS-Lys-~~~-Glu-ARG-~~-Asp-Leu-g4

-~~-Ala-Tyr-Leu-Lys-ser L’S-Alo-Thr-!$-G&COOH

FIG. 1. The amino acid sequences of horse and bonito cytochrome c. Where two amino acids are shown for a given residue, the upper 0723 is horse and the lower is bonito. All other residues are identical in both species. Residues designated bv capital letters are invariant among the 29 species whose sequences are listed in the “Appendix.” The bonito sequence is a personal com- municat,ion of as yet unpublished work by T. Nakayama, K. Titani, and K. Narita. There may be a deletion of the 104th residue in bonito, as in tuna.

dog, rabbit, California gray whale, great gray kangaroo, chicken, turkey, pigeon, Pekin duck, snapping turtle, rattlesnake, bullfrog, tuna, bonito, dogfish, two moths, screw worm fly, Drosophila, bakers’ yeast, Candida krusei, Neurospora, wheat, mung bean, sunflower, sesame seed, and castor bean. (A con- venient tabulation of the proteins from 29 species from Reference 21 is given in the “Appendix”.l)

Cytochrome c from one species will react in vitro with cytochrome-oxidase preparations from distantly related species (2). When proper control is exercised over ionic strengths and specific anions present, all the cytochromes c tested appear to be func- tionally identical, in spite of considerable differences in amino acid sequences.2 This argues strongly for the retention of the same tertiary folding of the molecule during the course of evolution. It also means that by studying the three-dimensional structures of one or two cytochromes c alongside the entire corpus of amino acid sequence information, we can hope to determine what portions of the molecule are essential for its structural integrity and its operation as an electron carrier. We can use cytochrome c to study the process of evolution, and can also use the results of evolution to study the cytochrome c molecule.

CHEMICAL PROPERTIES OF CYTOCHROME C

The amino acid sequences of horse and bonito cytochrome c are shown in Fig. 1. The 104 amino acids are in one continuous polypeptide chain, with no disulfide bridges. In contrast to the globins, the heme group in cytochrome c is covalently bonded to the protein chain by thioether links to cysteinyl residues 14 and 17. Four of the octahedral ligands to the iron atom come from

1 References to the amino acid sequences of the cytochromes c listed can be found in Reference 21, except for those from bonito, mung bean, sunflower, sesame seed, and castor bean. The amino acid sequence of bonito cytochrome c was kindly provided in a personal communication by T. Nakayama, K. Titani, and K. Narita, and the plant cytochrome sequences were provided by Dr. D. Boulter. None of these unpublished results have been included in the tabulation in the “Appendix”. The comparisons in this paper are based on that 29.species tabulation, although none of the conclusions reached are altered in any way by the inclusion of the unpublished sequences.

2 L. Smith, personal communication.

TABLE I Variability of amino acid residues in comparison of horse with

28 other species

Data from “Appendix.”

Residue type

Rasic Lys

Arg 38, 91

Acidic Asp Glu

Ambivalentb Asn. Gln Ser. Thr

52, 70

78

His 18 Tyr 48, 67, 74 Trp. 59 cys. 14, 17 Ala. 51

Glycine. 1,6,29,34,41, 45, 77, 84

Hydrophobic Val Leu. Ile. Met Phe. Pro.

32, 68 75 80 10, 82 30, 71, 76

Residue nos. in horse cytochrome c -

--

-

27, 72, 73, 79, 87

-

.-

-!-

Conservative” Variable

13 5, 7, 8, 22, 25, 39, 53, 55, 60, 86, 88, 99, 100

2, 50, 93 4, 21, 61, 62,

66, 69, 92, 104

31 54, 103 42 12, 16

19, 40, 47, 49, 63, 102

26 97 (Phe)

28, 58, 89

15, 43, 96

23, 37, 56

83, 101

24

35, 64, 94, 98 57, 81, 85, 95

3, 11, 20

9 (Thr) 65

36, 46 Wyr) 44

a Conservative substitutions as recognized in this table involve changes within the classes shown at left, with the addition that Ala is also included in the hydrophobic class and Gly in the ambivalent class.

b An ambivalent residue is one which has been encountered often both in the interior and on the surface of globular proteins. This class includes the polar uncharged residues plus Ala.

the porphyrin ring itself. The fifth is an imidazole nitrogen of histidine 18. The sixth ligand was uncertain on chemical grounds. It was proposed to be a histidine by Theorell and Akesson (22), a lysine by Margoliash, Frohwirt, and Wiener (23), and methionyl residue 80 by Harbury et al. (24), Ando, Matsubara, and Okunuki (25), and Tsai and Williams (26, 27).

Vertebrate cytochromes c generally have 104 amino acids, with an acetylated amino terminus, as shown in Fig. 1. The inverte- brate proteins have as many as 8 extra residues at, the amino terminus, which is acetylated in higher plants but not in microorganisms or insects. One of the most striking features of cytochrome c is its high lysine content-19 residues in the horse protein (Table I). Horse cytochrome c also carries 2 arginines and only 12 acidic residues, or aspartic or glutamic acids. The result is a basic protein, with an isoelectric point near pH 10 (3).


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Both of the arginine residues and 5 of the lysines are totally residue ellipticity for a 100% helical structure as Flatmark. But invariant in all cytochromes of known amino acid sequence. with a different calibration value proposed by Aki et al. (33) The lysines are not uniformly distributed along the chain, but are the same data yielded 17% and 18% for ferric and ferrocyto- clustered, as can be seen in Table II. Moreover, when a lysine chrome c. Zand and Vinogradov regard these figures as an common to the cytochromes c of most species is missing in one upper limit because of the possibility that clusters of several particular species, another basic residue is often found nearby in amino acids with (Y helix-like Ramachandran angles (4, #) might the same cluster. The basic character of these regions of the add to the circular dichroic values. They estimate 10% helix chain is maintained throughout evolution even though the specific as a lower limit. As we shall see, this last figure is correct, and lysine positions are not (2, 9). for the reasons that they propose. However, what is even more

Acidic regions of the chain, with aspartic and glutamic acids, significant than the absolute values is the similarity of helix con- are also conserved, as the data in Table II indicate. This feature tent in the ferric and ferrous forms of the molecule. was overlooked in earlier sequence comparisons because no Several indirect lines of evidence indicate some conformational acidic group is absolutely invariant (Table I). Evolutionary changes in the cytochrome c molecule upon reduction and oxida- selection pressures evidently operate for a negative charge in tion. This evidence is discussed in Reference 2 but can be sum- these portions of the molecule and not for a specific size of side marized briefly. The reduced protein is less susceptible to chain. The regions containing residues 2 to 4, 21, 60 to 69, and digestion by trypsin (34-37), less easily spread at water-air 89 to 93 are particularly acidic. Equally impressive are the interfaces (38), and shows greater thermal stability than the long stretches of chain where acidic groups are never found. oxidized molecule (39), all suggesting a more compact structure. Even from amino acid sequence comparisons alone, it is apparent Nuclear magnetic resonance spectra (40-42) and spectrophoto- that basic and acidic side groups are segregated along the poly- metric tyrosine ionization curves are different in the two states peptide chain. (43). Finally, it is easier to replace the sixth iron ligand with

The hydrophobic residues (alanine, valine, leucine, isoleucine, an outside ligand such as cyanide in the oxidized molecule, sug- phenylalanine, and methionine) also occur in clusters along the gesting that the heme crevice is masked or closed in some way on chain (Table II). Although individual residues are seldom reduction (44). invariant (Table I), the changes which are observed are highly Cytochrome c interacts with the cytochrome reductase complex conservative from one hydrophobic residue to another (2, 6, 9), and with cytochrome oxidase. Considerable evidence now indi- All of the 12 glycines in horse cytochrome c are either invariant cates that it binds to cytochrome oxidase during electron trans- throughout all species, or very nearly so, and 9 of the 12 occur in fer. The mode of interaction with the reductase is completely the first half of the chain. There is a high degree of conservatism unknown, but the outlines of binding to preparations of the among the hydroxyl-bearing and potentially hydrogen-bonding oxidase are at least partly clarified (2, 45-47). One mole of serine and threonine residues (15). Aromatic groups are also cytochrome c binds per mole of oxidase heme, and the binding evidently important to the operation of the protein, with the sites are independent of one another. Oxidized and reduced essential aspect of tyrosine being the aromatic ring more than the cytochrome c bind equally well to the oxidase, and the spectrum hydroxyl group. Tryptophan 59, tyrosines 48, 67, and 74, and of the complex indicates a mixture of both oxidation states. phenylalanines 10 and 82 are totally invariant. Tyrosine 97 Binding is independent of pH between pH 6.0 and 7.5, but is becomes a phenylalanine in one species; phenylalanine 46 can inhibited by cations, which compete with cytochrome c for only be replaced by tyrosine (nine species) ; and phenylalanine binding to the oxidase. The basic lysyl side chains are essential 36 is replaced only by tyrosine (one species) or isoleucine (three for binding. If they are succinylated, changing the positive species). The heme-binding cysteines and the heme-liganding charges to negative, or if they are acetylated, removing the histidine 18 are completely unchanging, and a striking region of charge, then the oxidase reaction is destroyed. But if the lysines total invariance is that from residues 70 through 80, where are guanidinated, retaining the positive charge but with bulkier deviations from the sequence given in Fig. 1 are never observed side chains, then the molecule is as active or more active than in any species. native cytochrome c. Basic proteins such as salmine, clupein,

In summary, cytochrome c is a highly conservative protein or poly-L-lysine compete with cytochrome c for the oxidase. with certain key regions or groups invariant: the heme-binding (To what extent results obtained with detergent-solubilized region, residues 70 to 80, and some of the aromatic groups. preparations of the oxidase can be considered to represent the Other types of residues are either highly conserved (glycines, situation in the mitochondrial membrane is very hard to assess.) serines, threonines, and aromatics) or both conserved and clus- Finally, Wada and Okunuki (48) have recently shown that half tered along the chain (basic, acidic, and hydrophobic residues). of the reactivity of cytochrome c with its oxidase is lost if just one One of the tasks of x-ray analysis, obviously, is to give a satisfac- lysine is trinitrophenylated, and that this essential residue is tory explanation of these features. residue 13, and not lysine 72 or 73 as originally reported (49).

Estimates of a! helix content of cytochrome c by optical rota- The second residue to be trinitrophenylated, lysine 22, has a far tory dispersion and circular dichroism methods have steadily smaller effect. The reactivity to acetic anhydride is the reverse; fallen with time. Lumry3 estimated a 41 to 45% helix content lysine 22 is the first to be acetylated, and lysine 13 is probably in 1964 from the Cotton effect at 235 rnp. Urry and Doty next. The relative oxidase activity drops linearly with the (28-30) predicted 27 % helix in the oxidized form and 34 % in the number of acetylated lysyl residues, and activity is totally de- reduced from optical rotatory dispersion measurements. Flat- stroyed when 6 lysines are modified (50). As shown below, lysine mark and Robinson (31) found 27% helix in both oxidation 13 sits on the outside of the molecule, at the top end of the heme states from circular dichroism. Zand and Vinogradov (32) crevice. calculated 26% and 2Syc using the same value of the mean

The acetyl group on lysine 13 is much less effective in inhibiting reaction with oxidase than is the trinitrophenyl group,

3 R. Lumry, personal communication. nrobablv because the bulkv, hvdrophobic trinitrophenyl blocks “. I _

Issue of March 10, 1971 R. E. Dickerson et al. 1513


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TABLE II

Frequency of occurrence of jive classes of amino acids in the cytochromes c of 29 species

(Data from “Appendix.“)

Residue type

Basic (Lys, Arg) ..................... Acidic (Asp, Glu) .................... Hydrophobica. ....................... Glycine. ..............................

Ser-Thr .............................. Asn-Gln .............................. Tyr-Trp-Phe .........................

cys ................................. His .................................. Pro ................................... Alaa. .................................

Residue type

Basic (Lys, Arg) ..................... Acidic (Asp, Glu) .................... Hydrophobica. ........................ Glycine ...............................


cys ................................. His .................................. Pro ................................... Alac ..................................

Residue type

Basic (Lys, Arg) ..................... Acidic (Asp, Glu) ............... ... Hydrophobica. ...................... Glycine ...............................


cys ................................. His .................................. Pro. .................................. Alaa ..................................

Residue no. -

2 -

24

-

2 3

-

-

- 4

3 25

-

1

-

,

-

-

-

_

_

-.

-

-8 -7 -6 I I ---

-5 12 -3 -2 -1 1 --

1 1

5 29

1 1

3

5 1 6

10 11

4

24

-.

-.

26 25 26 2

5 27

29

1

1 1

Residue no.

- 24

2 27

-

-

-

- 15

-

1

- 8

-

20 -

- 35

-

29

-

- 17

-

.-

- 14

-

-

29

-

34

29

-

--

-

-

_-

. -

-

-

_-

_-

-̂

-

-

--

--

-_

-

.-

.-

-

-

_-

--

--

26

59 --

3

31 32 --

29

28

49 50 --

20

-- 29

4

--

5

--

1

51 52

-

-

-

.-

.-

-

29

-__

29

-

_-

-_

-.

13 16 20 -

1 27

18 19 --

1 --

28

--

29

21 22 --

20 28

1 1

2

-.

-,

-.

-

-

-

-

29 2 1

26 1

-

28

-

29

- 1

6

Residue no.

33 46 36 37 38 39 40 41 42 ---- ---

29 26

3 28 29

-----_-

1 29 2 1 27

26 --A----

2

43 44 45 ---

8

28 7 29

-__-

1 2

- -.-

12

-_

1 2 2

-

24

-

access to the heme crevice, or prevents a rearrangement of poly- applied to data from the bonito protein (51), whose crystals peptide chain near the crevice upon reduction. are isomorphous with those from horse.4

EXPERIMENTAL PROCEDURE 4 The bonito crystals are from “old” bonito in the terminology

of Reference 51. In Table II of that paper, Line 3, the left-hand

Cr&al Structure AnalysisA two-derivative isomorphous entry: “Young Bonito (Fe++)“, should read: “Old Bonito (Fe++)“.

replacement phase analysis was carried out with horse ferricyto- The a axis is doubled over that previously reported. The true

chrome c at a resolution of 2.8 A. These phase results were then space group is P212121, and the cell dimensions are a = 57.44 A, b = 84.61 A, c = 37.58 A, with 8 molecules per unit cell.


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Issue of March 10, 1971 R. E. Dickerson et al.

TABLE II-Continued

Residue no.

- 61

-

27

-

2

-

-

Residue type

53 -

Basic (Lys, Arg) ...................... 28 Acidic (Asp, Glu) ..................... Hydrophobic”. ......................... 1 Glycine ................................

- Ser-Thr ............................... Asn-Gln ............................... Tyr-Trp-Phe ..........................

-

-_

-_

cys .................................. His .................................... Pro .................................... Alaa ...................................

- -

Residue type

73

Basic (Lys, Arg) ...................... 29 Acidic (Asp, Glu) ..................... Hydrophobic”. ......................... Glycine ................................

Ser-Thr ............................... Asn-Gln ............................... Tyr-Trp-Phe ............... ..........

cys .................................. His ................................... Pro .................................... Alaa ...................................

-

- 59

-

-

29

-

- 57 -

29

-

-

-

- 58

-

2 7

20

-

- 60

-

2 3

18 -

5

-

1 -

- - 65 -

1

20

-

2

6 -

6 15 2

-

.-

.-

54 55 56 ---

2 28

27 -~-

2 21 1

-__-

4 1 1

62 63 --

25

64

29 :

.--

67 68 69

29

_-

29 _-

27 1

.__- 1

Residue no. -

74 -

-

29 -

-

75 76 --

29

--

29

77 78 79 80 -_--

29

29 29

--___

29

85 86 87 ---

28 29

29

---

1

---

88 89 --

20 4 2 5

8 --

1 5 5

--

3 3 2

90 91 --

29 29

--

--

Residue no.

Residue type

93 96 97 --

--

3

29 --

26

-

--

--

--

-

98 99 100 101 102 ___----

28 15 1 8

29 1

-~-~- 4 2 26 2

-~-~-

2

26 1

103 104 ___-

6 6 1 12

~- 6 2

11

~-

5 1

Basic (Lys, Arg) ...................... Acidic (Asp, Glu) ..................... 27 Hydrophobic”. ........................ Glycine ................................

Ser-Thr. ............................... Asn-Gln. ............................. Tyr-Trp-Phe .........................

2

cys .................................. His ................................... Pro .................................... Alao ...................................

= Hydrophobic = Val, Leu, Ile, Phe, Met. Ala is counted as hydrophobic if at least one other hydrophobic residue occurs at that position.

The preparation of horse crystals, search for heavy atom derivatives, and two-dimensional analysis have been described in References 52 and 53. In brief, ferricytochrome c was ex- tracted from horse hearts, chromatographically purified, and crystallized from nearly saturated ammonium sulfate solution at pH 6 to 7. The two suitable heavy atom labeling compounds, K2PtC14 and mersalyl, HO-Hg-CH.z--CH(O-CH8)-CH2 NH-CO-(o-CGHJ-0-CH-COONa, were added in solution

to separate vials of crystals after growth. Greater binding of both derivatives to the protein could be produced by transferring previously grown crystals from ammonium sulfate to 4.3 M

K2HPOrNaH2P04 buffer at pH values near 6.2 before adding heavy metals, and this improvement was used for both derivatives at high resolution (53, 54).

The space group is tetragonal, P43, with 1 molecule per asym- metrical unit or 4 molecules per unit cell of dimensions: a = b =


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1516 Ferricytochrome c Xtructure at 2.8 A Resolution

TABLE III

Vol. 246, No. 5

Observed structwe factor data and phase analysis results for horse heart ferricytochrome c analysis Data are on same relative scale as coefficients of Table IV. To place on absolute scale of electrons per unit cell, multiply by 0.18.

The symbol -1 indicates no data. The complete Table III and electron density map (Fig. 3) have been deposited with the National Auxiliary Publication Service. For copies of these data, order NAPS Document 01341 from ASIS National Auxiliary Publications Service, CCM Information Sciences, Inc., 909 Third Avenue, New York, 10022. Remit $4.00 for microfiche or $9.50 for photocopies, payable to CCMI-NAPS.

h k

-

1 (nBb Fld Fz

0 0 4 130 159 860 8430 8706 0 0 8 20 22 990 5825 5941 0 0 12 55 80 791 -1 1324 1 0 2 240 247 814 4638 4236 1 0 3 55 115 504 852 755 1 0 4 285 350 391 3620 3506 1 0 5 105 103 989 2443 2188 1 0 6 170 169 933 2002 2004 1 0 7 20 26 945 2899 2901 1 0 8 180 164 705 1974 2238 1 0 9 235 188 605 3812 3886

Fa MI M2 Ma

8876 9177 9278 9633 6524 7975 5939 6398 6522 6680 6642 5689 1494 -1 2397 2321 -1 1187 4172 4921 4767 4889 4313 4413 1351 952 1446 1316 2172 2568 3683 3469 3587 3714 3662 3747 2394 2206 1560 1875 3179 2247 1884 1934 1500 1704 1883 1997 2801 3131 2896 2780 3363 3334 2033 2061 2076 1897 1635 1691 3861 3883 3681 3682 3372 3364

-

-

(Data continued on microjilm; total of 3697 rejZections)

Tf

7535 6696 7304 5561 3307 3226 1449 368 857 4370 4183 3418 2395 1029 893 3935 3570 3831 2730 2293 2660 1923 1470 1540 3054 2627 3448 1635 1412 1556 3282 2998 3447

a pi, most probable phase in degrees, from horse phase analysis. b qB, centroid or “best” phase in degrees, from horse phase analysis. c m, figure of merit X 1000. d Horse data: F1, native protein in ammonium sulfate, 4 A resolution; F 2, native in ammonium sulfate, 2.8 A, F(h, k, 1) ; Ft. same,

Friedel pair, F(E, ,&, t); M1, mersalyl in ammonium sulfate, 4 A; Mz, mersalyl in phosphat,e, 2.8 A, F(h, k, 1); Ma, same, Friedel pair, P(&, f, I); P1, platinum chloride in ammonium sulfate, 4 A; Ps, platinum chloride in phosphate, 2.8 A, F(h, k, 2); P:, same, Friedel pair, F(fi, I%; t).

e Bonit,o data: B, native bonito cytochrome in ammonium sulfate, 2.8 A. 1 Tuna data: T, native tuna in ammonium sulfate, 2.8 A. (Collected by Ron Jevning and Paul Simpson, Stanford University.)

58.24 A, c = 41.86 A. Heavy atom positions were located inde- ever, the mean value of the cytochrome structure factors at the pendently for the platinum and mercury derivatives from two- center of the pattern is 2910 and at the 2.8 A limit is 1080, and and three-dimensional (AF)z Patterson maps, and then cross- has an average value of 1777. The mean per cent error in F checked with (AP) difference Fourier maps with intensity data values from scaling films together was 3.6 to 3.7y0 for all three from one derivative and signs or single isomorphous replacement data sets. The mean deviation of Friedel pairs from their phases from the other (52, 53). Mersalyl binds to a single site average F was 1.7 y0 for both the parent crystals and the platinum which proved to be histidine 33. Platinum binds to methionine and mercury derivatives. The mean change in protein F pro- 65, with a minor second site near histidine 33 which makes little duced by the heavy atoms was 20.4y0 for mercury and 19.8% contribution to the phase analysis. for platinum.

For the 2.8 A resolution analysis, 7400 reflections were collected on 21 sets of precession camera films for the parent horse cytochrome c crystals and for each of the two derivatives. These included all 3400 reflections within the 2.8 A limiting sphere, and their Friedel conjugates for anomalous scattering analysis. In- tensities were measured with a Joyce-Loebl microdensitometer and scaled by a least squares procedure using the maximum overlap of data on different film sets. The final mutually scaled data are given in Table III (microfilm version). Since the inner- most reflections would be sensitive to a change of medium, the extent of salt changes produced in going from ammonium sulfate to phosphate buffer was examined as a function of sin 0. For safety, all data from the mercury and platinum 2.8 A sets in phosphate were suppressed within a sphere of resolution 9 A around the origin. Phases for these inner reflections were determined only with the mercury and platinum 4 A ammonium sulfate data.

A search for secondary binding sites was made with three- dimensional difference Fourier (AF) maps with a two-derivative phase set based on one spherical heavy atom per derivative. The platinum major site, which was elongated at 4 A, proved from the 2.8 A difference map to be a double site (54). The two sites, each of roughly half-occupancy, had the correct location and spacing to be the mutually exclusive alternate coordination sites to the two lone electron pairs on the sulfur atom of methionine 65. There was no suggestion of asymmetry in the mercury heavy atom, or of minor mercury binding sites.

Radial distribution statistics for the data of Table III after scaling are shown in Fig. 2. The mean structure factor curve would run off the top of the plot and has been omitted. How-

Although the mean Friedel differences in mercury and platinum were little greater than those in the parent protein (which can be taken as a measure of the experimental error in their measure- ment), enough large changes were present to make an unam- biguous determination of the absolute handedness of the molecule. Samples of the greatest 50 and greatest 200 Friedel pair differences in both heavy atom derivatives all gave a uniform preference for space group P43 rather than P41. This choice produces a right-handed a! helix in the final structure, which is reassuring since a left-handed (Y helix has never yet been found


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Issue of &larch 10, 1971 R. E. Dickerson et al. 1517

in any globular protein. Anomalous scattering contributions were included in the phase analysis by the method proposed by Matthews (55).

The heavy atom parameters were refined by alternate cycles of phase determination and least squares refinement as described in Reference 56. Final values of these parameters are given in Table IV. An approximate absolute scale was obtained by assuming a full weight contribution of 146 electrons in the two PtClb2- sites on methionine 65, taken together. This produced

600-

-1.00

m

- .90

. .80

- .?O

- .60

- .50

- .40

- .30

- .20

oAF,(ano)

o- I 1 8 .02 .04 .06 .dS ,,b ' I ' s 8 ' I ' s .20 .30 .34

f t t 10% 68 5%

t 48 3% t 2.88 t 2s t

S2= (2 sin Q)2-

FIG. 2. Radial distribution plot of statistical data from the final 2.8 A phase analysis of horse oxidized cytochrome c. m, mean figure of merit, from centroid phase determination; AFH,, A8'pt, mean changes in protein structure factors produced by the mersalyl and PtC1k2- derivatives; AFN(B - H), mean difference between native cytochrome c data from horse and bonito. eHg, tpl, mean lack of closure errors in heavy atom derivatives; A~N(ano), AFH, (ano), A8'pl (ano), mean differences between Friedel pairs for native cytochrome and the two derivatives. The numbers at the left may be put on an approximate absolute scale of electrons by multiplying them by 0.18.

a scale factor of 0.18 for the data of Table III, and occupancies of 68 and 78 electrons for the platinum double site, 17 electrons for the minor site, and 73 electrons for the mercury site. Individual phases and figures of merit are listed in Table III.

The over-all figure of merit for the 2.8-A analysis was 67.4%, with the radial distribution shown in Fig. 2. An electron density map was calculated, weighted with figures of merit, in a series of sections of spacing AZ = 0.02 along the c axis. Eight of these are shown in Fig. 3, and the entire map is available on microfilm from ASIS along with Table III. The root mean square error in electron density in this map, as calculated from Equation 10 of Reference 57, was 0.27 e per Aa, or 1.5 contours in Fig. 3. The mean lack of closure errors in the phase triangles, c, is plotted for both derivatives in Fig. 2. The disappointing aspect of these results is that the mean lack of closure error is roughly independent of sin 0, and that the observed heavy atom changes approach this value at the outer edge of the 2.8 A data set. A large proportion of the observed AF is evidently not accounted for by the heavy atom model. Experimental errors in intensity data are well under control, and are considerably smaller than this lack of closure error. Neither is there any indication from various types of difference maps or least squares refinement that additional heavy atom sites are called for, or that the ones now used should be modified in shape. Preliminary results with experiments now in progress suggest that the change of crystal medium is likewise not the cause. We have been forced to conclude that the isomorphism of the heavy atom derivatives and the parent crystals is defective. In view of the inherent tendency of the horse crystals to show growth disorder, and their sensitivity to thermal, radiation, and mechanical damage (53), this is not an unexpected conclusion. Crystals of bonito cytochrome c, in contrast, are stronger and less disordered, and produce a better x-ray diffraction pattern. The mersalyl derivative is lost with bonito crystals, for residue 33 is tryptophan instead of histidine. If suitable new derivatives can be found, the 2 A analysis will be carried out on bonito crystals.

Interpretation of Electron Density Map-The electron density map was plotted on Plexiglas sheets at a scale of 2 cm per A, and a model was constructed with Kendrew skeletal units in a Richards box (58). In this device the map, illuminated from behind, is viewed through a half-silvered mirror set at a 45” angle. The image of the molecular model being constructed to one side is reflected in this same 45” mirror. I f the illumination of map and model is properly balanced, the illusion is created that the model is being built inside the electron density map. Model building is much more accurate than if dimensions are transferred from map to model-building frame with ruler and calipers. The Richards box is especially advantageous at this resolution, where what one

TABLE IV

Heavy atom parameters after refinement

z Y s Aa f' f" B 811

Mersalyl - 0.0208 0.4007 0.3581 405 16 -27 2.1

ptc14*- -0.2180 0.2004 -0.0002 377 16 -25 0.00312 -0.2631 0.2035 -0.0326 440 16 -25 0.00312

-0.0129 0.4127 0.3675 a7 4 -6 3.9

a The A and f data may be placed on an approximate absolute scale by multiplying by 0.18.

0.00184 0.00600 0.00184 0.00600

381

362 362 362


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1518 Ferricytochrome c Structure at 2.8 A Resolution Vol. 246, No. 5


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FIG. 4 (upper). View of map sections z = 0.06 (nearest) through 0.20 in the Richards box, with a reflection of the model superimposed. The heme (upper center) is surrounded by a region of van der Waals packing of hydrophobic side chains and less than average electron density whose contours are omitted. surrounding salt solution.

The left, bottom, and right margins of the frame show the one- to three-contour features of the A neighboring molecule related by the 4a screw axis intrudes at the upper left corner. The methionine 80

side chain is visible at the left of the heme iron, and the nearly edgewise ring of histidine 18 is less well illuminated on the right. sweep of density by which cysteine 14 connects to the heme can be seen at the upper right edge of the heme.

The To the right of the heme

and below histidine 18 are the side chains of phenylalanine 10, leucine 32 (fainter, to left) leucine 98 (below IO), and phenylalanine 36 (ring visible below leucine 98). Immediately to the left of the label 95c is the side chain of leucine 64. tyrosine 67, and to its left are proline 71 and lysine 72.

Just below methionine 80 is

at the left channel. Lysine 73 is poorly defined, and tyrosine 74 lies on the surface of the molecule

the upper left.) (The sheets have been rotated 180” in the box, so that the lower right corner of each section in Fig. 3 appears at

FIG. 5 (lower). Closeup of map sections z = 0.16 through 0.28, with reflection of model superimposed. the bottom half of the heme are shown; section 0.14 would have contained the heme iron atom.

Only those sections displaying The geometrical arrangement of tyro-

sine 67 (below methionine SO), tryptophan 59 (lower right), and tyrosine 74 (lower left) is visible. leucine 35, and closer to the viewer is leucine 64 (model side chain not illuminated).

To the right of tryptophan 59 is

left margin. The ring of Proline 76 is visible halfway up the

Also visible is the Type II 310 bend of which it is a part, with glycine 77 at the other corner and with the connecting hydrogen bond. Threonine 78 is hydrogen-bonded to tyrosine 67. The salt region at the lower left corner of the frame gives a good impression of the difference in map density between regions of protein molecule and regions of interstitial salt solution.

observes in the map are not resolved atoms, but chemical groups was continuous from one end of the sequence to the other. The with characteristic shape and orientation.

With the aid of a knowledge of the shapes of the side chains, orientations of roughly half of the amide planes were fixed by the

there was no difficulty in tracing the polypeptide chain, which bulges of the carbonyl groups, and another third of the orientations was fixed by the geometry of the main chain and side groups


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on either side. The highest feature on the map was the center of the iron atom, 13 contours or 2.3 e per A. The sulfurs of residues 14, 17, 65, and 80 had peak heights of 1.5, 1.5, 1.3, and 1.9 e per A, respectively. Main polypeptide chain was typically around 0.7 to 1.2 e per A. (No F(O,O ,0) term was added to the Fourier synthesis, so all of these figures represent densities above the all cell average density of approximately 0.41 e per A for 95% saturated ammonium sulfate and for the entire protein molecule

(53) J

TABLE V Features in the bonito minus horse difference map

Side chain

nos.

- ! Features in difference map”

Bonito HOM --

4 Ala Glu 9 Thr Ile

Strong (-) on outside of molecule (f) and (-), Ile more buried in sur-

face than Thr

It was easy to identify aromatic rings, and usually possible to orient them, although only for the ring of phenylalanine 10 did dimples appear on either side of the flattened disk. Interior tyrosines were distinguishable from phenylalanines by their hydrogen bonding. All of the hydrogen bonds discussed subse- quently were visible in the map as streaks of density. At the risk of missing some, we have not yet built hydrogen bonds into the model on the basis of proximity alone. Several sections through the center of the molecule and the heme, including the sections of Fig. 3, are shown in the Richards box in Figs. 4 and 5 with the image of the model superimposed.

22 Asn LYS 28 Val Thr

Strong (-) on outside of molecule (-), anion bound near hydroxyl in

horse? 33 Trp His (+) and (-), His extended into sur-

roundings, Trp tucked into surface of molecule

44 Glu Pro (+) and (-), Pro ring visible as (-), Glu chain as a weaker (+). Some main chain readjustment on either side

As a check on the entire analysis, diffraction data from bonito cytochrome c crystals were collected on a four-circle diffractom- eter, and a bonito minus horse AF difference map was calculated with the horse protein phases. The results were as expected from the root mean square error in horse cytochrome density. The map was a sea of noise peaks of heights up to 0.22 e per A3, with meaningful features up to 0.55 e per A3. The positive and negative peaks over 0.22 e per A3 were plotted on the same Plexi- glas sheets as the horse cytochrome map, and both maps were compared in the Richards box with the known sequence differences (Fig. 1) tagged on the horse cytochrome model.

46 Tyr Phe 47 Ser Thr 54 Ser Asn 58 Val Thr 60 Asn b-s 62 Asn Glu

89 GUY Thr 92 Gln Glu 95 Val Ile

100 Ser LYS

Hydroxyl oxygen atom visible as (+) No changes visible (-), anion bound near Asn in horse? No changes visible, as expected (-) as expected (+) and (-), may be local chain con-

formation change IJnambiguous (-), as expected No changes visible, as expected Clear (-) Ambiguous, Lys poorly defined even

in horse 103

104

Ser Asn

The main features of the bonito minus horse difference map are listed in Table V. There are no appreciable differences in the folding of the main chain. In 12 of the 18 sites of sequence difference between the two proteins, we see exactly what is expected, knowing the nature of the changes. Residues 4, 9, 33, 44, 46, and 89 are especially striking. Of the other six predicted changes, two involve small differences not visible in the map (residues 47 and 54), and one represents a possible local change in chain conformation (residue 62). The remaining three involve side chains near the carboxyl terminus which are poorly defined in the horse map and presumably not well fixed in one conformation on the outside surface of the molecule (residues 100, 103, and 104). Even a single CH3 group change is visible when the side chain is packed or constrained in one position, as residue 95 in the interior of the molecule illustrates. Other peaks are seen throughout the solvent, probably representing differences in ion positions. This agreement between the difference map and the expected changes based on amino acid sequence alterations and the horse protein model is probably the most stringent test of the correctness of the horse cytochrome c analysis.

? Glu

Ambiguous, Asn poorly defined even in horse

Ambiguous, Glu poorly defined even in horse

- a (+), positive peak (feature peculiar to bonito) ; (-), negative

peak (feature peculiar to horse).

ligand, extending from the left wall. The propionic acid side chains on the heme point toward the bottom of the crevice as shown in Fig. 6, and not directly out into the surroundings as they do in myoglobin and hemoglobin. One propionic acid is hence in a polar environment at the surface of the molecule, and the other is deeply buried in the hydrophobic interior. As with buried polar groups in other proteins, this interior propionic chain is involved in a network of hydrogen bonds. One carboxyl oxygen is hydrogen-bonded to tyrosine 48, and the other to tryptophan 59 and the main chain carbonyl of residue 40.

RESULTS

Histidine 18 is coordinated to the heme iron by its e nitrogen atom. But the &nitrogen atom is also hydrogen-bonded, to the carbonyl oxygen of proline 30. The probable purpose of this bonding to the main chain is to hold the imidazole ring in a rigid conformation for interaction with the heme. An identical bonding of the F8 histidine d-nitrogen atom to a main chain carbonyl oxygen is also found in sperm whale myoglobin (61).

Folding of Molecule-The horse ferricytochrome c molecule is a The heme normal makes an angle of 71.5” with the z axis, in prolate spheroid, 30 x 34 x 34 A, including side chains. An excellent agreement with the value of 72” =t 3” obtained by Eaton a-carbon map of the molecule is shown in Fig. 6. The over-all and Hochstrasser (62) from the electronic spectra of single picture of the molecule obtained at 4 A has been verified (IQ, 59, crystals. The projection of this normal vector on the xy plane 60), although the attempts to fit polypeptide chain to the 4 A lies 14” from the +J: axis, positive angles being measured toward model were only partially correct. The heme group sits in a the +y axis. In Fig. 6, the +Z direction (Space Group P4,) is crevice, with cysteines 14 and 17 and histidine 18 connected to horizontally to the right, the +y axis comes directly toward the the right wall of the crevice and methionine 80, the sixth iron viewer, and the fz direction is vertically downward.


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FIG. 6. Stereoscopic a-carbon diagram of the horse heart oxidized cytochrome c molecule, with the 12 glycine residues emphasized by heavy circles. These and subsequent stereo drawings were carried out at Caltech on an IBM 360-75 computer with a Calcomp plotter, using the ORTEP program written by Dr. Carroll N. Johnson. Atomic coordinates used in preparing these drawings were measured from the skeletal model of horse cytochrome c in the Richards box. For a discussion of the significance of these glycine positions, see the text.

I III FIG. 7. The two alternate sterically permissible variants of the 310 bend, as described by Venkatachalam (64). Type I on the left is

close to being an excerpt from the 310 helix. Type II differs in having the amide plane on the left side flipped over. Numbered circles are a-carbons, and circled R are side chains. Because of close approach of the carbonyl oxygen, Type II is possible only when the side chain on or-carbon 3 is missing, or for glycine.

The molecule gives the appearance of being constructed in two 14 to 18,49 to 54, 62 to 70, and 71 to 75. But the only true (Y halves: residues 1 to 47 are to the right of the heme crevice and helix is the sequence 91 to 101 near the carboxyl terminus. The 48 to 91 are to the left, with 92 to 104 back across the top to the last 2 residues, 103 and 104, are ill-defined, and may not have a right again like the strap on a suitcase. There are several short fixed conformation. The total helix content, therefore, agrees chain segments which are gently helical and which probably have with the estimates of Zand and Vinogradov (32). Ramachandran (4, #) angles close to those of an cy helix: 9 to 13, At the left and right in Fig. 6 appear openings in the poly-


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TABLE VI

310 Bends in horse cytochrome c

Residue nos.

C==O . H-N

TYP@

21 24 II 35 38 II 61 64 I 66 69 I 67 70 I 75 78 II

Amino acid sequence5

1 2 3 4

Glu-Lys-Gly-Gly Leu-Phe-Gly-Arg Glu-Glu-Thr-Leu Glu-Tyr-Leu-Glu Tyr-Leu-Glu-Asn Ile-Pro-Gly-Thr

a From Reference 64. b The numbers refer to the or-carbons of Fig. 6.

peptide chain skeleton that have been called “channels,” although this terminology probably errs in suggesting openness to solvent. The left channel is enclosed by residues 52 to 74, and the right channel is bordered by residues 6 to 20 and the cr helix. These channels contain a great many hydrophobic side chains, and lead to the hydrophobic interior and the heme.

Most of the molecule is best described as extended chain wrapped around the heme. There are several sharp bends, with abrupt reversals of chain direction. Examples of this occur at residues 22, 37, 44, and 77. Some of these changes of direction are associated with the prolines at positions 30, 44, 71, and 76. (None of the four prolines have the cis configuration.) Others are produced by a particularly tight hydrogen bonding which resembles one connection of a 310 helix (Fig. 7). This structure, which we call a 31~ bend, was first seen in lysozyme (Reference 63

FIG. 8. a-Carbon diagram of cytochrome c, viewed from the top as the molecule was oriented in Fig. 6. The heme crevice, seen ful face in Fig. 6, now opens to the right. a-carbons of the 21 hydrophobic side chains, valine, leucine, isoleucine, methionine and phenyl alanine, are heavily circled. The apparently hollow center-is actually packed with these hydrophobic chains, surrounding the heme

FIG. 9. Bottom view of the molecule, with the 19 lysine residues emphasized. Note the invariable occurrence of these residues on the periphery of the molecule.


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Fig. 8), and has since been encountered in other proteins. Ven- katachalam (64) has studied the contact distances in polypeptide chains and has found that two variants of the 310 bend are steri- tally possible. In type I, the carbonyl group of the central amide plane (Fig. 7) points away from the side chains on either side of it, whereas in type II the carbonyl group points to the same side of the ring formed by the hydrogen bond as to the side chains. Type II is possible only when the 3rd residue is a glycine, without a side chain. A 310 helix can be thought of as a polypeptide chain in which every carbonyl and -NH group is involved in a type I 3ir, bend. But an isolated 31~ bend can relax its Ramachandran angles somewhat and avoid the strain and bent hydrogen bonds found in a full 310 helix. Type II is obtained from type I by flipping the amide plane 180” and making other compensating changes in the angles at a-carbons 2 and 3. Six such 310 bends are encountered in cytochrome c, as listed in Table VI. Three are of type I, and all three of the type II bends have glycine in the third a-carbon position, as predicted by Venkatachalam.

Most of the evolutionary invariance observed in the glycine residues (Table II) occurs because glycine is found in such tight corners that no room exists for a side chain (Fig. 6). This is so for residues 6, 29, 34, 41, and 84, all of which are invariant in all species. Residues 24 and 56 could accommodate side chains with small rotations of the main chain, and each of these positions is occupied by residues other than glycine in two species: alanine

FIG. 10. Heme packing diagram of the cytochrome c molecule. Heavy circles indicate side chains that are buried on the interior of the molecule, and attached black dots mark residues whose side chains pack against the heme. Light circles indicate side chains on the outside of the molecule, and dark half-circles show groups that are half-buried at the surface. Arrows from tryptophan 59 and tyrosine 48 to the buried propionic acid group represent hydrogen bonds. Residues designated bv capital letters are totallv invariant among the proteins of i9 species listed in the “Appendix.”

or leucine at 24 and alanine or asparagine at 56. Residues 23,37, and 77 are apparently glycine to accommodate the presence of a type II 310 bend. (Residue 77 is invariant, 23 is asparagine in one species, and 37 is serine in another.) It is difficult to see why residues 1 and 45 are invariably glycine. Both appear to point out from the surface of the molecule. It may be that one or more of these are located at binding sites of cytochrome c to other macromolecules such as cytochrome oxidase and that a side chain would interfere with the intermolecular contact. It may also be that close intramolecular contacts forbidding side chains occur in these positions in the ferrous form of the protein.

Packing of Hydrophobic Side Chains-To a first approximation, the molecule can be described as a shell one layer thick surrounding the heme. The heme is enveloped in closely packed hydrophobic side chains. Around this is the framework of polypeptide chain, and the surface is covered with charged groups. Cyto- chrome c is the simplest example yet of the “oil drop” model of a protein.

The clustering of hydrophobic groups can be seen in Fig. 8, a top view with the heme crevice opening to the right. The appearance of a hollow center and open top is illusory, and is a defect of such a-carbon maps. The side chains of leucines 32, 35, 64, 68, 94, and 98, isoleucines 81, 85, and 95, phenylalanines 10 and 46, prolines 30 and 71, tyrosines 48 and 67, and tryptophan 59 all point inward toward the heme and pack tightly around it. The top of the molecule is filled in by the side chains of valine 3 and isoleucines 9 and 85.

The distribution of charged groups on the surface may be appreciated from the bottom view of Fig. 9, with the 19 lysine residues emphasized. One invariant arginine, 91, occurs at the upper end of the a-helix. The only other arginine, residue 38, is also invariant and appears to be involved in a hydrogen bond across the bottom to glutamine 42. The bend produced by proline 44 is particularly clear in this view.

The environments of the heme and the individual side chains are summarized in Fig. 10, where outside, inside, and surface residues are distinguished. Note the tendency for interior hydrophobic residues to be invariant. Those which do vary, as pointed out earlier, do so in a conservative manner. The hydrophobic residues, apparently, are invariant or conservative because they play a vital role, that of providing the proper non- polar environment for the heme. In no case is an interior hydrophobic residue ever replaced by a charged group or even by a polar uncharged one. When isoleucine 9, half-buried in the top surface, is replaced by a threonine in bonito, the threonyl side chain is observed to be less buried than the isoleucine had been. Conversely, when histidine 33 in horse cytochrome is replaced by tryptophan in bonito, the less polar tryptophan ring tucks more tightly into the surface of the molecule than did the exposed histidine.

Evolutionary Invariance-The 35 residues which have not been observed to change in the proteins of the 29 species listed in the “Appendix” are marked in Fig. 11, a view in which the heme plane is seen on edge. It is true that some of these sites will be only accidentally invariant, and may be found to vary in the next species examined. However, statistical calculations based on an increasing number of species suggest that the number of residual invariant positions will not fall below a minimum value of about 32 (13, 19, 65). Most of these residues are probably invariant because they are essential to the operation of the cytochrome c molecule. The heme connections to residues 14,17,18, and 80 are of course unchanging. The long constant region,


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FIG. 11. Stereo drawing with the molecule rotated 14” about a vertical axis from Fig. 6. The 35 invariant residues among the sample of 29 cytochromes c are marked with heavy circles. Note the invariance of the left side of the heme crevice.

FIG. 12. View from the left side, with the molecule rotated 135” from Fig. 6. Invariant groups are marked. Note how these groups are clustered around the heme at the right, and how variable the back of the molecule is (left in this view). Note also the opening in the polypeptide chain framework formed by the loop from residue 52 to 74. This opening is filled with hydrophobic side chains, including the invariant tryptophan 59 and tyrosine 74 with aromatic rings parallel.

residues 70 to 80, is now seen to be folded to build the left, side of the hydrophobic box around the heme, or the heme crevice. Residues in the immediate vicinity of the heme on either side have the greatest tendency to be invariant. The back of the molecule is the most changeable region, as is seen particularly clearly in Fig. 12. Two of the three invariant prolines, 71 and 76, appear in the tightly knit left side.

The left “channel” mentioned above is especially visible in Fig. 12. The main polypeptide chain swings in a broad loop from residue 52 to 74, to enclose what appears to be an opening or channel reminiscent of that shown at the top of the molecule in Fig. 8. But this opening, like that on top, is filled with hydrophobic side chains, with the invariant and roughly parallel aromatic residues tryptophan 59, tyrosine 67, and tyrosine 74 being prominent. This left channel is one of the avenues from the outside surface to the heme that may possibly be involved in the transfer of electrons or of excited electronic energy states.

Distribution of Charge on Molecular Surface-In most globular proteins, those charged side chains that are not, directly involved

in catalytic or other active sites appear to have two roles: to ensure that the portion of polypeptide chain bearing them re- mains on the outside of the protein during folding, and by a proper over-all balance of acidic and basic groups to maintain an optimum pK for the molecule. In cytochrome c it is surprising

to find that acidic and basic groups are also segregated on the molecular surface into two positively charged patches with a negative patch between them. In Fig. 13, showing the 19 lysine positions, residues 86, 87, 88,72, 73, 79, 39, 53, and 55 are in one long stripe curving to the right, of and below the aromatic “channel” mentioned in the previous section. On the opposite side of the molecule, residues 5, 7, 8, 13, 27, 25, 22, 100, and 99 encircle a second opening, the right channel, described below. Between these two positively charged regions lies a zone, facing the viewer in Fig. 13, in which basic side chains are never encountered. (Lysine 60, at the bottom of this zone, is commonly glycine, asparagine, or glutamine. It is basic only in horse and donkey cytochromes c, and acidic in bakers’ yeast, Neurospora, and some higher plants.)


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FIG. 13. Backview, 180” rotation about vertical axis from Fig. 6. The 19 lysines are marked. Not,e how these lysines are distributed over the outer surface to the right and left sides, and how the back of the molecule nearest the viewer is almost totally devoid of positively charged side chains. Compare this with Fig. 14.

FIG. 14. Same backview as Fig. 13, but with the 12 acidic side chains, aspartic or glutamic acid, marked instead. Except for residues 21,50, and 104 at the sides, all of the negatively charged side chains are localized at the top rear of the molecule, which Fig. 13 showed to be totally empty of positive charge. Positive and negative charges are sharply localized on the surface of the cytochrome c molecule.

This blank zone in the lysine map is seen from Fig. 14 to be Why should this segregation be found in cytochrome c and not filled with acidic groups. Residues 2, 4, 61, 62, 66, 69, 90, 92, in other globular proteins studied to date? The answer is almost and 93 are all clustered at the top rear of the molecule. While surely that cytochrome c interacts with several other macro- none of these loci is totally invariant, and only residue 90 is molecular complexes-oxidase, reductase, mitochondrial mem- uniformly acidic, this region of the surface retains its negative brane-and that the distribution of charge plays a role in recogni- character in all species. In baker’s yeast,, Neurospora, and wheat tion and binding to these complexes. One of the two clusters cytochromes c, for example, residue 62 is neutral (asparagine of basic groups is almost surely a binding site to the oxidase, rather than aspartic or glutamic acids), but, in compensation, judging from the evidence listed in the section on chemical residue 60 is acidic. No species has fewer than 6 acidic residues properties. The critical lysine 13 is just above the heme crevice, in this region, and none has more than 5 acidic residues scattered lending weight to the idea that interaction with the oxidase anywhere else on the molecule. The only other sites which are occurs directly via the exposed edge of the heme. The functions acidic in a majority of species are residue 21 (28 species) and of the second basic patch and the acidic patch at, the rear are residue 50 (2’0 species). Thus, the x-ray analysis confirms what unknown, but presumably they must, be associated with the was suspected from comparisons of amino acid sequences. Acidic reductase or the membrane. Definitive chemical experiments and basic side chains are separated on the molecular surface, and are now needed in the light of the x-ray analysis to establish these regions have remained similarly charged in all species even which side chains are involved in binding to the other macro- though the individual residues change. molecular surfaces.


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FIG. 15 (upper). Stereo pair closeup of the loop from residue 52 to 74, as seen in Fig. 12. The sequence lysine 55-glycine 56-isoleucine 57-threonine 58tryptophan 59 can be seen along the front chain ascending from the bottom right to left. Directly above the vertical side chain of lysine 55 is the six-membered ring of tyrosine 74, with its hydroxyl group pointing out (and clamped to a supporting bracket in this model). Behind the tyrosine 74 ring is tyrosine 67, and behind it is methionine 80 and the heme. Below and to the right of tyrosine 74 is the side chain of isoleucine 75. Tyrosine 67 is hydrogen-bonded on the right to threonine 78. Tryptophan 59 is hydrogen-bonded to one oxygen of the buried propionic group, which is also bonded to the carbonyl oxygen of residue 40. Behind and to the right of this vertical hydrogen bond is the aromatic ring of tyrosine 48, which is hydrogen-bonded to the other oxygen of the buried propionic group. heme crevice.

Behind and slightly above tyrosine 48 is phenylalanine 46, exposed to the surface at the bottom end of the In front of tyrosine 48, threonine 40 is hydrogen-bonded to the main chain carbonyl of residue 55.

FIG. 16 (lower). Stereo pair closeup looking into the heme crevice as in Fig. 6. right, with the heme connection at cysteine 17 just below it.

Glutamine 16 extends toward the viewer at the upper Threonine 28 extends out just below the center of the picture, with glycine

29 and proline 30 visible as the chain recedes into the interior of the molecule. The hydrogen bond between the main chain carbonyl of residue 28 and the amide of residue 18 is visible, and behind it, the bond from the &nitrogen atom of the histidine 18 ring to the main chain carbonyl30. Behind the heme is the double ring of tryptophan 59. a bifurcated hydrogen bond to tryptophan 59 and to main chain carbonyl 40.

One oxygen of the buried heme propionic group forms

the bottom center. The other oxygen is bound to tyrosine 48, visible at

Phenylalanine 46, at the bottom end of the heme crevice, appears in ,front of tyrosine 48. heme, the side chain of lysine 13 sits atop the heme crevice.

At the upper left of the To the left of the heme is methionine 80.

hm+atic Groups-The aromatic groups in cytochrome c have a remarkable tendency to occur in approximately parallel pairs,

are parallel and 6 A apart, in the left hydrophobic “channel”

and it is tempting to speculate about the transfer of electrons or (Figs. 12 and 15). Tyrosine 67 is 4 A from tryptophan 59 and

excited electronic states via the overlap of aromatic rr electron 6 A from tyrosine 74, and is tilted more toward the heme plane.

clouds. (All distances have been measured roughly from the Kendrew

Tyrosine 74 and the six-membered ring of tryptophan 59 model between centers of six-membered rings, and checked


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FIG. 17. Stereo drawing of the molecule seen 76” to the right of Fig. 6. heme, bounded by residues 6 to 21 and the a helix.

Note the large opening (the “right channel”) leading to the This channel is filled with hydrophobic groups (heavy circles).

against the electron density map in the Richards box.) Tyro- sine 67 is 4.8 A from the heme plane. It is completely buried in the left side of the hydrophobic box surrounding the heme, and its hydroxyl group is hydrogen-bonded to threonine 78.

On the front of the molecule, phenylalanine 46 and tyrosine 48 are nearly parallel and 4.9 A apart (Fig. 15). They are roughly normal to the heme plane and parallel to histidine 18, but they are both 9 A from the histidine ring. The hydrogen-bonding pattern of tyrosine 48, tryptophan 59, and the main chain car- bony1 of residue 40 to the buried propionic acid group of the heme is shown in Figs. 15 and 16 and described in the legends. The other, exposed propionic group is probably hydrogen-bonded to threonine 49, although the pattern of density in this portion of the map, including the side chain of asparagine 52, is confusing. The bonito minus horse difference map reveals the oxygen atom of tyrosine 46 in the bonito protein, and suggests that it may be bonded to the exposed propionic group in that species.

The right side of the molecule is more open than the left, with what appears to be a second hydrophobic channel to the heme. This opening, seen in Fig. 17 and in closeup in Fig. 18, is outlined by residues 6 to 20 and the a: helix. In the center, phenylalanine 10 and tyrosine 97 stand parallel and 5.6 A apart. Around them in the channel are other hydrophobic groups: valines 11 and 20, leucines 32, 35, 94, and 98, and isoleucines 9 and 95. Even considering the van der Waals radii around the wire skeleta in Fig. 18, it is hard to imagine that the right channel is completely filled like the left channel (loop of residues 52 to 74). Indeed, a comparison of the two channels in the 4 A model shows how much more open the right one is (Figs. 19 and 20). There appears to be room for a hydrophobic side chain from another macromolecule to fit into the right channel, which could in this way form a hydrophobic binding site. The basic residues en- circling this channel, lysines 5, 7, 8, 13, 22, 25, 27, 99, and 100, then may play a role in recognition and binding as well.

Of all of the nine aromatic groups, only phenylalanines 36 and 82 are isolated. Residue 36 is the only aromatic residue that is ever replaced by a nonaromatic group. It fills a hydrophobic slot at the rear of the molecule, and apparently is important only for its size and hydrophobicity. As mentioned earlier, it is replaced by tyrosine in one species and by isoleucine in three.

Phenylalanine 82 extends to the left of its a-carbon atom in

Fig. 6, and is exposed to the external medium. The main chain from residues 80 to 85 is not anchored to its neighbors and is possibly loose. Trials with the Kendrew models show that this part of the chain can be swung to the right, displacing lysine 13, moving phenylalanine 82 against the heme, and blocking the heme crevice. It is not inconceivable that some such mechanism operates when the molecule is reduced. Direct evidence to test this hypothesis will have to await the analysis of ferrocytochrome c.

In summary, of the 4 tyrosines, residue 67 is totally buried with its hydroxyl group hydrogen-bonded and residue 48 is similarly buried and hydrogen-bonded, but has one edge of the ring at the surface. Tyrosines 74 and 97 have their aromatic rings mostly in hydrophobic environments, but extend their hydroxyl groups out into the surrounding medium.

Hydrogen-bonding Side Chains-Of the 10 threonyl residues, 5 (residues 28,47, 58, 63, and 89) are on the surface of the molecule and not hydrogen-bonded, 4 are internal (residues 40 and 78) or at the surface (residues 19 and 49) and are bonded, and only one (residue 102) is inside but not obviously a participant in a hydrogen bond. Threonine 40 connects two polypeptide chains by a bond to the main chain carbonyl of residue 55. It is replaced only by a serine in yeast, Candida, and the higher plant cytochromes. Threonine 49 probably bonds to the outermost propionic group of the heme. It is replaced by a serine in two moths and the higher plants. The invariant threonine 78 is bonded to tyrosine 67.

Threonine 19 provides an interesting example of what may represent internal compensation in the Neurospora protein, in which simultaneous changes at two sites counteract one another. In all but Neurospora cytochrome c, residue 19 is threonine, and the structure of the horse protein shows it to be hydrogen- bonded to the amide of residue 25. This bond and the 310 bend at residues 21 to 24 help to hold the hairpin loop in the chain from residue 17 to 28 (Figs. 6 and 17). In the Neurospora protein, this threonine is absent, but residue 25, which in nearly every other species is lysine, becomes threonine. The possibility then exists of a similar cross-chain hydrogen bond going the other way but serving the same function. The adjacent residue 26 is also particularly interesting in Neurospora cytochrome c. In all other species it is a histidine, and from the horse protein


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Issue of March 10, 1971 R. E. Dickerson et al.

FIG. 18. Closeup of the right channel, seen as in Fig. 17. Tyrosine 97 is at the upper right, with its hydroxyl group pointing toward the viewer. Parallel to it on the left is phenylalanine 10. Behind and to the right of phenylalanine 10 is the heme connection at cysteine 14. In the left foreground is aspartic acid 21, and behind it are valine 20, threonine 19 to the left, and histidine 18. Just above the bottom clamp on the right vertical support wire is threonine 102, with alanine 101 above it and halfway to the tyrosine 97 ring. In bullfrog cytochrome c, residues 20 and 102 are joined by a disulfide bridge.

FIG. 19. The low resolution (4 A) model of horse cytochrome c, viewed a few degrees to the left of Fig. 17. Notice the size of the opening below the aromatic groups phenylalanine 10 and tyrosine 97, and compare this with the more compact left channel in Fig. 20. All identifica- tions of side chains are based on the 2.8 A analysis.

FIG. 20. The low resolution model of cvto- chrome c, viewed approximately 20” to the”Zeft of Fig. 12. Note the relative positions of tyrosines 74 and 67, tryptophan 59, and the heme.


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map it apparently holds two folded stretches of chain together by a hydrogen bond to the carbonyl of residue 44. Only in Neuro- spora does this histidine vary, and here it is glutamine, another hydrogen-bonding group of similar over-all dimensions. In several other ways, Neurospora cytochrome c has its peculiarities, and it would be a particularly interesting structure to examine.

Threonine 102, although it points inward, has no obvious hydrogen-bonded partner from the electron density map. In one cytochrome, that of the bullfrog, residues 20 and 102 are cysteines, and are connected by a disulfide bridge. The spacing in horse cytochrome c between valine 20 and threonine 102 is exactly right for such a bridge (Fig. 18). This is apparently an example of a fossilized disulfide chain conformation similar to that found in comparing chymotrypsin and trypsin (see Fig. 6, Reference 66).

Of the 5 asparagines, 4 (residues 31, 54, 70, and 103) point outside the molecule and are not hydrogen-bonded. The invariant asparagine 52 is deeply buried in the left side of the hydrophobic heme box, and is probably involved in some form of hydrogen bonding with threonine 49 and the outermost heme propionic group. Glutamine 16 points out and is unbonded, while glutamines 12 and 42 interact with arginines 91 and 38 as mentioned above.

DISCUSSION

Theoretical models of protein structure have almost always turned out to be oversimplifications of reality. This is so in part because nature is far more subtle than we have been, and in part because we quite understandably tend to think of the corpus of knowledge to date as a sure guide to the future. Rut with cytochrome c, one of the oldest pictures of protein structure-the oil drop model-proves to be surprisingly accurate in describing the forces holding the molecule together. In this model, the hydrophobic side chains are thermodynamically more stable when segregated in the interior of the molecule rather than extended into the aqueous surroundings. This stability arises in the folding process because of the gain in entropy when ordered water molecules around the exposed hydrophobic groups are dispersed. The polypeptide chain therefore folds spontaneously in aqueous solution so that its hydrophobic side chains are buried and its polar, charged chains are on the surface. This, in essence, is the structure of cytochrome c.

Cytochrome c has another strong folding influence, the heme group. The data tell us only that the polypeptide chain bends back to the heme again and again to bring 16 hydrophobic side chains up to pack against it. Yet it is difficult to avoid the extrapolation that this is related to the way in which the molecule folds after synthesis-that the heme itself is the template for the

TABLE VII Predictions of 01 helical regions in horse cytochrome c

hm

15 to 21 9 to 19 9 to 17 31to 38

5t3to70 62 to 70 80 to 87 83to88 80to 85

fat0 101 93 to 101 92to 101

ham

9to 17 8to 20

64 to 69 53 to 73 80to85 k30to 100

90 to 97

construction of the molecule. One could imagine that the heme was attached first near the amino terminus, that the particular arrangement of hydrophobic side chains induced the folding of residues 19 to 91 first on the near side and then on the far side of the heme, that the carboxyl-terminal (Y helix was formed only after the heme was covered, and that residues 1 to 9 then lay down atop the 01 helix to complete the molecule.

The actual folding of the molecule may be the result of a com- petition between the tendency of the chain to wrap around the heme or to coil into an a! helix. It is interesting to compare the various theories for predicting QI helical regions of proteins with the x-ray results for cytochrome c. Most of these use the idea of helix-forming and helix-breaking side chains, either to estimate over-all helix content (67, 68) or actually to predict the locations of helical regions in the chain (69-74). Another technique looks for regions which would have one side hydrophobic and one polar if they were o( helical (75). A third approach involves searching known proteins for the occurrence in helices of typical amino acid pairs, 2 to 7 residues apart (76), or alternatively for dipeptide to hexapeptide sequences typical of helices (77).

The results of such methods, in Table VII, show that the prediction of helix locations has almost become a science. Prothero’s method (71) is based in part on regarding proline, aspartic, and glutamic acids and histidine as helix breakers, and looking for sequences of 6 residues or more without such groups. Kotelchuck and Scheraga’s procedure is similar in principle but more detailed (72, 73). They define the helix-forming residues (denoted by h) as arginine, glutamic acid, glutamine, cysteine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, and tryptophan. The helix-breaking residues (denoted by c) are lysine, histidine, aspartic acid, asparagine, serine, threonine, tyrosine, and proline. An Q helix then is assumed to begin when four h residues occur sequentially, and to grow toward the car- boxy1 terminus until ended by two c. Glycines are ignored, even when one glycine intrudes in the four h initiation zone, or one or more glycines separate the two terminating c. This can be called the “hac2” model, in an obvious notation. Lewis et al. (74) use a statistical partition function method (the Zimm-Bragg model) to calculate the probability that a given portion of the chain would coil spontaneously into an o( helix, starting from the completely unwound and denatured state. Schiffer and Ed- mundson (75) look for possible Q! helices with one side hydrophobic and one side polar. This theory works best with proteins in which the (Y helices are packed together around a hydrophobic core with one side exposed, as in myoglobin, hemoglobin, lysozyme, and ribonuclease. It would not be so successful with larger proteins having buried a! helices, such as subtilisin and lactic dehydrogenase. The method of Low, Lovell, and Rudko (77) looks for di- to hexapeptides which have been found in helical regions of other proteins of known structure.

All of the methods except that of Kotelchuck and Scheraga identify the (Y helical 91 to 101 region correctly. The latter method succeeds as well, if the rules are relaxed so that only three h residues are necessary for initiation (the “h3~2” model). Ex- amination of the sequence comparisons in the “Appendix” shows that the unmodified h4c2 criterion predicts (Y helix at residues 89 to 97 in the cytochromes of man, monkey, pigeon, and turtle. The helix prediction breaks down completely in this region for yeasts, microorganisms, and plants. This may be a hint of structural differences in the proteins of these distantly related


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organisms, but it is more probably a comment on the over- simplicity of the helix-predicting theories to date.

Most of the methods predict helix in the regions of residues 9 to 21 and 80-88. The first of these has some elements of reality. Residues 9 to 13 and 14 to 18 do resemble single a! helical turns, although there is a sharp change of direction at residue 13 and helical hydrogen bonding is absent. This segment 9 to 18 might be considered as a potential a helix which was aborted by the requirements of covalent bonding to the heme. The prediction for residues 80 to 88 is completely wrong. As Fig. 3 shows, this segment is virtually a fully extended chain running across the top left of the molecule. Region 31 to 38, predicted as helical by Schiffer and Edmundson (75), is another straight chain with no helicity.

The region in the 60’s is predicted as helical by Prothero, by Schiffer and Edmundson, by Lewis et al., and by Kotelchuck and Scheraga if the rules are relaxed to an h3cZ model. This is a portion of chain that is almost helical, and probably would be so if it were not near the heme. But the positions of the side chains and the path of the backbone in the 2.8 A map seem wrong for the accommodation of an ideal a helix. Lysine 68 is pushed too close to the heme, the bend at residues 66 to 69 is too tight, and the turns at 64 to 65 and 67 to 69 are spread too far apart for a true OL helix (Fig. 12).

Thus, for cytochrome c, the helix-predicting methods that have appeared to date have a considerable element of validity, their chief defect being that they are not sufficiently discriminating, with a tendency to predict more helix than is actually present. The partition function method of Lewis et al. (74) is particularly bad in this respect. But if the heme were not present, there is a possibility that two more predicted regions in cytochrome c would be helical, which emphasizes again the strong formative influence that the heme exerts on the shape of the molecule.5

In the behavior of basic, acidic, hydrophobic, aromatic, and hydrogen-bonding residues, and the special chain configuration of glycine and proline, cytochrome c is a model system for studying protein folding and evolution. The hydrophobic groups are located in such a way as to keep the protein folded around its heme. Glycine is used for close chain contacts and confined corners, and proline is found at other bends. Lysine and aspartic and glutamic acids not only serve to keep their part of the chain outside, but are also grouped into zones on the molecular surface which probably interact with the oxidase, reductase, and mitochondrial membrane. Serine, threonine, tyrosine, and to a lesser extent asparagine, are all used to hold the chain together with hydrogen bonds. The aromatic rings are hydrophobic, and also occur in parallel pairs which may play some part in electron or

energy transfer. All of these characteristics are sufficiently important to the operation of the molecule to have been main-

s An unpublished method by John Petruska appears to be slightly more successful in predicting 01 helix in cytochrome c than any of the foregoing methods, Its application to cytochrome c was shown to one of the authors, Richard E. Dickerson, in 1965, but was forgotten until this manuscript was completed. Even in its most elementary version, Petruska’s method predicts a good 01 helix from residues 87 to 104, and poorer helices with half of the probability from 0 to 16, 50 to 54, and 62 to 72. All other regions are predicted as nonhelical. A note on Petruska’s method is in preparation. This method suggests that the imperfection in the helix from residues 62 to 70 may arise from the presence of the invariant threonine 63 and nearly invariant methionine-se&e 6.5 as much as from the nearness of the heme.

tained through more than a billion years of molecular evolution. The highly variable loci-residue 89 with nine different side chains, residues 60 and 92 with seven alternates, and residue 44 with six-are all on exposed corners of the molecule where apparently anything can extend harmlessly into the surrounding solvent.

Four surface features are probably important for the function of the molecule: the heme crevice, the left channel, the right channel, and the back negative patch. Lysine 13, essential for oxidase activity, adjoins the heme crevice. Each of the two channels is a hydrophobic pathway to the interior with parallel aromatic rings, and each channel is surrounded on the surface with a ring of positively charged lysine side chains. While we cannot demonstrate the function of these features yet, there are some obvious possibilities. One of these channels may well be involved in recognition and binding to the oxidase. The other channel and the negative patch are probably associated with the reductase and the mitochondrial membrane, although what this association might be is unclear as yet. The positive charges and hydrophobic patch where the channel opens on the surface could bind to negatively charged phospholipid molecules, or the negative patch could bind to the choline part of phosphatidylcholine lipid. This last alternative would lead to the greatest degree of specificity in binding because of the uniqueness of the negative area in the cytochrome c molecule. Alternatively, phospholipids and the negative patch could be held together by cations such as Ca2+ or Mg2+. Much chemical work needs to be done to test these and other hypotheses suggested by the structure.

Some ambiguity exists regarding the chemical reactivity of the tyrosines. Evidence from spectrophotometric titration of tyrosyl hydroxyls has been presented to show that two (78), three (79), or four (43) of the four tyrosines have an unusually high pK and are presumably buried in the interior. Ishikura (80) found that two tyrosines are easily diiodinated, but at the price of loss of electron-transferring ability. Ulmer (81) observed that acetylimidazole modifies two tyrosines in the oxidized protein and essentially no tyrosine in the reduced state.

The balance of the evidence from the work above suggests that two tyrosines are buried and two are exposed in ferricytochrome c. From the x-ray analysis, one might expect that the external tyrosines (residues 74 and 97) would be iodinated and acetylated, and have a normal pK, whereas the buried tyrosines 48 and 67 would have a high pK and be unreactive. Yet Skov et al. (82, 83) have reported that tetranitromethane causes the nitration of tyrosines 48 and 67, the two buried residues, while McGowan and Stellwagen (84) find that under their conditions tyrosines 67 and 74 are iodinated. Clearly then, with both tetranitromethane and KII, it is the buried rather than the surface tyrosyl residues that are preferentially substituted. This unexpected reactivity of the buried residues could arise from the effect of the dielectric constant of the medium or environment on the reactions. Such a dependence has been observed for the reaction of tetranitromethane with substituted phenols (85). It could also merely reflect the preferential solubility of the reagents in the lipophilic interior of the protein. Tetranitromethane reacts with phenols with deprotonated -OH groups (83, 85), implying a preferential attack on those tyrosines with the lower pK. Yet it may be hasty to identify these as the external tyrosines, since both internal tyrosines are hydrogen-bonded and sharing their hydroxyl protons with other atoms, whereas the external tyrosines are largely un-ionized.


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Making allowances for van der Waal’s packing radii on the Kendrew skeletal model of cytochrome c, it appears possible for tetranitromethane to approach tyrosine 67 through the loop formed by residues 70 to 82 (Fig. 11). The polypeptide chain is sharply kinked inward to point proline 71 into the hydrophobic interior next, to Tyrosine 67. If this proline 71 were pulled back and the chain of residues 75 to 79 were moved down, then there would be room for a tetranitromethane molecule to slip inside next to the -OH group of the tyrosine, “dissolved” in the hydrophobic interior of the molecule. Tyrosine 48 is even more accessible to attack through the loop formed on the bottom by residues 41 to 52 (Fig. 9), assuming that some chain motion to open up the loop is possible (86).

This preferential reactivity of cytochrome c tyrosyl residues provides a vivid example of the extreme caution that must be exercised in equating availability of a side chain for reaction with its actual location on the surface of the protein. What is being tested in the tetranitromethane reactions is apparently not the surface exposure of the tyrosines but the flexibility of the protein. Globular proteins in general appear to be, not rigid, and not floppy, but elastic. They have a unique minimum energy conformation to which they will return after per- turbation if permitted to do so, but the energy required to de- form them temporarily without breaking covalent bonds or a significant number of hydrogen bonds is small. Resilience is apparently more advantageous in a protein molecule than either brittleness or plasticity.

Acknowledgments-We would like to thank Professor Masao

Kakudo, of the Protein Institute of Osaka University, for his generous gift of the bonito cytochrome crystals. We would like also to thank Drs. T. Nakayama, K. Titani, and K. Narita for their unpublished bonito cytochrome c sequence, and Dr. D. Boulter for the unpublished sequences from four higher plants. We are very much indebted to Mr. Joseph Dailey and the Cal- tech Computing Center for their support and patience while we calculated and plotted (many things), and to Miss Lillian Casler for preparing many of the finished drawings.

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APPENDIX

Amino acid sequence of cytochrome c from 29 species (data from Reference $1 with minor corrections)

Man, chimpanzee Rhesus monkey Horse Donkey Cow, pig, sheep

Dog Rabbit California grey whale Great grey kangaroo

Chicken, turkey Pigeon Pekin duck Snapping turtle Rattlesnake Bullfrog Tuna Dogfish

Samia cynthia Tobacco horn worm moth Screw worm fly Fruit fly (Drosophila) Baker’s yeast (iso-I) Candida krusei Neurospora crassa Wheat germ

Man, chimpanzee Rhesus monkey Horse Donkey Cow, pig, sheep Dog Rabbit California grey whale Great grey kangaroo

Chicken, Turkey Pigeon Pekin duck Snapping turtle Rattlesnake Bullfrog Tuna Dogfish

Samia Cynthia Tobacco horn worm moth Screw worm fly Fruit fly (Drosophila) Baker’s yeast (iso-1) Candida krusei

NeurospoEcrassa Wheat germ

-8-7-6 -5 -4 -3 -2 -1 1 5 10 15 20 25

aGDVEKGKKIFIMKCSQCHTVEKGGKHKTG aGDVEKGKKIFIMKCSQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTVEKGGKHKTG

aGDIEKGKKIFVQKCSQCHTVEKGGKHKTG aGDIEKGKKIFVQKCSQCHTVEKGGKHKTG aGDVEKGKKIFVQKCSQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTVEKGGKHKTG aGDVEKGKKIFTMKCSQCHTVEKGGKHKTG aGDVEKGKKIFVQKCAQCHTCEKGGKHKVG aGDVAKGKKTFVQKCAQCHTVENGGKHKVG aGDVEKGKKVFVQKCAQCHTVENGGKHKTG

hGVPAGNAENGKKIFVQRCAQCHTVEAGGKHKVG hGVPAGNADNGKKIFVQRCAQCHTVEAGGKHKVG hGVPAGDVEKGKKIFVQRCAQCHTVEAGGKHKVG hGVPAGDVEKGKKLFVQRCAQCHTVEAGGKHKVG

hTEFKAGSAKKGATLFKTRCELCHTVEKGGPHKVG hPAPFEQGSAKKGATLFKTRCAECHTIEAGGPHKVG

hGFSAGDSKKGANLFKTRCAECHGEGGNLTQKIG aASFSEAPPGNPDAGAKIFKTKCAQCHTVDAGAGHKQG

30 35 40 45 50 55 60

PNLHGLFGRKTGQAPGYSYTAANKNKGI IWGEDTL PNLHGLFGRKTGQAPGYSYTAANKNKGI IWGEDTL PNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETL PNLHGLFGRKTGQAPGFSYTDANKNKGI TWKEETL PNLHGLFGRKTGQAPGFSYTDANKNKGITWGEETL PNLHGLFGRKTGQAPGFSYTDANKNKGITWGEETL PNLHGLFGRKTGQAVGFSYTDANKNKGITWGEDTL PNLHGLFGRKTGQAVGFSYTDANKNKGI TWGEETL PNLNGIFGRKTGQAPGFTYTDANKNKGI IWGEDTL

PNLHGLFGRKTGQAEGFSYTDANKNKGITWGEDTL PNLHGLFGRKTGQAEGFSYTDANKNKGITWGEDTL PNLHGLFGRKTGQAEGFSYTDANKNKGITWGEDTL PNLNGLIGRKTGQAEGFSYTEANKNKGITWGEETL PNLHGLFGRKTGQAVGYSYTAANKNKGI IWGDDTL PNLYGLIGRKTGQAAGFSYTDANKNKGI TWGEDTL PNLWGLFGRKTGQAEGYSYTDANKSKGIVWNNDTL PNLSGLFGRKTGQAQGFSYTDANKSKGI TWQQETL

PNLHGFYGRKTGQAPGFSYSNANKAKGITWGDDTL PNLHGFFGRKTGQAPGFSYSNANKAKGI TWQDDTL PNLHGLFGRKTGQAAGFAYTNANKAKGITWQDDTL PNLHGLIGRKTGQAAGFAYTNANKAKGITWQDDTL PNLHGIFGRHSGQAQGYSYTDANIKKNVLWDENNM PNLHGIFSRHSGQAQGYSYTDANKRAGVEWAEPTM PALHGLFGRKTGSVDGYAYTDANKQKGITWDENTL PNLHGLFGRQSGTTAGYSYSAANKNKAVEWEENTL

A, Ala; C, CYS; D, ASP; E, Glu; F, Phe; G, Gly; H, His; I, He; K, Lys; L, Leu; M, Met; N, -4sn; P, Pro; Q, Gln; R, Arg; S, Ser;

T, Thr; v, Val;W, Trp; Y, Tyr; X, r-N-trimethyllysine (coded as Lys) ; a, acetylated end, CH3CO-NH-; h, free amino end, HrN-.


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Issuct of March 10, 1971 R. E. Dickerson et al. 1535

APPENDIX--CO&.

Man, chimpanzee Rhesus monkey Horse Donkey Cow, pig, sheep Doe

65 70 75 80 85 90 95 100 104

MEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE MEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE MEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE MEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE MEYLENPKKYIPGTKMIFAGIKKKGEREDLIAYLKKATNE MEYLENPKKYIPGTKMIFAGIKKTGERADLIAYLKKATKE

RaGbit MEYLENPKKYIPGTKMIFAGIKKKDERADLIAYLKKATNE California grey whale MEYLENPKKYIPGTKMIFAGIKKKGERADLIAYLKKATNE Great grey kangaroo MEYLENPKKYIPGTKMIFAGIKKKGERADLIAYLKKATNE

Chicken, turkey MEYLENPKKYIPGTKMIFAGIKKKSERVDLIAYLKDATSK Pigeon MEYLENPKKYIPGTKMIFAGIKKKAERADLIAYLKQATAK Pekin duck MEYLENPKKYIPGTKMIFAGIKKKSERADLIAYLKDATAK Snapping turtle MEYLENPKKYIPGTKMIFAGIKKKAERADLIAYLKDATSK Rattlesnake MEYLENPKKYIPGTKMVFTGLSKKKERTNLIAYLKEKTAA Bullfrog Tuna

MEYLENPKKYIPGTKMIFAGIKKKGERQDLIAYLKSACSK MEYLENPKKYIPGTKMIFAGIKKKGERQDLVAYLKSATS-

Dogfish RIYLENPKKYIPGTKMIFAGLKKKSERQDLIAYLKKTAAS

Samia Cynthia FEYLENPKKYIPGTKMVFAGLKKANERADLIAYLKESTK- Tobacco horn worm moth FEYLENPKKYIPGTKMVFAGLKKANERADLIAYLKQATK- Screw worm fly F EYLENPKKY I PGTKMI FAGLKKPNERGDL IAYL<KS ATK- Fruit fly (Drosophila) FEYLENPKKYIPGTKMIFAGLKKPNERGDLIAYLKSATK- Baker’s yeast (iso-1) SEYLTNPXKYIPGTKMAFGGLKKEKDRNDLITYLKKACE- Candida krusei S DYLENPXKY IPGTKMAFGGLKKAKDRNDLVTYMLEASK-

NeurospoEssa FEYLENPXKYIPGTKMAFGGLKKDKDRNDIITFMKEATA- Wheat germ YDYLLNPXKYIPGTKMVFPGLXKPQDRADLIAYLKKATSS


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Samson, Angela Cooper and E. MargoliashRichard E. Dickerson, Tsunehiro Takano, David Eisenberg, Olga B. Kallai, Lalli

PROTEINS AT 2.8 A RESOLUTION : I. GENERAL FEATURES OF THE HORSE AND BONITOcFerricytochrome

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Ferricytochrome c - Journal of Biological Chemistry · THE JOUXNAL OF ~~IOLOGICAL CHEMISTRY Vol. 246, No. 5, hue of March 10, pp. 1511-1535, 1971 Printed in U.S.A. Ferricytochrome