THE JOURNAL OP BIOLOGICAL CHEMISTRY

“0, 252, No. 2, Issue “f January 25, pp. 159-775, 1977

Printed L,I U S.A.

Tuna Cytochrome c at 2.0 A Resolution I. FERRICYTOCHROME STRUCTURE ANALYSIS*

(Received for publication, April 29, 1976, and in revised form, August 31, 1976)

ROSEMARIE SWANSON,* BENES L. TRUS,~ NEIL MANDEL,~~ GRETCHEN MANDEL,~] OLGA B. KALLAI, AND RICHARD E. DICKERSON

From the Norman W. Church Laboratory of Chemical Biology, California Institute of Technology, Pasadena, California 91125

The crystal structure of oxidized cytochrome c from tuna hearts has been solved by x-ray diffraction to a resolution of 2.0 13, using four isomorphous heavy atom derivatives. The crystals, space group P4:,, have 2 independent cytochrome molecules in the asymmetric repeating unit. No significant difference is seen between these 2 molecules, aside from conformations of a few surface side chains. The molecular folding observed is essentially that reported for tuna ferro- cytochrome c. In particular, the ring of phenylalanine 82 lies against the heme group and closes the heme crevice, and is not swung out into the surroundings as had been believed from the 2.8 A horse ferricytochrome c structure.

Cytochrome c is a member of the mitochondrial respiratory

chain found in all eukaryotes. It functions as an electron

carrier, with its heme iron accepting a single electron from

cytochrome cI and then passing this electron on to the cyto-

chrome a + a:, (“cytochrome oxidasc”) complex. It obviously is

of interest to examine the cytochrome c molecule in both its

oxidized and reduced states, to see whether any conformation

change in the protein occurs and to try to learn how the

electron travels from the surface of the molecule to the heme

iron and out again. This paper is a report of the 2 A resolution

structure analysis of ferricytochrome c. Subsequent papers

present the equivalent analysis of ferrocytochrome c’, refine-

ment of coordinates against the multiple isomorphous replace-

ment electron density map, and comparison of structures.’

The crystal structure of horse heart ferricytochrome c was

* This work was performed with the support of National Science Foundation Grant BMS’Il-00825 and National Institutes of Health Grant GM-12121. This paper is Contribution No. 5314 of the Norman W. Church Laboratory of Chemical Biology. California Institute of Technology, Pasadena, California 91125. .‘”

? Present address, Department of Chemistry, Texas A & M Uni- versity, College Station, Texas 77843.

$ Present address, Laboratory of Biochemistry, National Institute of Dental Research. National Institutes of Health. Bethesda, Md.

‘ Present address, Department of Medicine, The Medical’College of Wisconsin, Veterans Administration Hospital, Wood (Milwau- kee), Wisconsin 53193.

’ Takano, T., Trus, B. L., Mandel, N., Mandel, G., Kallai, 0. B., Swanson, R., and Dickerson, R. E. (19771 J. Biol. Chem. 252, 776- 785.

’ N. Mandel, G. Mandel, B. L. Trus, J. Rosenberg, G. Carlson, and R. E. Dickerson (1977) manuscript submitted to J. Biol. Chem.

solved to a resolution of 2.8 A using two isomorphous heavy

atom derivatives in 1969 (1) and this revealed the overall

architecture of the molecule: a heme group inserted into a

polypeptide “glove,” with one edge of the heme exposed to the

external environment (2). The quality of the horse crystals

was so poor that there was no point in trying to extend the

resolution beyond 2.8 A. That analysis accordingly was inter-

rupted while the ferrocytochrome c structure was solved. Since

horse ferrocytochrome crystals could not be obtained, but good

crystals of tuna cytochrome were relatively easy to grow, a

change of species was made. Tuna ferrocytochrome c was

solved to 2.45 A resolution in 1971 using three heavy atom

derivatives (3).

The horse ferri- and tuna ferrocytochrome maps of 1971

appeared to show considerable orientation changes in aro-

matic side groups between oxidation states and one major

difference in main chain orientation: phenylalanine 82 seem-

ingly extended out into the surrounding solution in the oxi-

dized form, but packed against the heme in the reduced.

Bonito-minus-horse difference maps for ferricytochrome

showed that these differences were not attributable to species

changes (11, so the differences, if real, could only reflect the

oxidation state of the heme iron.

The “if real” qualification is an important one. Almost as

soon as the ferrocytochrome results were published in 1973,

doubts began to arise about the validity of the striking confor-

mation changes observed. Tuna ferrocytochrome crystals

could be 67% reoxidized as measured spectrophotometrically

and x-ray data could be collected from them. Yet a reoxidized-

minus-reduced difference map showed no features above the

level of experimental noise.’ Similar results were reported for

the rcoxidation of bonito cytochrome crystals by Kakudo and

co-workers (4). In particular, no difference peaks could be

observed which would correspond to a shift in position of

phenylalanine 82. It could be maintained that in the reoxi-

dized crystals the bulky aromatic ring was constrained in its

ferrocytochrome conformation by molecular packing, but at

best this was an argument from the absence of an effect.

At about this same time, the structures of ferricytochrome cZ

from Rhodospirillum rubrum (5, 6) and ferricytochrome ciJo

from Paracoccus (Micrococcus) denitrificans (7, 8) were com-

pleted. These both showed that the phenylalanine ring corre-

sponding to position 82 was packed against the heme group as

in tuna ferrocytochrome c. Furthermore, the oxidized and

759

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Ferricytochrome c at 2.0 A Resolution

reduced crystals of cytochrome c2 were reported to be isomor-

phous (6). It became obvious that the external position of

phenylalanine 82 in horse ferricytochrome c was suspect; and a

new ferricytochrome structure analysis became a matter of

high priority.

All attempts to obtain better quality horse ferricytochrome

crystals by changing buffers, salts, pH, and other crystalliza-

tion conditions were unsuccessful. In contrast, a new crystal

form was found for tuna ferricytochrome which gave a good x-

ray pattern extending beyond 2 A resolution. The apparent

initial disadvantage that this crystal form possessed 2 inde-

pendent molecules per asymmetric unit was counterbalanced

by the advantage that, once the analysis was completed, there

would be 2 independent protein molecules to study, as in cy-

chymotrypsin. As will be seen in the structure comparison

paper,2 this turned out to be an important consideration.

The crystallization, derivative search, and 2 A structure

analysis of tuna ferricytochrome in the new crystal form are

the subject of this paper. The accompanying paper1 describes

the extention of the tuna ferrocytochrome study from 2.45 to 2

A. A third paper’ discusses the refinement of tuna ferri- and

ferrocytochrome coordinates by a combination of hand adjust-

ment of positions on miniature electron density maps and

rectification of bond lengths and angles by means of model-

building programs (“mini-map refinement”). Mini-map refine-

ment coordinates are published in that paper, although fur-

ther refinement of coordinates is planned using real-space

methods.

MATERIALS AND METHODS

Extractam, Purification, and Crystallizataon of Tuna Ferricyto- chrome c ~ Cytochrome c was extracted from frozen tuna hearts obtained from local Los Angeles fisheries, using methods developed by Takano et al. (3) who observed the precautions cited by Margo- liash and Schejter (9). From 20 pounds of hearts a typical yield was

2.7 g of cytochrome, with an optical ratio of AL$/A;$, = 0.95 before purification, Purification was carried out chromatographically on IRC-50 resin, using 0.2 M ammonium phosphate buffer and lo-’ M

potassium ferricyanide for elution. Fractions with optical ratios of 1.00 or better were used for crystallization trials.

Three different crystal forms of tuna ferricytochrome c could be

obtained by varying crystallizing conditions and these are shown in Table I. Type A crystals were obtained under conditions paralleling those for horse ferricytochrome (1). They were isomorphous with

horse, but were always small and of poor quality. Type B crystals, subsequently used for structure analysis, had the same space group as type A, but 2 molecules per asymmetric unit and a cell edge which

was nearly equal to the ab cell diagonal of type A. This suggested that the two crystal forms were related and differed only in the manner of packing of 4:, screw axes of molecules, a possibility that had been suggested 12 years ago by Blow and co-workers (10). Type C crystals always were small and were examined only in passing.

The best type B crystals for x-ray work were obtained by the following procedure.

1. Prepare a ‘7 to 11 weight per cent cytochrome solution (using E,,,, = 29,500) in 1.0 M ammonium phosphate buffer, 15% saturated in ammonium sulfate at pH 7.0.

2. Add powdered NaNO,, slowly to approximately 15% saturation. Then slowly add powdered ammonium sulfate to bring the ammo- nium sulfate concentration to 45 or 50% saturation, stirring after each addition. Watch for the point where tiny cytochrcme crystals

persist in the solution (visible under lo-power magnification). Pass- ing this point results in the formation of a gelatinous mass of fine crystals that do not grow larger even after standing for weeks.

3. Store the cytochrome solution in vials made by sealing the ends of Pasteur pipettes just at the point where they start to narrow.

Loosely stopper the vials or cover their mouths with Scotch tape. Allow the vials to sit at room temperature, while crystals grow larger, until the supernatant becomes colorless or ceases to lighten in color, usually about a month. Slow evaporation of solvent seems to be an important part of this procedure.

Cytochrome crystals grow on the glass walls of the vials under these conditions, with well formed faces, and up to 0.4 x 0.4 x 1.0 mm in size. At higher phosphate concentrations the crystals grow

more slowly and larger. In the complete absence of phosphate, crys- tal growth is complete within 2 days, but these crystals are long, thin needles, 0.1 mm or less in cross-section. If no salt other than ammo- nium sulfate is present, then type C crystals grow instead under the

conditions described above. Preparation of Heavy Atom Derivatives- Soaking conditions for

the four successful heavy atom derivatives are summarized in Table

II. Platinum chloride and platinum cyanide derivatives were pre- pared by methods described for ferrocytochrome (31. The gold cya- nide and iridium nitrite derivatives were found during a search

using metal salts at 0.1 to 2.0 mg/ml concentrations in solutions 70 to 90% saturated in ammonium sulfate and 30 to 10% in sodium nitrate. Cytochrome crystals added to these solutions were allowed to soak from 1 day to 3 months, All mercury salts tried either produced no

intensity change or damaged the crystals. Besides the four usable derivatives, other agents tried included palladium, silver, tin, os-

mium, iridium, gold, and uranyl, in a variety of oxidation states and complexes. The iridium nitrite derivative required several months of crystal soaking to produce intensity changes and occupancies varied from one preparation to the next. All of the 2 A iridium nitrite data

came from the same soaking batch, but from crystals removed and mounted at 2-week intervals. For this reason, the three concentric shells of iridium data were treated independently during phase

refinement, thus allowing for changes in degree of substitution. The pH of the gold cyanide derivative drifted downward with time and had to be readjusted once or twice during the 1st week of soaking to avoid crystal damage. For a warning about the pH drift in gold cyanide and long soaking times in iridium nitrite, we are indebted to Bunn’s and Baumber’s work:’ collating the derivative search experi-

ence of many protein structure groups. Data Collection and Scaling-Data were collected in spherical

shells of increasing 2~ on a Syntex Pi and a modified General

Electric XRD-490 automatic diffractometer, using CuKa radiation and a graphite monochromator in the perpendicular mode, with w scan techniques, The scan width (0.4-0.6” in w) was determined for

each crystal by measuring the full width at half-height of a few wide reflections and multiplying by 2.5. Background counts were collected for 10 to 20 s on either side of the peak, at positions offset from the

peak center by the total scan width. Smoothed backgrounds were calculated from these raw data by a least squares procedure (11). Total counting times per reflection were chosen to give counting

statistics such that two-thirds or more of the reflections had net intensities greater than four standard deviations. Thus the counting time per reflection was increased as intensities decreased at higher

resolution, ensuring more accurate data in these shells than would have been obtained at a constant counting time. Data were collected on a series of crystals in shells of 20, each containing approximately

3000 reflections. During data collection in any one shell, standard check reflections were observed to decline in intensity by about 15% at high angles (36-42” in 20, or 2.5 to 2.1 A resolution) and half this at

lower angles. Time-dependent decay curves obtained from check reflections were used to correct the other intensities in the 3000. reflection shell. Empirical absorption corrections were applied by the

method of North et al. (12). Statistics on the changes in cell dimensions and in structure

factors produced by the heavy atom substitution are given in Table III, in the miniprint appendix.’ The different 3000-reflection shells in

one derivative first were scaled together to make up a complete derivative set, before this derivative was scaled to the native data set. Low angle scaling, out to about 30” in 20 (3 A resolution), was

carried out using overlapping reflections as described in Ref. 13.

‘I C. W. Bunn and M. E. Baumber (1973) private communication. ’ Some of the data are presented as a miniprint supplement imme-

diately following this paper. Figs. 1 to 8, 10, and 11 and Tables III,

IV, and VI are found on p. 775. For the convenience of those who prefer to obtain the supplementary material in the form of 13 pages of full size photocopies, these same data are available as JBC Docu- ment Number 76M-571A. Orders should specify title, authors, and

reference to this paper, the JBC Document Number, and the number of copies desired. Orders should be addressed to the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014, and

must be accompanied by a remittance to the order of the Journal in the amount of $1.95 per set of photocopies.

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Ferricytochrome c at 2.0 A Resolution 761

TABLE I

TYPO space group

Crystal forms observed with tuna ferricytochrome c

Cell Dimensions Molecules Weight V /hhi~

per asym- metrical percent chz;;;’ Major faces

a b c unit protein

A

k P4., 58.24 58.24 41.86

B” p4:, 74.51 74.51 36.31

C P&2,2, 63.1 91.4 36.7

” The type studied in horse ferricytochrome c.

li The type used in this work.

Ai gem-” B

142,000 1.26 1 46 Prism IlOO~, {W, Illl}

201,600 1.37 2 60 Prism {ilo}, {iio), {loi}

211,700 1.35 2 58 Tabular {W

The pH was maintained at 7.0 i- 0.5

Abbreviation Heavy atom salt

A NaAu(CN),

C (NH,),Pt(CN), I K,,Ir(NO,),,

P K,PtCl,

TABLE II

Heavy atom derivative preparation

Preparation medium Metal salt concentration

90% A.S.“/lO% NaNO,, 0.3 mgiml

100% A.S. 0.2 mgiml

90% A.S.IlO% NaNO, Saturated (less than 3 mgiml)

90% A.S./lO% NaNO., 0.08 mgiml

Soaking time

3 weeks

4 weeks

5-13 weeks

2 days

U A.S., ammonium sulfate.

However, at high resolution, semilog plots of mean F versus (2 sin HY

sometimes revealed small discontinuities or changes of slope at the shell boundaries. In such cases, the linear (k) and exponential (B)

scale parameters necessary to produce a smooth, continuous distri- bution of mean F with (2 sin 0)’ were calculated and applied. Each internally scaled derivative data set then was scaled exponentially

against the native data (14). An exception to this procedure was the iridium derivative, in which the x to 2.7 A, the 2.7 to 2.35 A, and the 2.35 to 2.1 A data from separate crystals were treated separately

because of changes in extent of heavy atom substitution with soak- ing time.

A radial distribution plot of the fractional change in structure

factors produced by each of the four derivatives is shown in Fig. 1 in the miniprint appendix. Platinum cyanide produced the greatest changes and, as is seen later, maintained good isomorphism out to high diffraction angles. Gold cyanide and platinum chloride caused

smaller changes and iridium nitrite produced the least of all around 15% in F. The differences in extent of substitution which led us to

refine the three iridium data sets separately are clearly visible in Fig. 1.

Location of Heavy Atom Sites -The 5 A resolution difference Pat-

terson maps for three of the four derivatives are shown in Figs. 2 through 4 of the miniprint appendix. Sections chosen are the Harker sections for space group P4,,, showing self-vectors between sites related by the screw axis; but they also show slices through cross-

vector peaks between unrelated sites in a given derivative. The iridium Patterson map was essentially identical to that of platinum chloride except for peak heights, reflecting a difference in relative

occupancy of the same two sites. Difference Fourier maps at 3 A resolution are shown in Figs. 5

through 7 (miniprint appendix), in each case using phases which did not include contributions from the derivative being examined. The

two platinum derivatives were the first to be found and, whereas the platinum chloride Patterson map (Fig. 2) could be solved by inspec- tion, the platinum cyanide map (Fig. 4) was uninterpretable. Only

after the iridium derivative had been obtained could the platinum cyanide positions be found from a difference Fourier map (Fig. 7).

Because the two derivatives used in phasing for this map (platinum chloride and iridium nitrite) share a common major site, a spurious peak appeared at this position in Fig. 7 (see Ref. 15). Subsequent

addition of the gold derivative and least squares phase refinement eliminated this ghost peak (Fig. 8, miniprint appendix). The four derivatives were placed on a common origin of coordinates from difference map comparisons. Final heavy atom parameters as deter-

mined by phase refinement (to be discussed) are given in Table IV (miniprint appendix).

Low Resolution Pilot Map and Crystal Packing-The number of

molecules per asymmetric unit, coupled with the fact that the a and b cell dimensions were approximately Jytimes those of the old horse crystal form, su gested that the horse and tuna crystal forms were

related. The 5 s resolution pilot map using all four derivatives

showed a simple but unexpected relationship. In horse cytochrome crystals, all of the 4, screw axes are parallel, having the same sense along the c direction. In tuna crystals, neighboring 4,, screw axes are

antiparallel, or turned in opposite directions. Hence, the tuna crys- tals have twice as many molecules per asymmetric unit and the former ab cell diagonal becomes the new cell edge. The actual

molecular packing is shown in Fig. 9. Platinum chloride sites are marked specifically because they locate the methionine 65 residue.

This packing does not quite represent a simple inversion of alternate screw axes; in the interest of better intermolecular packing, the two 4:, axis units are oriented differently with respect to the a and b axes. In Fig. 9, a line from the iron atom to the screw axis relating outer

molecules makes an angle of 6” with the crystal axes, whereas the corresponding line in the inner molecules from the iron to the central screw axis makes an angle of 30” with the crystal axes. In spite of this

rotation, the local environment of both kinds of molecules with neighbors up and down their own 4:, axes is the same and is identical to that encountered with horse ferricytochrome (1). Only the con-

tacts between molecules in nonequivalent 4:, units differ. This sug- gests that when cytochrome c crystallizes the head to tail associa- tions around one screw axis form first, and that the association of

parallel or antiparallel axes into a crystal lattice is a secondary process. The nature of these intermolecular contacts and the forces active in crystallization of cytochrome are discussed later, along

with the reasons for heavy atom binding. Phase Refinement - Isomorphous replacement phase analysis was

carried out by the method of Blow and Crick (16), as adapted for

machine computation (17), and heavy atom parameters were refined by the least squares method of Dickerson et al. (14, 18). At low resolution, coordinates and occupancy factors (A values) were re-

fined, but isotropic temperature factors (B values) were not. These were corrected between cycles from Wilson semilog plots of the ratio of mean heavy atom contribution to observed changes in F versus (2

sin 0)‘. Only with the inclusion of data beyond 2.1 A was it felt that a sufficient range of sin 0 existed for meaningful refinement of temper- ature factors. At lower resolution the interaction between A and B

was too strong, although limited success was obtained by alternating refinement ofA and B in successive cycles, holding the other param- eter fixed.

With 2 A data it was possible to refine isotropic temperature factors to convergence, along with heavy atom coordinates and occu-

pancy factors, and to conclude with anisotropic temperature factor refinement for all heavy atoms except iridium. Convergence was considered complete when the mean change in phase angle from one cycle to the next fell below 1”. Final refined heavy atom parameters

are listed in Table IV (miniprint appendix). Table V and Figs. 10 and 11 (miniprint appendix) present statistics on phase refinement. Fig. 10 also shows the radial distribution of native protein F data.

The mean figure of merit (16) for all 14,276 reflections fell smoothly from 0.95 at low resolution to 0.45 at the 2 A data limit. The ratio of mean lack of closure error (14) to the changes produced by

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762 Ferricytochrome c a :t 2.0 A Resolution

FIG. 9. Packing diagram of the complete unit cell, showing the crystallographically different 4, screw axes at the center and corners of the cell. The 4 molecules around the central 43 axis (the “inner” molecules) are viewed from the conventional top of the molecule, near the NH,-terminal (Y helix. The 4 molecules around the corner 43 axis (the “outer” molecules) are viewed from the bottom, near the 50’s helix. The heavy line is the edge view of the heme, with histidine 18 (H) and methionine 80 (S) ligands to either side. Circled crosses are platinum chloride site 1 and circled dots are platinum chloride site 2, both at methionine 65. Decimal fractions are z coordinates of iron or platinum atoms.

TABLE V

Phase refinement statistics-final refinement

Mean figure of merit: all 14,276 reflections = 0.633, 1,111 centric

reflections = 0.816. Mean change in ‘~a between cycles = 0.93”. Mean relative error + number of derivatives = 0.473.

Derivative Kraut R factors Cullis R factors (all reflections) (centric reflections)

Pt(CN),*- 0.167 0.660 571 ptc1,*- 0.150 0.760 424

Ati(C 0.111 0.613 360

Ir (1) (~0 - 2.7 A) 0.105 0.618 447

Ir (2) (2.7 - 2.35 A) 0.088 0.772 279 Ir (3) (2.35 - 2.1 A) 0.113 0.766 293

heavy atoms is shown in a radial plot in Fig. 11. This quantity is a useful measure of the quality of the phasing contributed by each derivative. Phasing contributions from platinum cyanide and gold cyanide remained strong to the 2 A limit, but platinum chloride and iridium nitrite shared a high lack of closure error as well as common binding sites at methionine 65. As is discussed later in connection with the mode of binding of heavy atoms, these large errors probably arise from lack of true isomorphism between these two derivatives and the native protein crystals.

The mean residual error or MRE per derivative (17) was close to its expected value of 0.50, indicating that the root mean square error for each derivative in the denominator of the exponential phase probability expression had been properly chosen. Hence, the mean figure of merit in this specific structure analysis is a meaningful assessment of the quality of phasing, which is not always the case when figures of merit are quoted.

Table VI (miniprint appendix) shows the format in which the complete native and derivative structure factor data, most probable phases f&l, centroid phases ($s), and figures of merit are stored for all 14,276 reflections on magnetic tape, available upon request from the Protein Data Bank, Brookhaven National Laboratory.

The Electron Density Map - A least root mean square error elec-

tron density map was calculated by using centroid phases and weighting individual reflection F values with figures of merit. The map for publication was calculated in 48 equally spaced sections from z = 0 to z = 1, with x and y in each section running from 0 to l/z. Several sections of this map in the vicinity of the hemes appear in Figs. 12 and 13.5

The map was contoured at equal intervals corresponding to 1.4 times the root mean square error in electron density as calculated from Equation 10 of Ref. 17. All positive contours are shown, but the zero contour, indicating the mean electron density throughout the crystal, has been omitted. Data were placed on an approximate absolute scale by assuming that the largest occupancy factor, A = 845 for site 1 of iridium nitrite, represents full occupancy of 77 electrons. On this scale the contour interval in the map is 0.18 e/A3, the root mean square error over the entire map is 0.13 e/AZ, and the iron peak height is 5.1 e/A3. A check of this scaling is provided by the observation that the main polypeptide chain density is five to seven contours or about 1.1 e/A3 above average density, a value typical of other protein maps reported at 2 A resolution. The occupancy factors in Table IV and the F data in Table VI can be placed on an absolute scale of electrons per molecule by multiplying by 0.09.

The two space group-independent molecules were treated sepa- rately throughout the analysis. They are designated as the “inner” molecule, with iron at z = 0.013 in Fig. 9, and the “outer” molecule, with iron at z = 0.004. For model building purposes (Kendrew wire models, 2 cm/A), the electron density region containing each mole- cule was calculated and displayed in its own two-mirror Richards box of the design shown in Ref. 19. The use of two half-silvered mirrors permitted two maps of the same molecule, sectioned at right angles to one another, to be studied simultaneously as the model was being built. This frequently was useful when a particular feature was oriented unfavorably for viewing in one of the maps.

RESULTS

Overall Molecular Folding-The polypeptide chain path can

be followed through each molecule without difficulty or ambi-

guity and carbonyl oxygens generally are well defined. The

difference in appearance between protein molecule and inter-

stitial solvent is demonstrated in Fig. 13, A to D, showing the

clear separation between neighboring molecules. The shapes

of bent groups such as main chain and most side chains are

best appreciated in stacked sections, but planar groups such as

the heme can be examined easily in two dimensions. Fig. 14

shows the plane section and a cross-section through the li-

gands and nitrogens of rings 2 and 4 for the inner molecule,

and Figure 15 shows the plane section and a cross-section

through nitrogens 1 and 3 of the outer molecule, The five-

membered pyrrole rings are not resolved suffticiently to show a

depression at the center, but each of the four six-membered

rings formed by the iron, 2 nitrogen, and 3 carbon atoms has a

prominent central depression. Side groups, whether methyl,

propionate, or thioether attachments to the main chain, are all

visible. In the cross-sections, the sulfur ligands are prominent

because of their density and the heme planes show no obvious

sign of nonplanarity.

The main chain folding in tuna ferricytochrome c is illus-

trated by front views of the 2 molecules in Fig. 16 and by top

views in Fig. 17. These stereo pair drawings have been di-

vorced from the crystallographic axes so the molecules can be

compared more easily; in each case the coordinate system is

defined by the atoms liganded to the iron. There are no signifi-

cant differences in main chain conformation from the earlier

horse ferricytochrome structure (2, 31, with the exception of

5 The complete map is available as JBC Document No. 76M-571B, in the form of one microfiche or 48 pages. Orders should specify the title, authors, and reference to this paper and the JBC document number, whether microfiche or photocopy is wanted, and the number of copies desired. Address orders to the Journal of Biological Chemis- try, 9650 Rockville Pike, Bethesda, Md. 20014, and enclose remitt- ance to the order of the Journal in the amount of $2.50 per microfiche or $7.20 per set of photocopies.

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Ferricytochrome c at 2.0 A Resolution 763

FIG. 12. Four sections through the 2 A electron density map near the iron atom of the inner molecule. at 2 values of (A) 0.042, (B) 0.021, (C) 0.000, and (D) 0.979. Origin at upper left, x vertical from 0 to r/z, y horizontal from 0 to ‘/a. The 4:, axis relating inner molecules is at the lower right corner. Sections through the heme are indicated by a thick diagonal line. with the iron atom appearing at z = 0.00.

Density corresponding to prominent side chains is marked. S = sulfur atom of cysteine or methionine. The outlines of the inner molecule are in solid line, and those of the outer molecule are dashed. Dashed lines within a molecule represent polypeptide chain paths, Site 1 of the platinum chloride derivative appears on the z = 0.00 section.

the position of phenylalanine 82, which is discussed in a later

section.

Heavy Atom Binding Sites and Intermolecular Contacts -

As has been observed previously for horse ferricytochrome and

tuna ferrocytochrome (3,201, the PtCl,Lm complex binds speciti-

tally to 1 residue/molecule, methionine 65. In the present

analysis, platinum chloride sites 1 and 2, on the outer and

inner molecules, respectively, have 42 and 53% occupancy.

Iridium nitrite binds to the same two positions, but with

widely different occupancies: 100 and 14% for sites 1 and 2,

respectively. These drastically different substitutions make

both derivatives useful in phasing, even though they occupy

the same positions in the crystal.

In contrast to the chemically specific binding of these deriv-

atives, platinum cyanide diffuses into many different sites of

varying occupancy. The packing diagram of Fig. 18 shows that

these are all interstitial sites, where 2 molecules lie in close

proximity in the crystal. Platinum cyanide sites define every

intermolecular contact region in the crystal but one. Each of

the three gold cyanide positions corresponds to one of platinum

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764 Ferricytochrome c at 2.0 A Resolution

FIG. 13. Four sections near the iron atom of the outer molecule, at z values of (A) 0.458, (B) 0.479, (C) 0.500, and (D) 0.521. Same origin and axis conventions in Fig. 12. The 4:] axis relating outer molecules passes through the origin at the upper left corner. Boundaries of outer molecules are dashed; those of inner molecules are solid. Platinum chloride site 2 appears on section z = 0.50. Note the extensive solvent region at the bottom of each section.

cyanide, so an examination of the latter implicitly includes the serted between the chains of alanine 51 and lysine 55, and the

gold sites as well. The different heavy atom sites, their occu- carbonyl of residue 79 lies against serine 54. All of these are panties and correspondence between derivatives, and nearby hydrophobic or nonpolar contacts and no interactions of oppo- protein side chains on the outer and inner molecules, are listed sitely charged side chains can be seen. This seems to provide in Table VII. Since the two cyanide derivatives bind by inter- an energetically stable assembly that dictates to a great extent calating between molecules, the heavy atom sites are best the final crystal structure.

discussed in the framework of molecular packing in the crys- These two types of 4:, units consisting of outer and inner tal molecules then are packed against one another as dia-

Packing about the 4:, screw axis is almost identical in the grammed in Figs. 9 and 18. The tightest contact is between outer and inner molecules. In each case, as in horse ferricyto- NH,-terminal N helices in an outer and inner molecule, with chrome crystals (l), the left edge of the heme crevice in 1 inverted orientation with respect to the z axis. Platinum cya-

molecule is packed against the lower left flank of the next. nide is trapped in this intermolecular contact zone at sites 1

Alanine 83 is nested against proline 76, isoleucine 81 is in- and 3. The midpoint of this contact surface occurs atz = 0.250,

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Ferricytochrome c at 2.0 rf Resolution 765

He I-I

FIG. 14. Plane section and cross-section through the heme group of the inner molecule. The centers of the six-membered rings above, below, and to either side of the iron atom are depressions or minima, not peaks. Numbers14 and1 7 indicate attachments of cysteine residues of the polypeptide chain and P indicates a propionic acid heme substituent. Methyl substituents are marked by a straight line extending from a pyrrole ring.

He 1-O

Fro. 15. Plane and cross-sections through the heme of the outer molecule. Same general comments as Fig. 14. The I-fold symmetry, with maxima at pyrrole rings and minima between rings, is even more pronounced here than in Fig. 14.

in the center of Fig. 18. The outer and inner molecules in- volved in this contact (iron atoms at 0.504 and 0.013 in Fig. 18) are related approximately by a noncrystallographic a-fold screw axis along a cell diagonal in the z = 0.250 plane, with a 10 A screw displacement. Platinum cyanide in site 1 is close to glutamine 16 on the outer molecule and to lysines 7 and 8 and valine 11 on the inner molecule. Site 3 is close to lysines 7 and 8, valine 11, and glutamine 12 on the outer molecule, and to serine 100 and alanine 101 on the inner. Intermolecular pack- ing at this contact interleaves the side chains of the beginning of the NH&rminal helix of the inner molecule, with the end

of the corresponding helix in the outer molecule. The packing is nonpolar, with no specific charge interactions.

A much looser contact between inner and outer molecules occurs halfway down the z axis, at z = 0.750, again seen in the center of Fig. 18. This also is a nonpolar interaction, with the side chain of asparagine 22 on the inner molecule inserted between histidine 26 and the residue 23-24 main chain of the outer. This contact surface is small and no heavy atoms are trapped between molecules at this point.

I f the two contacts just described are labeled A and B, respectively, then tuna ferricytochrome has A-A and B-B con-

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766 Ferricytochrome c at 2.0 A Resolution

FIG. 16. Stereo pair drawings of the

(A) inner and (B) outer molecules, in each case using the heme atoms as a coordinate frame in order to obtain mo-

lecularly equivalent orientations. For simplicity, the only atoms drawn are cy carbons, the heme and its ligands, aro- matic side chains, prolines, and a few

other key groups.

tacts. The same contact regions are employed in horse ferricy-

tochrome crystals, but because all 4, screw axes run in the

same direction, unlike contact surfaces are paired, A to B,

around a true crystallographic 2, axis. Tuna cytochrome c also

can be induced to crystallize in this manner, and the apparent

ease of forming either like (A-A, B-B) or unlike (A-B) contacts

using the same portions of the molecular surface argues in

favor of nonpolar, hydrophobic interactions without charge

attractions or “polarity” (in the logical sense) to the mating

surfaces.

The outer and inner molecules in Fig. 18 that have iron

atoms at z = 0.254 and 0.263 are packed back to back and are

approximately related by a 2-fold axis near the dot-dashed

line separating them in the figure. Platinum cyanide is

trapped at several points in this contact zore, at sites 2, 4, and

8. The side chains proximal to these three sites on both mole-

cules are listed in Table VII. A common requirement in all five

of these platinum cyanide sites, in addition to a constrained

crevice between molecules, seems to be the nearby presence of

positively charged lysines. Both the crevice and the positive

charge seem to be necessary; the cyanide complexes do not

bind in crevices without positive charges, nor to lysine side

chains extending free into solution.

This explanation of platinum and gold cyanide binding is

reinforced by site 5. It lies close to methionine 65 and lysine 88,

but is occupied only in the outer molecule, for which this is a

cnntact zone with a neighbor. Because of differences in molec-

ular orientation about the screw axes (the 24” rotation men-

tioned earlier), methionine 65 on the inner molecules is not in

contact with a neighbor and so there is no platinum cyanide

binding at that point. Binding to the outer molecule at site 5 is

strengthened because the neighboring inner molecule has 2

lysines at this contact surface, residues 25 and 27.

Iridium nitrite binding appears to be intermediate between

the coordinate covalent link of platinum chloride and the

interstitial packing of gold and platinum cyanides. While

iridium nitrite is specific for methionine 65, it cannot bind well

unless held in place by another protein molecule. At site 1,

where it is packed against lysines 25 and 27 of the inner

molecule, it has full occupancy; but at site 2, with no neighbor-

ing molecule to hold it, it has only 14% occupancy. In contrast,

platinum chloride has approximately half-occupancy at each

site, indicating that its interaction with methionine 65 is

independent of the presence of a neighboring molecule.

The only heavy atom position lying between two symmetry-

related molecules is platinum cyanide site 6. It sits in the

heme crevice of an inner molecule, between lysine 13, gluta-

mine 16, and phenylalanine 82; and at the left side of the next

molecule up the 4, axis close to lysine 55, glutamic acid 66, and

tyrosine 74. There is no platinum cyanide-binding site at the

equivalent position on the outer molecule, which at first sight

is surprising. However, the orientations of the two independ-

ent molecules about their 4, axes are not quite identical, as

can be seen by differences in glutamine 16 side chain orienta-

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Ferricytochrome c at 2.0 A Resolution 767

FIG. 17. Stereo drawings of the mol- ecules, viewed from the top in Fig. 16. A, inner molecule; B, outer molecule.

tions. The local packing environments apparently differ just

enough to make platinum cyanide binding unfavorable at this

position in the outer molecule.

In summary, contacts between molecules in crystalline tuna

ferricytochrome c all seem to be hydrophobic and nonpolar.

Platinum chloride binds specifically and covalently to the

sulfur atom of methionine 65. Platinum and gold cyanide

complexes fit into hydrophobic crevices between molecules, in

regions not too far from lysines. Both the crevice and the

charge appear to be required for binding. Iridium nitrite is

intermediate in binding, with a specificity for methionine 65,

but with an inability to bind well unless held in place by a

neighboring molecule.

These differences in modes of binding account satisfactorily

for the differences in behavior of lack of closure errors in

phasing with sin 0 portrayed in Fig. 11 (miniprint appendix).

The two derivatives that slip into interstitial crevices without

otherwise disturbing the protein (gold and platinum cyanide)

retain a small lack of closure error even at high resolution. In

marked contrast, the two derivatives that bind specifically to

methionine at a region of intermolecular contact (platinum

chloride site 1) have high errors which suggest failure of

isomorphism at higher resolution. It is probable that the bind-

ing of heavy atoms to methionine 65 on the outer molecule

disturbs the molecular packing and makes these derivatives

poorly isomorphous with the parent crystals. No such deterio-

ration of isomorphism with increasing sin 0 was observed with

the platinum chloride derivative in either horse ferricyto-

chrome or tuna ferrocytochrome, in which the methionine site

faced interstitial solution and not a neighboring molecule (1,

3).

Reinforcing the idea of the relative gentleness of binding of

the two cyanide derivatives is the observation that platinum

and gold cyanide never bind just at the point of closest inter-

molecular contact, but adjacent to it instead. In Fig. 18, the

closest approach of molecules around platinum cyanide sites 1

and 3 occurs at z. = 0.250, but the heavy atoms bind above and

below this, at z = 0.100 and 0.335. Similarly, the closest

contact of molecules around sites 2, 4, and 8 also occurs at z =

0.250, but the heavy atoms are located to either side and atz =

0.037, 0.105, and 0.324.

An implicit goal in many previous protein structure analy-

ses has been the finding of amino acid-specific reagents, which

would label the protein in a small number of chemically

defined positions. Multisite and nonspecific derivatives have

been somewhat denigrated, although never scorned if found.

The experience with tuna ferricytochrome c suggests that this

attitude may be wrong. Nonspecific reagents such as gold and

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768 Ferricytochrome c at 2.0 A Resolution

Inner

FIG. 18. Packing diagram of tuna ferricytochrome c crystal, showing heavy atom positions. Circled crosses and circled dots, respectively, are platinum chloride sites 1 and 2. Circled numbers are platinum cyanide sites. Iridium sites are identical to those of platinum chloride and gold sites correspond to certain platinum cyanide positions as listed in Table VII. Dot-dashed lines mark contact regions between symmetry-independent molecules. Decimal fractions are z coordinates of heme irons, heavy atoms, or of points of closest approach in contact regions. See text for discussion of heavy atom binding.

TABLE VII

Occupancy and environment of heauy atom sites

Side chains in parentheses are more distant from the heavy atom site.

Deriva- tive

site Per cent Nearby side chains Nearby side

occupancy on outer molecules chains on inner

moleculea

a. PtCl 1 42 Ir 1 100 PtCN 5 29

b. PtCl 2 53 Ir 2 14

c. PtCN 1 68 Au 3 13

d. PtCN 3 41 Au 1 67

e. PtCN 2 50 f. PtCN 4 44

Au 2 17

g. PtCN 8 5

h. PtCN 6 21

M65, (K88) M65, (K88) (M65), K88 (Distant) (Distant) Ql6 Ql6 K8, Vll, Q12 K7, K8, Vll F36, G37, K99 V3, K99, SlOO V3, K99, SlOO A96, K99 (Distant)

K25, K27 K25, K27 K25, K27 M65, (K88) M65, (K88) K7, K8, Vll K7, K8, Vll

SlOO, Al01 SlOO, Al01 D62 G37, V58, N60 G37, V58, N60 V58 K13, QlS, F82 K55, E66. Y74

a One-letter amino acid code and residue number.

platinum cyanide may be especially valuable precisely be- cause they do not bind strongly to the protein and hence do not introduce distortions and impaired isomorphism. Derivatives with few sites may be necessary in the early stages of an analysis in order to interpret the multisite derivatives, but the latter may be of more value in phasing at high resolution.

Orientation of Aromatic Rings - Cytochrome c is distin- guished by a remarkably conservative set of aromatic resi- dues, which have retained their aromatic character through- out evolutionary history even though the specific amino acids have changed. All positions at which aromatic rings are found in any of the 60 eukaryotic cytochrome sequences from 67 different species are listed in Table VIII, which also shows how many times each position is encountered as phenylalanine,

tyrosine, tryptophan, or a nonaromatic group. The 10 aromatic groups in tuna cytochrome are designated in bold face type.

These same key positions tend to remain invariant in the bacterial cytochromes that are known to be structurally ho- mologous with cytochrome c: respiratory cytochrome cssO from Paracoccus denitrificans (8) and photosynthetic c2 from Rhodospirillum rubrum (5) and other purple non-sulfur bac- teria. The highly conserved aromatic residues occur in clusters in the molecule, visible in Figs. 16 and 17. These clusters include positions 10 and 97 at the right side of the molecule, 46 and 48 at the bottom, 59, 67, and 74 at the left rear, and isolated rings at positions 82 and frequently 36. Rings occur only rarely at other positions in the structure.

The pronounced evolutionary conservatism of aromaticity around the heme group suggests that these rings have an important role. Attempts have been made to involve them in electron transfer mechanisms (20, 23), although these mecha- nisms now seem unlikely. No matter what their role, the orientation of these rings and the degree of confidence that can be placed in the published orientations, are matters of interest when the final structures of ferri- and ferrocytochrome c are studied. Numerical comparisons will be deferred until the third paper in this series,2 after the structure of the reduced form of the molecule has been presented.’ The immediately comprehensible measure of confidence is the appearance of the electron density map itself. For this reason, miniature in- plane sections and cross-sections are shown in Fig. 19 for all histidines and prolines, and in Figs. 20 and 21 for all phenylal- anines, tyrosines, and tryptophans.

One generalization is quickly apparent. At 2 A resolution, a five-membered ring appears as a flat disc without a central depression. Histidine 18 in both inner and outer molecules (labeled H18-I and H18-0) are good examples. The angular shape of the disc is correct and the disc cross-section is satisfy- ingly flat, but there is no central hole or dimple. Prolines 71 and 76 in the outer molecule (P71-0 and P76-0) may be considered especially favorable exceptions to this principle, but the general rule still holds.

In contrast, a six-membered ring is large enough to show a central depression at 2 A resolution, as Figs. 20 and 21 indi- cate. Phenylalanine 10 in the inner molecule (FlO-I) and tyro- sine 46 in both molecules are particularly clear examples and the tyrosines also illustrate the clarity with which an -OH group stands out. Tryptophans 33 and 59 are consistent with the above principles: their six-membered rings are dimpled in the center but their five-membered rings are not.

The ring orientations in the stereo drawings of Figs. 16 and 17 and the plane sections of Figs. 19 to 21 are the results of independent minimap fitting to the inner and outer molecules: hand adjustment of chain positions to map density, alternated with least squares optimization of bond lengths and angles. The ring cross-sections indicate the degree of confidence that can be placed in ring orientation comparisons. For phenylala- nine and tyrosine, the uncertainties in rotation about the CD- C, axis should be no more than 5-10”. A comparison of the stereo drawings in Figs. 16 and 17 shows that the two symme- try-independent molecules have virtually identical conforma- tions. Aromatic ring orientations are the same within the limits just mentioned. Although some external side chains have slightly different conformations in the inner and outer molecules,2 there is no indication that main chain conforma- tions are influenced by the different local environments that the 2 molecules have in the crystal. The degree of similarity or difference observed in the main chain and internal side chain

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Ferricytochrome c at 2.0 A Resolution 769

TABLE VIII

Occurrence of aromatic residues in cytochrome c Number observed out of a set of 60 sequences from 67 eukaryotic species. Bold face numbers are the aromatic residues present in tuna

cytochrome c.

Position 1CP 33 35 36 46O 4W 5P 61 54 65 61° 14 320 97’

Tyrosine 1 2 33 60 1 23 59 58 59 Phenylalanine 60 1 2 53 27 1 8 1 2 60 1

Tryptophan 4 60 Other 54 58 5 59 59 29

D Also invariantly aromatic in bacterial c JJ0 from Purucoccus denitrifiuns and all known cg sequences from purple non-sulfur bacteria.

(Phe 20 structurally equivalent to Phe 10 in Rhoabspirillum rubrum.) See Ref. 19, 21, and 22.

Fe

P7I-I P76-I P7k0 P76-0

FIG. 19. Plane sections and cross-sections through histidine (H) and proline (P) rings for the inner (I) and outer (0) molecules identified by residue number. Chain link lines in plane sections indicate the cutting plane for corresponding cross-sections.

portions of these 2 oxidized molecules furnishes a calibration standard or control, against which any presumed folding changes with oxidation state must be compared.

Position of Phenylalanine 82 -Phenylalanine 82 is an espe- cially interesting ring in tuna ferricytochrome c because of previously published reports indicating that this residue was positioned differently in the oxidized and reduced molecules (1, 3). These reports were wrong, as can be seen from the composite electron density drawings of Figs. 22 to 24. The sections through the electron density map should be viewed as if they were stacked, jigsawed pieces of plywood, each piece eclipsing from view those below it. Each such piece is outlined by a thick line in Figs. 22 to 24 and higher density contours within a section are represented by thinner lines. All line thicknesses are decreased in sections farther removed from the viewer, as a means of introducing perspective. The view in each figure is from the top, close to that of Fig. 1’7, with the

heme crevice opening at the bottom of the figure. The heme plane is represented as a square fading into the paper and portions of main and side chains are superimposed on the composite sections. The xy plane sections are spaced 0.84 A apart in z. Section numbers counting upward from the heme iron section are given in parentheses.

It is clear from Figs. 22 and 23 that the aromatic ring of phenylalanine 82 lies next to the heme group and roughly parallel to it. Since these chain positions were only sketched in by hand from viewing the models in a Richards box, the stereo drawings of Figs. 16 and 17 actually give a more accurate

impression of ring positions. The phenylalanine position is the same as that reported for tuna ferrocytochrome c (1). For comparison purposes, similar sections through the new 2.0 A reduced tuna cytochrome c map are given in Fig. 24. The molecular tilt relative to the crystal axis chosen for sectioning is not quite the same as for the oxidized crystal form, but the

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770 Ferricytochrome c at 2.0 A Resolution

F IO-1

\/----’

F36-I -.-- -~

FIG. 20. Plane and cross-sections through aromatic rings in the inner (I) and outer (0) molecules. F = phenylalanine; Y = tyrosine. Chain link lines in plane sections indicate the cutting plane for cross-sections. Note in this figure and the following one that six-membered rings generally are well resolved enough to show a central minimum at 2 A resolution, while five-membered rings are not (Fig. 19).

same features can be seen, At present there is strong crystallo- drawing of the equivalent region of the two-derivative, 2.8 A graphic evidence to indicate that the phenylalanine 82 portion resolution horse ferricytochrome map. The resolution of de- of the cytochrome c molecule does not undergo any kind of tails is markedly poorer in this map. This is attributable to conformation change when the iron atom is reduced or oxi- three causes: the lower nominal resolution of the map, the dized. smaller number of derivatives used in phase analysis, and the

Where did the horse ferricytochrome analysis go astray? poorer intrinsic quality of the horse crystals. Two alternatives The answer can be seen in Fig. 25, which shows a composite were open in the 2.8 A map for the phenylalanine 82 ring

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Ferricytochrome c at 2.0 A Resolution 771

FIG. 21. Plane and cross-sections through six-membered rings in the last half of the amino acid sequence and through tryptophans 33 and 59. Same identification system and cross-section conventions as in Figs. 19 and 20. In tryptophans, as elsewhere, the six-membered rings have a central depression whereas the five- membered rings are not quite large enough. In all cases the ring plane orientation is unambiguous and well defined.

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772 Ferricytochrome c at 2.0 A Resolution

FIG. 22. Composite of electron density sections through the inner molecule in the vicinity of phenylalanine 82 and the heme. See text for details of preparation. The curved dashed line at the bottom indicates the molecular surface. The methyl groups on the heme connections to the sulfurs of cysteines 14 and 17 are unambiguously located by peaks labeled with section numbers (2) and (6). The set of peaks marked (6) at upper right is an edge view of the phenylalanine 10 ring.

FIG. 23. Electron density composite similar to Fig. 22, but through the outer molecule. (Atomic positions in Figs. 22 to 24 have been sketched in by hand from the Richards box model and are not comparable with the accurate coordinates used in Figs. 16 through 21.)

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Ferricytochrome c at 2.0 rf Resolution

773

/

a

FIG. 24. Similar electron density composite for the reduced tuna cytochrome molecule at 2 A resolution. These areyz sections in space group P2,2,2 and 0.84 A apart in 3~. The molecule is tilted somewhat differently relative to the sectioning directions from Figs. 22 and 23, but the same features can be seen.

FIG. 25. Equivalent sections through horse ferricytochrome c from a two-derivative map at 2.8 A resolution. This should be compared vith Figs. 22 and 23, from which an approximate polypeptide chain path can be followed. The edge of the heme group is --J--~ I-- ---“-- ruza1-nau vy Stxbi”II lumber (5) at the right. The (7) directly above it is the sulfur of cysteine 14 and the (10) to the right is the beginning 01 ‘the lysine 13 side . . I

cham.

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774 Ferricytochrome c

position: an external location marked by section number (9) at

the far left of Fig. 25, and an internal position to its right

marked by an (8). The internal position density was weaker

and this site was felt to be close to a lysine 13 side chain

coming across the top of the heme crevice. This site therefore

was rejected in favor of the outer position and the inner peak

later was proposed tentatively as a bound ion in the heme

crevice. This was an error; the “bound ion” was a weak image

of the phenylalanine ring. The external position, (9) in Fig. 25,

has no counterpart in any of the other density maps shown in

Figs. 22 to 24. If it is not simply an error in the 2.8 A map, then

it may be a bound ion on the molecular surface, unique to the

horse ferricytochrome c crystal form.

Further discussion of ferricytochrome c will be deferred

until the presentation of the ferrocytochrome c structure in the

accompanying paper.’ At the moment it can be stated simply

that the tuna ferricytochrome structure resembles that previ-

ously reported for ferrocytochrome c (3) and that the two

independent molecules in the ferri.cytochrome crystals show no

appreciable differences in structure aside from the conforma-

tions of a few external side chains.

1.

2. 3.

4.

5.

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775

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R Swanson, B L Trus, N Mandel, G Mandel, O B Kallai and R E DickersonTuna cytochrome c at 2.0 A resolution. I.Ferricytochrome structure analysis.

1977, 252:759-775.J. Biol. Chem. 

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