THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 19&( by The American Society of Biological Chemists. Inc.

Vol. 259, No. 21, Issue of November 10, pp. 13027-13036 1984 Printed in d.S.A.

Crystal Structure of Yeast Cytochrome c Peroxidase Refined at 1.7-A Resolution*

(Received for publication, March 26,1984)

Barry C. Finzelt, Thomas L. Poulos$, and Joseph Kraut From the Department of Chemistry, University of California, San Diego, La Jolla, California 92093

The crystal structure of cytochrome c peroxidase (EC 1.11.1.5) has been refined to an R factor of 0.20 com- puted for all reflections to 1.7 bi. The refined molecular model includes 263 bound water molecules and allows for x-ray scattering by amorphous solvent. The mean positional error in atomic coordinates is estimated to lie between 0.12 and 0.21 A.

Two factors are identified which may account for the ability of the enzyme to stabilize high-oxidation states of the heme iron during catalysis: 1) the proximal histidine forms a hydrogen bond with a buried aspartic acid side chain, Asp-235; and 2) the heme environment is more polar than in the cytochromes c or globins, owing to the presence of the partially buried side-chain of Arg-48 and five water molecules bound in close proximity to the heme. Two of these occupy the pre- sumed peroxide-binding site.

Two candidates are likely for the side chain that is oxidized to a free radical during formation of Com- pound I: 1) Trp-51, which rests 3.3 A above the heme plane in close proximity (2.7 A) to the sixth-coordina- tion position; and 2) Met-172, which is 3.7 A from the heme. Nucleophilic stabilization of the methionyl ca- tion radical may be possible via Asp-235.

His-181 is found to lie coplanar with the heme in a niche between the two propionates near the suspected cytochrome c-binding site. A network of hydrogen bonds involving this histidine may provide a preferred pathway for electron transfer between hemes.

Cytochrome c peroxidase (ferrocytochrome c:HzOz oxido- reductase, EC 1.11.1.5) is a single-chain 294-residue heme enzyme found in yeast mitochondria where it catalyzes the oxidation of ferrocytochrome c by peroxides.

2H+ + ROOH + 2 cyt c (Fez+) -P 2 cyt c (Fe3’) + ROH + Hz0

The enzyme reacts rapidly with hydroperoxides, donating 2 reducing equivalents for the cleavage of the peroxy oxygen- oxygen bond, to generate Compound I (alternatively, Complex ES (1)). Like the corresponding Compound I of horseradish peroxidase (2), the iron atom of this oxidized intermediate is in the Fe(1V) oxidation state (3), but in cytochrome c perox- idase the second electron is abstracted from an amino acid side chain rather than from the heme (2, 4). In addition,

* This work, based on the Ph.D. thesis research of B. C. F., was supported by Research Grants GM 10928, AM 07233, and RR 00757 from the National Institutes of Health, and PCM-820S414 from the National Science Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Genex Corporation, Science and Technology Center, 16020 Industrial Dr., Gaithersburg, MD 20877.

cytochrome c peroxidase Compound I is considerably more stable than its counterpart in plant peroxidases.

Cytochrome c peroxidase Compound I is readily reduced by 2 equivalents of ferrocytochrome c to regenerate the native enzyme. This strong preference for cytochrome c as a reduc- tant is unique among peroxidases (1) and bestows upon this redox system a special significance. Since the water-soluble cytochrome c peroxidase is a relatively small monomer of 294 amino acid residues (5), which is easily purified and crystal- lized (61, this enzyme can serve as a simplified model for multisubunit membrane-bound cytochrome c oxidases which are much more difficult to study. Moreover, study of the cytochrome c peroxidase:cytochrome c couple has much to contribute to an understanding of how two macromolecular redox partners recognize and bind to one another, and how long-distance electron transfer is controlled in biological sys- tems.

We have previously reported the structure of yeast cyto- chrome c peroxidase as determined by interpretation of a 2.5- A electron density map (7, 8). With the aid of this model, we have proposed a stereochemical mechanism for the formation of Compound I (9) and presented a hypothetical model for the cytochrome c peroxidase:cytochrome c electron-transfer complex (10). In the present paper, we describe an improved structural model obtained by high-resolution crystallographic refinement and reassess these earlier proposals in light of the new, more accurate model.

EXPERIMENTAL METHODS AND DETAILS OF REFINEMENT

Cytochrome c peroxidase was purified and crystallized from pressed bakers’ yeast as described previously (8). Crystals, grown in 30% (v/ v) 2-methyl-2,4-pentanediol buffered to pH 6.0 with 50 mM potassium hosphate, belopg to space group P21212~ with a = 107.43 A, b = 76.78 1, c = 51.43 A, with one cytochrome c peroxidase molecule per

asymmetric unit. X-ray diffraction data to a Bragg spacing of 1.7 A were collected on the multiwire area detector diffractometer of Xuong and co-workers (11,12). Details of data collection on this instrument have been presented elsewhere (12). The resulting data set consists of 44,888 unique reflections derived from 181,721 observations. Data from six crystals were combined with an overall weighted internal R., of 0.07.’

The structural model used to initiate refinement was originally obtained by interpretation of a 2.5-A multiple-isomorphous-replace- ment phased electron-density map which had been subjected to density modification procedures (7). Because interpretation of this map had been carried out with the aid of a conventional half-silvered mirror comparator, the original model was first readjusted using an

1 c c I W h , k, 0 ) - I(h, k, 01 I N

R e , - - h*l 6 1

I(h, k, 0;

N

hkl i=l

where N is the number of symmetry-related reflections.

13027

13028 Refinement of Cytochrome c Peroxidase Evans and Sutherland Picture System I to improve electron-density fitting. Although backbone chain density for 27 amino acids and many side chains could not be identified in this map, the resulting model was judged adequate for a first round of F, calculations, which gave an R factor of 0.54. A revised electron-density map was obtained using computed F, phases and observed amplitudes, and the entire model was reconstructed into this improved map, incorporating the complete polypeptide sequence information which had just become available (13). All but 6 residues at the N-terminal tail of the molecule could now be located, but it was still necessary to assume the precise conformation of several poorly defined segments of chain.

The best general strategy, for refinement is to alternate visual inspection of maps and rebuilding of the model with application of the automated parameter optimization procedure of Hendrickson and Konnert (14). The electron-density maps are computed using IFJ, (2F, - F J , and IF,, - FJ as amplitudes and phases derived from F, calculations on the previous model. This process was greatly facili- tated by employing an Evans and Sutherland Picture System 11, using software developed locally? Model rebuilding was by far the most time-consuming activity, even though a single cycle of refinement with all data to 1.7 A required -17 h of processing time on a VAX 11/780 computer.

Early in the refinement procedure, only an overall temperature factor was refined, but later, individual isotropic thermal parameters were adjusted as well. Ordered solvent molecules were added to the model throughout the procedure whenever the two following criteria were met: 1) a peak larger than -4u appeared in the IF, - FJ maps, where u is the estimated root mean square difference density com- puted over the asymmetric unit; and 2) the position of this peak allowed favorable hydrogen bonding or ionic interaction with some existingportion of the molecule or solvent structure. Potential solvent sites with an occupancy less than 0.3 were discarded.

To correct for the contribution of amorphous solvent within the crystal lattice, the nonlinear scaling function used by Bolin et al. (15) was applied throughout the refinement. Optimum values for kz and Bz at the end of refinement were 0.60 and 210 A’ comparable to values found by Bolin et al. (15) during the refinement of two species of dihydrofolate reductase.

Progress in refinement was irregular during the early stages. The automated parameter-optimization procedure converged rapidly enough, typically in two or three cycles of least squares, depending upon the magnitude of the shifts in the preceding round of model rebuilding. However, progress most often depended on our ability to locate and correct erroneous portions of the model through visual inspection, and this became increasingly more difficult. After 38 cycles of refinement utilizing progressively higher resolution data (eventually incorporating all reflections to 1.7 A) and including eight rounds of model rebuilding, the lowest R-factor attained was 33%. Several long sections (10-20 residues) of the polypeptide chain were identified which clearly had been modelled incorrectly, but electron- density maps phased with this incorrect model failed to provide adequate information to enable an assignment of the correct confor- mation. The analysis of “partial difference Fouriers,” using coeffi- cients F, - Fc’, where F,‘ is computed by omission of questionable portions of the model, was helpful in resolving smaller ambiguities. However, this method failed when larger sections of the chain were involved, since the partial difference Fouriers tended to reproduce features of the incorrect model even though they had been omitted from F, calculations. In retrospect, this failure can probably he attributed to compensating errors distributed throughout the model, as it was occasionally possible to improve the quality of the electron density by proceeding with refinement of the remaining atomic pa- rameters in the absence of the incorrect segment. To overcome this cooperative effect, it became necessary to discard approximately 15% of the molecular model all at one time prior to cycle 40, and to replace model F, phases by centroid phases derived from a combination of m . i ~ . ~ and model-phase probabilities (16). Because m.i.r. phases were available only to 2.5 A, higher-order model F, phases were used but weighted in Fourier syntheses as suggested by Sim (17). Details of the implementation of an independently devised but similar phase combination procedure have been described elsewhere (18). Ideally, this procedure provides the best combination of the m.i.r. and model- phase information. Weighting of Fourier coefficients with the figure

S. A. Dempsey, unpublished programs. The abbreviation used is: m.i.r., multiple isomorphous replace-

ment.

of merit proved to be quite important in generating improved electron- density maps. Electron density computed in this way exhibitedbetter connectivity in areas where large sections of the model had been omitted and subsequent rebuilding and refinement using these maps eventually resulted in the unambiguous assignment of an acceptable conformation to those sections of chain. We regard the use of the phase-combination technique as critical to the eventual success of this refinement.

The refined model, obtained after 90 cycles of least squares and 17 separate rounds of model analysis and rebuilding gives an R of 20.2%, computed over all 44,888 unique reflections to 1.7 A’, or 18.1% for 32,089 reflections with intensities greater than twice the measured standard deviation. The final model consists of 2,346 non-hydrogen protein and heme atoms and 263 bound water molecules. Atomic coordinates and high resolution x-ray data have been deposited with the Brookhaven Protein Data Bank (19) and are available from that source.

The position of the N-terminal threonine residue remains unde- termined, as do some parts or all of the following 24 side chains: Thr- 2, Leu-4, Val-10, Glu-17, Lys-21, Glu-32, Glu-35, Asn-38, Lys-74, Lys-90, Glu-93, Lys-97, Glu-120, Lys-183, Asp-210, Lys-212, Asn- 219, Lys-226, Lys-243, Lys-249, Lys-260, Lys-264, Lys-278, and Asp- 279. Each of these side chains extends into the solvent, and is not observed in any single conformation, even at low electron-density levels. Two side chains, Thr-63 and Leu-161, are modelled in two different conformations, each rotated around the terminal torsion angle by about 120”. The second conformation was added when it became apparent that neither model alone sufficiently accounted for the observed electron density. The quality of the final model might be gauged by the refinement parameters given in Table I.

To estimate error in these atomic positions, we apply the method of Luzatti (20) in which the crystallographic R factor is plotted uersus sin e and the distribution compared to curves obtained assuming various levels of error. We observe a poor correlation between the distribution of our data and that predicted for any coordinate error, particularly a t high sin e, but a mean positional error of 0.21 A was obtained by least-squares minimization. Because of the underlying assumptions of the Luzatti treatment, this is necessarily an upper bound of the true coordinate error. As an alternative to this overall estimate of error, we have obtained estimated standard deviations of all positional parameters from the diagonal of the least-squares normal matrix. Radial errors in position for protein atoms computed from these deviations range between 0.1 and 0.3 A, exhibit a mean of 0.12 A, and, as expected, vary regularly with thermal parameters. These values are underestimates, as emphasized by the very small

TABLE I Summary of least-squares refinement parameters

Final model value is the root mean square deviation from expected geometry (14). u is the inverse square root of the weight applied in least-squares refinement and as such establishes an expectation value for the standard deviation desired for this type of geometry restraint. Target IJ and root mean square deviations vary during the course of the refinement. All parameters given are those found at the end of refinement.

Target u Final model

R Distances

1-2” 1-3 1-4

Planes Peptides Other

Chiral volumes Nonbonded contacts

Other

Main chain 1-2 Main chain 1-3 Side chain 1-2 Side chain 1-3

1-4

Thermal parameter correlations

1h 1 F, - F,I 0.030 0.050 0.050

0.030 0.030 0.400

0.500 0.500

2.00 3.00 2.00 3.00

0.202

0.022 0.042 0.044

0.030 0.012 0.368

0.193 0.187

1.36 1.97 1.68 2.47

The notations 1-2, 1-3, 1-4 refer to atom pairs related through a bond, a bond angle, or a dihedral angle, respectively.

Refinement of Cytochrome c Peroxidase 13029

radial error (0.02 A) computed for the iron atom, but are probably closer to the true coordinate error. than that obtained from the Luzatti plot since restraints imposed upon the coordinates during refinement will tend to reduce overall coordinate uncertainty.

THE MOLECULAR MODEL

Cytochrome c peroxidase is folded into two clearly defined domains so that the overall shape of the molecule .resembles a prolate ellipsoid. As described in our earlier analysis (9), the structure is dominated by 10 a-helical segments which com- pose about 50% of the molecule. The molecule also contains two antiparallel @-pairs and one small @-sheet.

Two stereoscopic views of the a-carbon backbone of the molecule are shown in Figs. 1 and 2. Domain I (top of Fig. 2) contains the N terminus and helices A through D, and is evenly distributed about helix B which forms the environment directly above the heme. A short section of extended chain and helix E connects this domain with domain 11, which contains helices F through I and most of the @-structure. C- terminal helix J begins in Domain I1 (below the heme, Fig. 2) and projects well into Domain I. A long section of extended chain then wraps around the utside of Domain I to the COOH terminus on the far side of the molecule, tying the two domains together. Such an arrangement of two well-defined domains with amino and carboxyl termini situated in the same domain is characteristic of many two-domain structures (21).

A large channel, formed at the interface between the two domains, connects the distal side of the heme crevice with the molecular surface. This channel presumably allows the trans- port of substrate peroxides and alcohol by-products into and out of the active site. The channel is about 10 A deep and 5 A wide at the mouth and is easily large enough to accommo- date long-chain alkyl-hydroperoxides known to be substrates (1).

Revisions to the Backbone Geometry-While all the essential features of the secondary structure were correctly described previously (9), the crystallographic refinement has necessi- tated several major modifications to the original description of the backbone conformation, an important correction to our description of the proximal histidine environment, and one correction to the sequence (13). In the following paragraphs we discuss these revisions in detail to minimize any confusion that may arise in the future.

The refined model shows that an asparagine residue must be inserted between positions 163 and 164 of the published sequence (13). The choice of asparagine over aspartic acid is made possible by hydrogen-bonding considerations and has

been confirmed by Kaput et al. (5), who have sequenced the cytochrome c peroxidase gene. A revised sequence numbering will be used throughout this discussion. Kaput et al. (5) also report two additional differences between the published amino acid sequence and the gene sequence; threonine-53 to isoleu- cine and aspartic acid-152 to glycine. Thr-53 lies near the C- terminal end of interior helix B and the density envelope clearly defines a threonine side chain. Asp-152 is on the molecular surface, but the side chain is well ordered owing to an ionic interaction with the side chain of Arg-155. Thus, our results agree with the original amino acid sequence in regard to the identification of these 2 residues. However, differences between the DNA and protein sequences require the change of only a single base pair and may be dependent upon the strain of yeast used (5).

Several segments of extended chain were poorly defined in the original m.i.r. map and required extensive modification. These include the first 14 residues of the N-terminal tail of the molecule which were not present in the m.i.r.-based model at all and chain segments 30-39,58-84, and 127-138.

Additional corrections have resulted from the clarification of four surface loops which were poorly defined in the m.i.r.- phased map. The bend following the C terminus of helix F, formerly thought to be composed of residues 180-183, has been lengthened by 3 residues, resulting in a correction to the identification of each of the residues in the short antiparallel @-strand which follows. Three residues were removed from the bend at the C-terminal end of the &structure so the original identification remains correct following residue 193. A similar change was made to the solvent-exposed loop fol- lowing the @-strand, comprising residues 223-225, which was the most poorly defined region of the m.i.r.-phased map. This turn was shortened by 4 residues and now forms a classical Type I hairpin turn (see discussion below). Identification of the following 50 residues (including residues which compose all of helices I and J) had been incorrect in the m.i.r.-based model. A disordered loop following residue 277 was lengthened by 4 residues to re-establish the original identification prior to the C terminus.

The last of these major changes, apart from their obvious effect on progress in the refinement, are of considerable importance since they force a reassessment of recent ideas abut how this enzyme functions. The 178-192 @-pair contrib- utes five hydrogen bonds to the outermost heme propionate (pyrrole IV) and may play an important role in the biological electron-transfer process (22). In addition, in our previous analysis of the catalytic mechanism, Gln-239 and Glu-187 were thought to play key roles since these residues were

FIG. 1. Stereoscopic view of the a- carbon backbone of cytochrome c peroxidase. The view is through the ligand access channel to the active site. Critical side chains of Arg-48, Trp-51, His-52, and the proximal histidine, His- 175, are shown. N and C termini are indicated by Nand C.

N N

13030 Refinement of Cytochrome c Peroxidase

FIG. 2. A second stereoscopic view of the a-carbon backbone of cytochrome c peroxidase (see Fig. 1). The view is to emphasize the domain structure (see text) and helical segments ('44).

TABLE I1 Helices

Helices are defined by hydrogen-bonding pattern and appropriate torsion angles. Criteria for the assignment for all hydrogen bonds were distance (2.4-3.2 A) and a qualitative assessment of linearity.

Helix

A B B' C D E F F' G H I J J'

Ending Beginning

R15 0 . . . N F19 K29 0 . . . N D33 Y42 0 . . . N L46 A50 0 . . . N S54 F73 0 . . . N F77 K74 0 . . . N N78 G84 0 . . . N N87" I95 0 . . . N F99 S103 0 ... N L107 A115 0 ... N M119 Dl50 0 . . . N V154 V154 0 . . . N H158 N164 0 . . . N V168 A174 0 . . . N L177" H181 0 . . . N S185 L182 0 . . . N G186 E201 0 . . . N N205 N205 0 . . . N E209 L232 0 . . . N Y236 S237 0 . . . N Q240" D241 0 . . . N L245 K249 0 . . . N N253 Q255 0 . . . N F259 K268 0 . . . N D272 T288 0 . . . N Q292 L289 0 . . . N G293

Number of resi-

dues

19 13 6 16 17 9 14 6 9 9 13 18 6

a Indicates a helix which is either initiated or terminated by 310- helical turns.

believed to interact with the proximal heme ligand (9). In the refined model, the two corresponding electron density features are identified as the side chains of Asp-235 and Trp-191, respectively. Reinterpretation of the functional significance of these structural features will be a principal topic in a later section.

Secondary Structure-On .the basis of backbone torsion angles and hydrogen bonding, 10 major a-helices have been identified in the structure of the peroxidase (Table 11). These helices (A-J) had been identified previously and we retain the nomenclature put forth in the earlier paper (7). In addition, three short a-helical segments (Bl, F1, and J l ) , comprising 6 residues each, have been found in the course of refinement. Average main chain torsion angles (4) and (I)) within the helices are (4) = -63" and (I)) = -42", consistent with values found in other protein molecules (15, 23, 24). The hydrogen- bonding pattern of helix C is disrupted by the presence of a proline at position 94. This helix runs across the surface of Domain I, and the proline enables the helix to bend inward to follow the contour of the molecule more closely.

As indicated in Table I, three of the helical segments are either initiated or terminated with 3,0-helical turns. A 310- helical turn is identified by an (n)O-(n + 3)N hydrogen bond and ideal torsion angles, 4 = -76" and I) = -50 (25). Mean torsion angles, (4 ) = -65" and (I)) = -18", are actually observed for the helix-breaking 310 turns in this molecule. The only 310 helix of more than one hydrogen bond occurs at the C terminus of a-helix F, where residues 171-176 form two

full turns of 310 helix just beneath the heme. This short segment contributes the proximal heme ligand (His-175). Close proximity of this compact helix to the heme leads us to speculate that perhaps the 310 conformation is the result of a distortion of helix F caused by binding of the heme to the apoenzyme. This distortion may be required to place His-175 in a position where it can serve as the heme ligand. A similar distortion of what might hae been an a-helical turn to form two 310 turns has been found in the high potential iron protein of Chromatium where the Fe4S4 cluster is bound (26).

Cytochrome c peroxidase has very little j3-structure, con- taining only two short antiparallel pairs and one small sheet (including connecting turns) which account for less than 12% of the structure. The first antiparallel j3-pair, formed by main chain atoms of residues 58 and 63, is the result of a sharp reversal in the direction of the chain following helix B. An- other antiparallel &pair is found between residues 180 and 189. Intervening residues form a tight loop which resembles a j3-pair in general appearance but is distorted so that only one of the hydrogen bonds (Thr-180 N . . . Gly-189 0,3.0 A) expected within such a pair is observed. The distortion from expected @-pair geometry may be a result of interactions made possible between the heme propionate and this region of the protein molecule (see below).

The only j3-sheet in the molecule forms a small plate of three strands which forms one side of Domain 11. Three antiparallel strands participate in this sheet: residues 212- 214,222-225, and 229-231. Type I j3-bends (discussed below) at residues 216-219 and 225-228 reverse the direction of the chain between the strands.

To discuss hairpin turns in cytochrome c peroxidase, we invoke the nomenclature of Richardson (27), which recognizes types I, 11, IV and VI, and classifies allo turns for which the distance Can - C,n+3 is less than 7.0 A. A turn is assigned to one of the categories if the main chain torsion angles, (#, I))n+l (4, I))n+2, are within 40" of the ideal values, provided the residues are not part of the same helix. Turns identified in cytochrome c peroxidase are summarized in Table 111. Types I and 11 (and mirror images I' and 11') are the turns recognized by Venkatachalam (28), where Type I includes the 3,,,-helical turn sometimes designated Type I11 (28). Type I1 turns strongly prefer glycine in position 3, and all three Type I1 turns in the cytochrome c peroxidase molecule have glycine at this position. Type VI turns involve cis-prolines, but no cis-peptides have been identified in the molecule and no turns of this type are found. Type IV is a miscellaneous category for the classification of any other turns with two dihedral angles more than 40" away from the ideal values defining other types.

Water Molecules-A total of 263 fixed water molecules have

Refinement of Cytochrome c Peroxidase 13031 1 C I I D I W L T J lHll I I I J I

m

=IO n TABLE 111

Hairpin turns T W O Residues Swuence Hvdromn bond’

I1 11-14 I 33-36 I’ 37-40 I I

53-56

I 54-57 58-61

I 59-62 I 66-69 I I

69-72

I 70-73

I1 79-82 82-85

I 99-102 I 117-120

IV 118-121 I 134-137 I 135-138 I 146-149 I 159-162

IV I

192-195

I 194-197 200-203

I 216-219 I 225-228 I 251-254

IV I

252-255 254-257

I1 271-274 I 277-280

EKGR DDEY DNYI TSGT SGTW DKHD KHDN SYGG GTYR TYRF DPSN NAGL FPWI QEMQ EMQG PEDT EDTT DADK QRLN GAAN ANNV NEFY NDAN SKSG YAND ANDQ DQDK EDGI PKDA

011 . . . N14

037 . . . N40

058 . . . N61

070 . . . N73 079 . . . N82 082 . . . N85 099 . . . N102

0177 . . . N120

0134 . . . N137

0159 . . . N162

0200 . . . N203 0216 . , . N219 0225 . . . N228

0271 . . . N274 0277 . . . N280

a The definition of a hairpin turn and the scheme for classifying

* Hydrogen bonds are listed only if both donor and acceptor are the turns according to type are given in the text.

main chain atoms belonging to 1 of the 4 residues within the turn.

been located in the crystal asymmetric unit and their posi- tions, temperature factors, and occupancies have been refined. The majority of these molecules assume positions on the molecular surface which enable intractions with polar protein atoms. There is also a tendency for water molecules to form bridges of 2-4 molecules which span crevices on the surface, thereby enhancing the ellipsoidal appearance of the molecular surface.

One notable exception to their generalization about the solvent structure is the presence in Domain I of a narrow channel or finger of solvent that penetrates and divides the upper portion of this domain in two. At least a dozen water molecules with varying occupancies and degrees of hydrogen bonding fill this channel so that no empty space remains. The prominence of this cavity came to our attention only upon examining solvent accessibility surfaces on the graphics sys- tem. This channel may be present to provide a means of hydrating several side chains (including Glu-76, Asp-79, and Asn-82) which are directed toward the molecular interior, rather than the exterior where they might be more conven- tionally solvated. The function, if any, of this solvent channel remains obscure.

As might be expected, the large ligand access channel is also filled with solvent molecules. The variation in thermal and occupancy parameters for these water molecules suggest that molecules closest to the active site are more tightly bound to the protein, while those near the channel entrance are more anamolously associated. The rapid exchange of this solvent with potential substrate molecules might be anticipated.

Five water molecules occupy positions within the molecular interior which are not accessible via channels to the external solvent. These water molecules have unit occupancies and temperature factors comparable to the protein atoms with

IO

o s IO n L r n 1 ~ 1 w m x a a s m m RESIDUE NUMBER

FIG. 3. Isotropic thermal parameter (B) variation in cyto- chrome c peroxidase. Individual temperature factors for main chain atoms (N, C,, C, 0) of each residue are averaged and plotted. Residues in helical segments are indicated at top.

which they are associated, and must be considered as integral parts of the structure. H20-408 fills a small gap formed at the intersection of helices B and D. Hydro en bonds are observed between H20-408 and Leu-46 0 (2.9 x ) of helix B and Phe- 108 0 (2.9 A) of helix D. H,O-432 occupies a site at the N- terminal end of helix D, where it hydrogen bonds with Gly- 105 NH (2.7 A), Asp-132 0,l (2.7 A), and Tyr-67 0 (2.8 A). This water molecule helps to stabilize the turn initiating the helix while simultaneously holding elements of two flexible sections of chain (the 60s and 130s) in place. Two other water molecules (H20-725 and H20-425) form a small “hydration pocket” in the distal domain. H20-725 bridges the buried Ar - 130 guanidinium (3.0 A) and Glu-271 carboxylate (2.8 d ) while interacting with the amide NH of Arg-127 (3.0 A). H20- 425 contacts H20-725 (3.0 A) and the carbonyl oxygen of Pro- 125 (2.6 A). These water molecules presumably provide a mechanism for delocalization of the buried charges originating at the polar side chains.

The last of the buried water molecules is H20-535, which occupies a hydrophilic pocket in the interior of the heme crevice, just 3.7 A below heme pyrrole I. This water molecule helps to fulfill the hydrogen-bonding requirements of the buried carboxylate of Asp-235 (2.8 A), which in turn hydrogen bonds with Na of the proximal histidine (2.9 A).

Thermal Motion and Disorder-A detailed interpretation of the B factors as thermal motion is complicated by the inability of the crystallographic experiment to distinguish between true thermal motion and static disorder within the crystal. While these are physically distinct properties which happen to affect the scattering of x-rays in a similar way, both properties reflect an inherent flexibility of the molecular structure which may play an important role in the enzymic activity.

In Fig. 3, the average individual atomic B factor for main chain atoms (N, Ca, C, 0) is plotted uersus residue number. As expected, residues in elements of secondary structure, particularly a-helices, have the lowest B factors and are more rigid. Solvent-exposed loops and long sections of extended chain are much more flexible.

An interesting generalization results from a consideration of the spatial arrangement of the more flexible portions of the molecule. Four segments of the chain, 2-14, 58-80, 125- 145, and 272-278, all converge to form nearly half of Domain I, making this portion of the molecule quite flexible. It is interesting to note that these chain segments are precisely the ones set apart from the rest of Domain I by the solvent penetration alluded to earlier.

HEME ENVIRONMENT AND GEOMETRY

The heme in cytochrome c peroxidase is inserted into a crevice between two antiparallel helices, helix B of Dopain I and helix F of Domain 11. The heme edge is nearly 10 A from the molecular surface and, even though the propionate side

13032 Refinement of Cytochrome c Peroxidase

FIG. 4. Cytochrome c peroxidase heme propionate environment. Stereoscopic representation shows the protein groups separating the heme from the nearest molecular surface (forefront), the putative cytochrome c binding site (involving Asp-37). Heme pyrrole rings are numbered with Roman numerals (I-ZV). Hydrogen bonds are indicated with dashed ines and tetrahedra mark the position of bound water molecules.

chains are fully extended, they are prevented from making extensive contact with the surrounding medium by a rigid network of hydrogen bonds (Fig. 4). The propionate of pyrrole IV is completely sequestered behind the a-helical turn F1. As noted earlier, this turn connects two strands of an antiparallel P-pair. The propionic acid forms hy-drogen bonds with Na of His-181 (2.5 A), Na of Asn-184 (3.2 A 0, of Ser-185 (2.6 A), the backbone NH of His-181 (2.8 A. ), and a buried water molecule (H20-348, 2.8 A). These interactions may account for the unexpected geometry of the 180-189 P-pair, since the presence of the propionate may cause a departure from the normal @-hydrogen bonding pattern. The propionate of pyr- role I11 occupies the bottom of a small recess in the molecular surface. It is hydrogen-bonded to the backbone NH of Lys- 179 but otherwise is surrounded by a matrix of fixed water molecules which inhabit the surface recess. The size and rigid architecture of this recess (the 6-structure of 180-189 and a left-handed helical turn of residues 36-40) would seem to prohibit direct contact between the propionate and any macromolecule as large as the physiological redox partner, cytochrome c.

Heme geometry is summarized in Table IV. The heme is not planar but slightly distorted into a saddle shape, with pyrroles I and 111 tilted toward the proximal side and pyrroles I1 and IV tilted toward the distal side, resulting in a root mean square deviation of all porphyrin atoms from the mean plane of 0.24 A. Distortion from planarity permits an expansion of the inner core of pyrrole nitrogens to accommodate the pre- dominantly high-spin iron atom in cytochrome c peroxidase at room temperature (29). Some distortion of heme planarity is observed in most heme proteins. Oxidized tuna cytochrome c also has a saddle-shaped heme (24), while the heme in aquometmyoglobin is bowl shaped (30). The plane determined from the four pyrrole aitrogens is well defined (root mean square deviation, 0.03 A). Our previous estimate of a 0.3-0.5 A out-of-plane displacement of the iron was erroneous since distortions of heme geometry were not taken into account. A more accurate postrefinement interpretation places the iron 0.2 8, out of the pyrrole N plane (see Table IV), probably not a significant displacement since it approaches the positional error of the pyrrole nitrogens. Model heme x-ray structures of hexacoordinate high-spin complexes also show that the iron is in the plane (31).

It should be noted that no restraints were applied during refinement to enforce overall porphyrin planarity or other preconceived notions of iron-coordination geometry. Only the planes of individual pyrrole units and of the propionate car- boxylates were restrained.

The fifth and sixth coordination positions are occupied by

TABLE IV Heme geometry

Distance A

Fe to pyrrole N plane" 0.2 Fe to His 175 N. 2.0 Fe to HzO 595 0 2.4 Fe to pyrrole N (average) 2.0 His 175 N. to pyrrole N plane 2.1 HZ0 595 0 to pyrrole N plane 2.2

Angles

Pyrrole I normal to porphyrin normal 5.7 Pyrrole I1 normal to porphyrin normal 7.8 Pyrrole 111 normal to porphyrin normal 8.9 Pyrrole IV normal to porphyrin normal , 5.9 Pyrrole N normal to porphyrin normal 1.7 His 175 imidazole plane to pyrrole N normal 6.8 HzO 595 0-Fe bond to pyrrole N normal 7.1 His 175 Ne-Fe bond to pyrrole N normal 5.7 His 175 imidazole plane to 1N-3N vector 14.2

Porphyrin plane (and the normal to that plane) is computed by least squares using all 33 atoms in the porphyrin core and one atom removed from that core. Pyrrole N plane is computed using only the four pyrrole nitrogen positions.

the side chain of His-175 and a water molecule (H20-595), respectively (Fig. 5) . The imidazole nitrogen of His-175 is 0.2 A from the expected position on the normal to the heme plane. As in the globins, the imidazole plane is oriented to eclipse the IN-Fe-3N plane of the heme. This geometry is maintained in two ways: 1) owing to the position of helix F (which contributes the histidine) relative to the heme, His- 175 must eclipse the 1N-Fe-3N plane in order for N, to assume a position axial to the iron; and 2) Na of His-175 is hydrogen bonded to the buried carboxylate of Asp-235 which, in turn, is held in place by a hydrogen bond with Trp-191 (see Fig. 5). Both constraints allow little conformational flexibility to the His-175 imidazole.

The position and orientation of the proximal histidine may be important to catalysis, since it appears to constrain the high-spin iron in-plane. In the globins (30, 32), the proximal histidine is oriented in a way which permits side chain move- ment directly away from the iron via rotation about the C,- CB bond. This type of coordination sphere expansion is not possible in cytochrome c peroxidase, since the histidyl side chain approaches the iron atom so that the C,-C@ bond is parallel to the heme plane (Fig. 5). Rotation about the C,-Cp (or any side chain dihedral angle) can only produce N, move- ment parallel to the heme plane. This architecture, which holds the iron atom in the pyrrole plane, could assist in

Refinement of Cytochrome c Peroxidase 13033

FIG. 5. Cytochrome c peroxidase heme crevice. Stereoscopic view of the enzyme active site. Important amino acids and bound water molecules on the distal (top) and proximal sides (below) of the heme are labeled. Distal side H20- 596 has been omitted from the figure, as its position is almost directly in front of the aguo ligand (H20-595). Hydrogen bonds connecting the sixth coordination position to the cytochrome c binding site (via H20-648, Arg-48, H20-348, pyrrole IV propionate, and His-181) are evident.

forming Fe(1V) of Compound I since low-spin Fe(1V) prefers the in-plane geometry.

The axial water ligand is displaced laterally away from the heme normal toward pyrroles I and IV, so that it remains nearly colinear with the Fe-N, bond. This displacement re- lieves steric crowdin around the aquo ligand, which is man- ifest by a close (2.7 R ) interaction with the indole nitrogen of Trp-51. This distortion from axial geometry, coupled with the similarly displaced proximal histidine, may account for the larger rhombic component obsrved in the EPR signal of cytochrome c peroxidase as compared to the globins (33).

The environment on the distal side of the heme, where the peroxide substrate must bind, is formed by 3 residues all contributed by one turn of helix B: Arg-48, Trp-51, and His- 52 (Fig. 5). The indole ring of Trp-51 is 3.3 A above and parallel to the most buried surface of the heme and is hydro- gen-bonded to the aquo ligand (Hz0-595, 2.7 A). Arg-48 extends across the top of the heme in the other direction to form part of a wall of the ligand-access channel. The side chain hen+ upward at the guanidinium group to hydrogen bond (2.7 A) with a highly ordered water molecule (H,O-348) situated between the two heme propionates. The imidazole ring of His-52 is nearly perpendicular to the heme and directly abov? the sixth coordination position. N. of this histidine is 3.2 A from the aquo ligand but the geometry for hydrogen bonding is not good, since the Ne-H bond is directed toward an unoccupied position in the solvent-access channel and not toward the heme ligand.

Two other active-site water molecules have been located as a result of the refinement. H20-648 hydrogen bonds to the aquo ligand (3.2 A) and the Arg-48 guanidinium (H20-648 . . . N,, 2.5 A; H20-648 . . . Nhl, 2.5 A). H20-596 (omitted from Fig. 5 for clarity) is the third species to form a hydrogen bond to H20-595. It too is close enough (2.9 A) to interact with N, of His-52, but, again, the geometry for hydrogen bonding is not good. The significance of the histidine orientation will be discussed below.

It is highly probable that a similar distal environment exists in plant peroxidases as well, since Arg-48, His-52, and an aromatic amino acid at position 51 are conserved in all se- quences so far elucidated (9).

STRUCTURAL BASIS FOR ENZYMIC ACTIVITY

Formation and Structure of Compound I-The mechanism for the formation of Compound I, proposed on the basis of solution studies and the unrefined m.i.r. model (9), remains

consistent with the above description of the enzyme’s active site. We envision that the peroxide substrate R-01-02-H‘ enters the active site through the access channel, displacing the aquo ligand at the sixth coordination site, H20-595, and two other distal-side water molecules, H,O-648 and H20-596, to form a Michaelis-Menten comlex (as yet unobserved ex- perimentally). This short-lived intermediate would contain an ionized peroxide molecule, R-01-02-, covalently bound to the heme iron a t 02 and would be formed with concomitant transfer of the 0 2 proton to His-52. The precise orientation for the peroxide substrate suggested by the geometry of the active site is with 01 occupying a position exactly between H20-648 and H20-596 in the native enzyme, so that 01 is directly below and hydrogen bonded to N, of His-52. In the next step, the negative charge on 0 2 is transfered to 01, resulting in the eventual heterolytic cleavage of the 01-02 bond. The presence of the positively charged Arg-48 guanidin- ium group at the active site promotes this electronic rear- rangement within the dioxygen complex by stabilizing the negative charge on 01. The distal histidine can then return its proton to the substrate, this time at 01, to complete the heterolytic cleavage of the 01 -02 bond. The R-01-H leaving group then diffuses away from the active site.

The importance of Arg-48 to the heterolytic cleavage of the 0-0 bond should be emphasized. The presence of a positively charged side chain to promote charge separation in the tran- sition state is essential. We believe that the absence of such a group in the Fe(II1) globins accounts, at least in part, for the failure of this class of proteins to promote efficient cleav- age of H20a despite the otherwise close resemblance of the distal-side environment of the heme in the globins to that of cytochrome c peroxidase. The Arg-48 side chain in the native enzyme is, however, directed away from the sixth coordination position along the side of the ligand access channel, with NhZ donating a hydrogen bond to a bound water molecule (H,O- 348). It is important to note, therefore, that the ability of the Arg-48 side chain to shift toward the active site in the presence of an anionic ligand has been demonstrated crystallographi- cally (34). This small conformational change enables both Ne and Nhl of the guanidinium group to interact with 01 in a manner mimicked by bound HzO-648 in the native structure.

We suggest that His-52 plays a critical role in effecting this conformational change during operation of the catalytic mechanism. By abstracting a proton from 0 2 of the iron-

“The numerical designations (1 and 2) on the oxygen atoms of hydrogen peroxide are used to provide unambiguous identification.

13034 Refinement of Cytochrome c Peroxidase

bound substrate, His-52 generates an anionic ligand at the sixth coordination psotion and precipitates the slight inward shift of the arginine side chain. Deprotonation of the peroxide (pK, - 12) by histidine (pK, - 6) need not be difficult as iron-peroxide chelation weakens the 02-H bond. Relevant to its role as acidbase catalyst, it should be recalled that the hydrogen-bonding geometry between His-52 and the 0 2 is somewhat strained. His-52 is ideally positioned, however, to interact with 01 of the peroxide substrate. This arrangement favors the transfer of a proton from 0 2 to 01 and is reminis- cent of the serine proteases, where the active-site histidine in the tetrahedral intermediate forms a better hydrogen bond to the leaving group which is to be protonated than to the catalytic serine side chain (35). Upon protonation of 01, the arginine is freed to return to its initial conformation, hydrogen bonded with H20-348, while the leaving group can be dis- placed by the normal flow of solvent molecules into and out of the access channel.

Following the sequence of reactions envisioned above, the activated enzyme is isoelectronic with Compound I. Formally, the iron atom is in the Fe(V) state, 2 oxidizing equivalents above the native enzyme. As stated earlier, cytochrome c peroxidase is unique among peroxidases in that it is capable of an electronic rearrangement of this species which results in the oxidation to a free-radical state of an amino acid side chain. The identity of this amino acid has been the center of considerable debate and we had hoped that the refined crystal structure would allow an unambiguous identification, on geo- metrical grounds, of the side chain on which the free radical must reside. Unfortunately, this is not the case. TWO side chains must be seriously considered as candidates. The first, Trp-51, was suggested originally on the basis of the correlation of the m.i.r. model with the available spectral and biochemical data (9) and remains a possible candidate. The indole nitrogen of Trp-51 is in intimate contact (2.7 A) with the sixth coor- dination position and it is easy to imagine the abstraction of a hydrogen atom by the oxene-like heme-bound oxygen atom to give a tryptophanyl radical. The choice of tryptophan is consistent with earlier EPR data (4) and accounts for the failure of other peroxidases to form this radical, since in all plant peroxidases for which the sequence is known, this residue is a phenylalanine (9). The x-ray structure of catalase (36) reveals that a phenylalanine is similarly positioned.

The other likely candidate is Met-172. Hoffman and CO- workers (37,38) have carried out extensive EPR and ENDOR studies on Compound I at liquid helium temperatures and have concluded that an aromatic radical, particularly trypto- phan, is inconsistent with the observed magnetic properties of the radical center (37). Comparison with model compounds has led these authors to suggest that a nucleophilically sta- bilized sulfur radical is more reasonable (37, 38). The most likely candidate is Met-172 which lies just below the heme (see Fig. 5). The sulfur of this methionine is close enough to (3.7 A) and correctly oriented for extensive d-orbital overlap with the porphyrin ?r-cloud. This assignment can also account for the failure of plant peroxidases to form the protein free radical, since preliminary sequence alignment suggests that none of these other enzymes have a methionine in the heme crevice (22).

An important requirement of a methionyl radical is the need for stabilization by a closely associated nucleophile (37). The nearby carboxylate of Asp-235 could fulfill this require- ment and it has been proposed that one-electron oxidation of Met-172 results in a shift of the carboxylate toward the sulfur cation (38). In light of the crystal structure, it is more likely that a methionyl cation woud shift toward Asp-235. By simple

rotation about the C&, bond of the methionine side chain, the sulfur atom can be repositioned to within 3.0 A of the Asp-235 carboxylate without encountering any major steric barriers. If such a conformational change does occur, it should be easily observed by difference Fourier analysis of Compound I itself.

Biological Control of Heme Chemistry-The hypothetical mechanism described thus far addresses the problem of 0-0 bond cleavage, but, as yet, we have not considered the role of the protein tertiary structure in modulating the chemistry of the heme moiety. The formation, at least transiently, of an Fe(V) species in the reaction pathway implies the existence of some structural feature(s) which can stabilize higher oxi- dation states of the heme. The same feature(s) presumably can account for the low oxidation-reduction potential (E'o = -194 mV) of the Fe(II)/Fe(III) coule in cytochrome c perox- idase (39).

Peisach (40) has proposed that interactions between the proximal imidazole (His-175 in cytochrome c peroxidase) and other protein groups may influence the electronic properties of the axial ligand, and, therefore, the reactivity of the heme iron. This proposal has been tested using model compounds. Mincey and Traylor (41) have demonstrated that model por- phyrins complexed with anionic axial ligands are more sen- sitive to oxidation than the same porphyrins complexed with neutral ligands. Fujita et al. (42) have observed significant spin delocalization to the axial ligand in synthetic models of ferry1 porphyrin *-cation radicals, confirming that axial li- gands do participate in the delocalization of the iron formal charge.

In cytochrome c peroxidasf,we find Na of the proximal histidine in close contact (2.9 A) with the carboxylate of Asp- 235 (see Fig. 5). The Asp side chain is buried and inaccessible to external solvent, and is also hydrogen bonded to the Trp- 191 side chain (2.5 A) and the internal water molecule (HzO- 535, 2.8 A). For purposes of comparison, it should be noted that in other heme proteins for which structural data are available (except cytochrome c ' (43)), the proximal histidine is hydrogen bonded to a peptide backbone carbonyl oxygen (44). These heme proteins, in contrast to cytochrome c per- oxidase, have positive reduction potentials ranging from 20 to 320 mV (45). A strong hydrogen bond between the proximal histidine and a buried carboxylate should impart greater anionic character to the imidazole than can be achieved by hydrogen bonding to a peptide carbonyl oxygen, and this anionic character should in turn aid in the stabilization of higher oxidation states of the heme.

The potential importance of the Asp-235 . . . His-175 inter- action may be evidenced by the observed pH dependence of the rate of formation of Compound I. It has been demon- strated that a protein group with pK, = 5.5 must be unpro- tonated to realize the maximum rate of reaction with hydrogen peroxide (46), and we believe this group is Asp-235 (34). Protonation of the Asp-235 carboxylate would weaken or disrupt the hydrogen bond to His-175 and slow reaction, since anionic character imparted to the histidine by the unproton- ated carboxylate would be lost. A pK, of 5.5 would be some- what high for an exposed aspartic acid but comparable to pK, values determined for two buried carboxylates in the active site of lysozyme (47).

The present crystal structure suggests that other processes may influence heme reactivity as well. Kassner (48, 49) has proposed that the polarity of the heme environment may dictate oxidation-reduction properties. The presence of the Arg-48 side chain in the active site, coupled with the accessi- bility of the heme by external solvent via the ligand access

Refinement of Cytochrome c Peroxidase 13035

channel, makes the heme crevice in cytochrome c peroxidase more polar than in either the globins or the cytochromes C. Takano and Dickerson (50) have examined .the structural effects of oxidizing tuna ferrocytochrome c to ferricytochrome C. They observe movement of an internal water molecule 1.0 A toward the heme following oxidation and argue (after Kas- sner (48)) that the increased polarity of the heme environment helps to stabilize the positive charge on the heme iron. A water molecule (H20-535) buried in the interior of the heme crevice (Fig. 5) in cytochrome c peroxidase may play an analogous role. Since the oxidation state of the native enzyme is Fe(III), the water is properly placed (just 3.6 A below pyrrole I) to help stabiize both the native enzyme and high-oxidation- state intermediates.

Electron Transfer-Following the formation of Compound I, the peroxidase catalytic cycle is completed by two-electron reduction of the activated complex to regenerate the native enzyme. This is accomplished by two sequential one-electron- transfer events in which ferrocytochrome c is oxidized to ferrocytochrome c (1). The two individual electron-transfer steps were thought to be identical, with the same Fe(1V) form of the heme serving as recipient of the transferred electron (1). However, recent kinetic data (38) indicate that the situ- ation is more complex and identification of the enzyme inter- mediate involved will require further study. The role of the enzyme in mediating the electron transfer is, we believe, to provide specificity, ensuring that only the intended redox partner is oxidized, rather than to accelerate an already nat- urally fast process (51).

Poulos and Kraut (10) have proposed a hypothetical model for the cytochrome c peroxidase:cytochrome c electron-trans- fer complex, based on model-building experiments using the prerefinement peroxidase model and the structure of tuna cytochrome c (52). With refinement of both of these structures now complete (24), a re-evaluation of this model complex has been made (22) to detail any changes necessitated by correc- tions to the protein structures. Despite important corrections to our original cytochrome c peroxidase model, no substantial modifications to the hypothetical model complex are neces- sary. Additionally, recent chemical modification (53) and cross-linking data (54) lend further support to the hypothet- ical model.

The model complex is based upon the observation that certain acidic side chains on the surface of cytochrome c peroxidase (Asp-33, Asp-34, Asp-37, and Asp-217) are posi- tioned to interact with a complementary set of lysine residues on the surface of cytochrome c. In the resulting model com- plex, the two hemes are parallel and as close to each other as the two structures will permit without causing severe confor- mational changes. As noted earlier, cytochrome c peroxidase residues 181-185 wrap around one of the propionates of the cytochrome c peroxidase heme, preventing direct contact be- tween that heme and the heme of the complexed cytochrome c. The closest approach of the two heme edges is approxj- mately 18 A, and the two iron atoms are separated by 25 A. The cytochrome c peroxidase:cytochrome c complex thus con- stitutes a detailed model for a biologically important long- distance electron-transfer complex.

Tunneling is often the preferred mechanism for long-dis- tance electron-transfer reactions (55). Parameters affecting tunneling rates include the distance between redox centers, height of the potential barrier, and the extent of vibronic coupling between hemes (56). Additionally, recent spectro- scopic evidence on the diheme nitrite reductase from Pseu- domonas aerugimsa indicates that heme-heme orientation is also quite important (56). It appears from the cytochrome c

peroxidase:cytochrome c model, as well as from other hypo- thetical models (57-59), that complementary surface inter- actions are indeed designed to bring the hemes into parallel alignment.

An important question left unanswered, however, is whether or not heme alignment alone is sufficient when the distance between the hemes is large. It may be that the bridging protein groups also participate in the electron-trans- fer process. The cytochrome c peroxidase:cytochrome c model complex suggests that such a bridge may play a role. An intriguing system of hydrogen bonds connects the distal side of the cytochrome c peroxidase heme with the surface of the molecule at the proposed cytochrome c binding site. Central to this system is His-181, the imidazole ring of which occupies a niche between the two hemes in the model complex. More- over, the imidazole ring is parallel to the two heme planes. His-181 lies in a particularly unusual environment in that it is surrounded by carboxylates (Fig. 4). The imidazole interacts with the pyrrole XV propionate (N, . . . 02, 2.5 A) and the surface aspartic acid-37 (Oal . . . Nal, 2.6 A). Asp-37 has been implicated both in earlier model-building experiments (10) and solution studies (54) as the participant in an ionic inter- action with Lys-13 of cytochrome c. It may be that this network of interactions, coupled with the previous identifi- cation of a hydrogen-bonding link between the heme propion- ates and the sixth coordination position via Arg-48, consti- tutes a conduit to assist in and control the electron-transfer process. Such a network of X-* interactions and hydrogen bonds may enhance the rate of electron transfer by lowering the potential barrier between the two hemes. More impor- tantly, the conduit might provide direction to the tunneling event to ensure that the electron is preferentially funneled into the peroxidase porphyrin.

While the above arguments are somewhat speculative, the hypothetical model complex and the involvement of the heme- heme conduit in the electron-transfer process are supported by the available experimental evidence (22). Moreover, Bos- shard et al. (60) have studied the photooxidation of the cytochrome c peroxidase histidines in the presence and ab- sence of cytochrome c and have obtained data which supports our assertion that His-181 is at the cytochrome c peroxi- dase:cytochrome c interface and that His-181 is likely to play a critical role in the electron-transfer process.

McGourty et al. (61) have recently demonstrated that long- distance through-protein electron transfer can be effected between hemes. These authors report rapid electron transfer between two hemoglobin subunits in which one heme has been replaced by a high-potential zinc protoporphyrin. The distance between redox centers in this artificial electron- transfer complex (24.7 A) is the same as in the cytochrome c peroxidase:cytochrome c complex and transfer occurs at near enzymatic rates without the presence of any specialized con- duit between them. We would emphasize, however, that the postulated conduit in the cytochrome c peroxidase-cyto- chrome c complex proposed above is not present to enhance the rate of transfer so much as to provide directional regula- tion. It is significant that McGourty et al. (61) also observed a competing spontaneous anomalous discharge of the oxidiz- ing equivalent of just the sort which could, presumably, not be tolerated in a physiological system. A conduit of the kind we describe may be precisely what is required to prevent such nonspecific electron transfer.

Acknowledgment-We are indebted to Dr. Stephen Edwards, who collected the diffraction data for this study.

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