Assignment of hyperfine shifted resonances in high-spin forms of cytochrome c peroxidase by reconstitutions with deuterated hemins

Biochimica etBiophvsica Acta, 743 (1983) 246 255

246

Elsevier Biomedical Press

BBA31546

A S S I G N M E N T OF HYPERFINE S H I F T E D R E S O N A N C E S IN H I G H - S P I N F O R M S OF C Y T O C H R O M E c PEROXIDASE BY R E C O N S T I T U T I O N S W I T H DEUTERATED H E M I N S JAMES D. SATTERLEE ~,*, JAMES E. ERMAN

b GERD

N. LaMAR ~, KEVIN M. SMITH " and KEVIN C. LANGRY ~

Department of Chemistry, University of New Mexico, AIbuquerque, N M 87131, h Department of Chemistry, Northern Illinois University, DeKalb, IL 60178, and ~ Department of Chemistry, Universi(v of California, Davis, CA 95616 (U.S.A.) (Received September 21st, 1982)

Key words." Cytochrome c peroxidase; Hyperfine shift; Protohemin IX," NMR," (Saccharomw'es cerevisiae)

Assignments of hypedine shifted proton resonances for the high-spin forms of cytochrome c peroxidase (EC I.II.I.5) have been made (cytochrome c peroxidase, cytochrome c peroxidase-F) employing the technique of reconstituting the apoprotein with specifically deuterated protohemin IX derivatives. The results show that the heme methyl group pattern differs significantly from similar assignments made for metmyoglobin. In cytochrome c peroxidase the methyl pattern is 5 > I > 8 > 3. For cytochrome c peroxidase-F the pattern is 5 > 8 > 1 > 3, but the resonances are not shifted as far downfield and they exhibit a narrower spread. For myoglobin the relative methyl ordering has previously been shown to be 8 > 5 > 3 > I. Several conclusions have been reached, including confirmation of the essential correspondence between the solution- and crystal-derived data for several heme crevice structural features. The pH dependence of the cytochrome c peroxidase-F methyl resonances is also presented and is shown to differ from native peroxidase. For cytochrome c peroxidase-F smooth, continuous titrations are observed with no evidence of the second conformation which was found for the native enzyme.

Introduction

In paramagnetic heine proteins the proton hyperfine resonances can be valuable, non-invasive, indicators for molecular-level events which occur in the heme crevice, at the protein active-site. Such utility is a direct consequence of the fact that the hyperfine resonances are shifted away from the complicated and often featureless protein envelope which dominates the diamagnetic portion of the proton spectrum. In general, the shifts are caused by heme-centered paramagnetism with two contributing mechanisms; the dipolar (pseudocontact) shift depends upon the existence of magnetic ani-

* To whom correspondence should be addressed. Abbreviation: DSS, 2,2-dimethyl-2-silapentane-5-sulfonate. 0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

sotropy; the contact shift depends upon spin delocalization via metal-ligand covalency [1]. However, for high-spin ferric heme proteins such as native cytochrome c peroxidase and cytochrome c peroxidase-F magnetic anisotropy is negligible, so that the observed resonances are due primarily to the contact shift. This result dictates that only protons originating from groups directly bonded to the heroin iron may experience substantial hyperfine shifts. The important prerequisite for deriving quantitative information is successful, correct assignment of the hyperfine resonances. Once unambiguous assignments have been made, conclusions can be drawn about hemin electronic structure [2], specific solution conformation [3,4], structure within the active site [5] and molecular-level fluctuations accompanying reactivity [6]. Much work has been

247 devoted to this end, but generally using indirect methods, and the results have been only partially successful [2,7,8]. A more direct approach has been to reconstitute b-type heme proteins with protohemin IX derivatives in which protons of the peripheral groups have been selectively deuterated [3-5,9-11] (Scheme I). Since protohemin IX is the I1,.t

H

.'C.

H

I ~H

H I

a2C=C

/

H

CH3. o

H

H3

,CHz

" Hz CO).

H

CH:,

cz

CO~

(;H2

extensive proton N M R studies have indicated interesting molecular-level fluctuations within the cytochrome c peroxidase heme crevice for a variety of ligation states (Satterlee and Erman, unpublished results; and Refs. 16-18). Interpretation of these events requires unambiguous assignments. Such assignments can be compared with horseradish peroxidase and evaluated in terms of the functional similarities or differences between the peroxidases. These assignments may eventually be useful for quantitating solution structural differences between the class of native ferriheme proteins and the class of native ferroheme proteins. Finally, cytochrome c peroxidase is the only member of the heme peroxidase class which, to date, has had its crystal structure reported. This final point allows the solution structural inferences made from the N M R assignments to be tested against known solid-state structure data. Methods

Scheme I naturally occurring prosthetic group in b-type heme proteins, deuteration is not expected to perturb the heme crevice structural integrity in comparison to native, unreconstituted proteins. This technique has proved valuable for making assignments in heme-globins [3-6,8], horseradish peroxidase [9] and the low-spin forms of cytochrome c peroxidase [12,13]. Here we present similar results for native cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) and cytochrome c peroxidase-F. Cytochrome c peroxidase is one of the peroxidase class of ferriheme enzymes. Its properties differ significantly from the other, extensively studied member of this class, horseradish peroxidase. The most notable difference is the fact that the oxidized intermediate, horseradish peroxidase-I, is a berne-centered radical, whereas cytochrome c peroxidase-I (compound ES) forms an amino acid based radical. Moreover, the horseradish protein readily binds several ligands which cytochrome c peroxidase does not. The enzyme's cellular function in yeast is to catalyze the hydrogen peroxide oxidation of ferrocytochrome c [13,14]. This work was stimulated by the fact that

Cytochrome c peroxidase was isolated from baker's yeast as previously described [13,14]. The deuterated hemins have been previously reported [4,19,20] as well, and are derivatives of protohemin IX (Scheme I) with abbreviations as follows. Protohemin IX with methyl protons at hemin positions 1 and 5 replaced with deuterium (2H) is designated [ 1,5- 2H6]hemin; deuteration of methyls 1 and 3 is [1,3-2H6]hemin. For deuteration of the vinyl group protons, we abbreviate: 13 methylene protons yield [2,4-13-2H4]hemin; a protons give [2,4-t~- 2H 2]hemin; perdeuteration of vinyl 4 yields [4-: H 3]hemin. Reconstitutions were carried out according to the method of Yonetani et al. [21-23] with the following modifications. The apoprotein was generated at 0 - 4 ° C with acidified butanone. Usually a 1-4 mM solution of apocytochrome c peroxidase (either freshly prepared or previously lyophilized) in a dilute potassium phosphate buffer (Mallinckrodt; 0.02-0.04 M, pH 7.0-7.5) was cooled on an ice bath. A 10% mole excess of hemin dissolved in a minimal amount of 0.1 M NaOH was added to the apoprotein solution and allowed to react for 15-30 min. Enough potassium phosphate was added to make the solution 0.5 M in phosphate and to adjust the pH to 6.0. The reaction mixture

248 was subsequently passed through a 1 x 10 cm DEAE-cellulose column equilibrated at pH 6 in a 0.5 M potassium phosphate buffer. The excess hemin and any denatured enzyme adsorbed to the column while the reconstituted enzyme passed through. For hemins in small supply (and control experiments with protohemin), a slightly less than stoichiometric amount of the hemin was added to the apoenzyme and the column step eliminated. The reconstituted enzyme from either procedure was then exchanged with ZH 2 0 solutions of an appropriate buffer for N M R experiments and concentrated, if necessary, by ultrafiltration. To minimize the number of manipulations, lyophilized apocytochrome c peroxidase could be dissolved directly into buffers made with 2H20 and reconstituted with heme dissolved in a minimal amount of 0.1 M deuterated potassium hydroxide. After reconstitution, the sample could be adjusted to the desired pH and used for N M R studies. All reconstitution procedures with protohemin IX gave products with identical N M R spectra between pH 7 and 7.5. Cytochrome c peroxidase samples were prepared with Merck 99.8% 2H20. Samples were ultimately unbuffered in 0.1 M KNO 3, the phosphate buffer removed by ultrafiltration. Solution pH was continuously monitored throughout sample handling and maintained within the region pH 7 to 7.5. Cytochrome c peroxidase-F was obtained simply by dissolving a 90-times mole excess of NaF (Alfa) in the solutions of native or reconstituted cytochrome c peroxidase. Proton N M R spectra were recorded on a Nicolet 8.45 Tesla instrument (360 MHz proton frequency) at the University of California, Davis, N M R Facility, or on a Nicolet 11.05 Tesla spectrometer (470 MHz proton frequency) at the Purdue University Biochemical Magnetic Resonance Laboratory. Instruments were operated with quadrature phase detection and decoupler suppression of the residual water resonance. Between 5000 and 1 000000 transients were required for each spectrum. Samples were run at 2 2 + I°C, unless otherwise indicated in the figure captions. pH values were measured before and after each spectrum using an Orion meter with a Beckman combination pH electrode which was standardized immediately before each use.

Results are reported as either observed or hyperfine (isotropic) shifts. The form is stated in the text or in figure and table legends. Observed shifts were calculated by referencing each resonance to the residual water peak, then reporting each position relative to external DSS (2,2-dimethyl-2-silapentane-5-sulfonate). This has been done because DSS has recently been shown to bind to proteins, so that its resonance position is actually not constant [24]. Hyperfine shifts are calculated by referencing the observed shifts to their corresponding positions in the diamagnetic zinc porphyrin [25]. Due to the broadness of these resonances the estimated maximum error in reproducing all shifts reported here is + 0.1 ppm. A nonlinear least-squares fitting program written by Darrow E. Neves was used to determine pK values from pH titration data for cytochrome c peroxidase-F. Theoretical lines from the Henderson-Hasselbalch equation were generated by a program written by J. Timothy Jackson. All computing was carried out on a Nicolet 1180 computer equipped with a Nicolet Zeta Plotter. Results and Discussion

A. Methyl resonance assignments Reconstituting apocytochrome c peroxidase with deuterohemin IX results in spectral duplication characteristic of heme-centered asymmetry (Satterlee and Erman, unpublished results; and Ref. 16) and makes it necessary to determine whether reconstitutions with protohemin IX would cause similar complications for this study. Such precaution was required prior to embarking upon reconstitutions with the specifically deuterated protohemin IX derivatives. As shown in Fig. 1, protoheroin IX-reconstituted peroxidase exhibits a spectrum identical to that of the native protein. Because identical hyperfine resonance patterns are observed in the two species, it can be concluded that the heme rotation geometry about the a-~ pseudo C 2 axis (Scheme I) is the same in each and that there is essentially a single form of the protein present in the protohemin reconstituted protein. Further, because the deuterium-for-proton substitution is a very minor perturbation, this result implied that only single-form, native-like structures would result from reconstitutions with

249

CCP CCP-F

c ''1

....

~ .... 60

7O

] .... 50

I .... 40

I .... 30

i .... 20

I 0

I 10

. . . . . . . . -rig

-2JO

CCP B •

,

•

,

"

,

~o I

]

eo

.

.

,

,

'r;o'

.

b

c

.

--r

.

.

I . . . .

7o

''~0 . . . . . . .I .0

";0

;0'

d

I . . . .

6o

k

I

5o

. . . .

~ . . . .

~o

I

3o p p M o

I

.

.

.

.

.

.

.

]lo

Fig. 1. (A) 470 MHz proton hyperfine N M R spectrum of native cytochrome c peroxidase; (B) apocytochrome c peroxidase reconstituted with protohemin IX; and (C) 360 MHz spectrum of cytochrome c peroxidase-F. Conditions: 22°C, 0.1 M KNO 3, pH (A) 7.55, (B) 7.2, (C) 6.95. The major protein envelope in the diamagnetic region has been omitted. Note different scales for A - C .

specifically deuterated protohemin IX derivatives. Therefore, unique, unambiguous assignments could be expected, which would directly correlate to the native enzyme. The native peroxidase spectrum and that of cytochrome c peroxidase-F (Fig. 1) are similar to those of other high spin ferric heme proteins. Downfield, the spectrum consists of four peaks with relative intensities corresponding to three protons each, accompanied by several overlapping single proton resonances [17]. The four three-proton intensity resonances correspond to the peripheral heme methyl groups (Scheme I). Reconstituting the protein with [1,5-2H6]hemin and [1,3-2H6]hemin results in assignment of the individual hemin methyl resonances. For native peroxidase, Fig. 2 illustrates these assignments, while similar results for cytochrome c peroxidase-F are shown in Fig. 3. In Fig. 2A, a trace of the methyl region in the unreconstituted enzyme shows that four methyl resonances are present. Fig. 2B and C shows the changes which occur when the apoprotein is reconstituted with the pairwise methyl-deuterated hemins. The peaks which decrease in intensity are attributed to deuteration of the 1,5 and 1,3 methyl groups, respectively. The relative intensity of the peak due to the methyl group at heme position 8 does not change, so that

F 90

. . . .

I 80

.

.

.

.

]

.

.

.

70

.

I 60

PPM

Fig. 2. Proton assignments of the home methyl groups in cytochrome c peroxidase at 360 MHz, 25°C and 0.1 M KNO 3. (A) Cytochrome c peroxidase (native), pH 7.5; (B) [I,52 H 6]hemin-reconstituted cytochrome c peroxidase, pH 7.2; (C) [ 1,3- z H 6 ]hemin-reconstituted cytochrome c peroxidase, pH 7.5.

the assignments are unambiguous. The extent of deuteration for methyl group 1 is not as great as for groups 3 and 5, causing residual proton intensity to appear at that position. Nonetheless, its

CCP-FLUOR

80

] DE

70

80

50

~0

PPH

Fig. 3. Proton assignments of the heine methyl groups in cytochrome c peroxidase-F at 360 MHz, 0.1 M KNO 3, 25°C. (A) Cytochrome c peroxidase-F, pH 7.55; (B) [l,5-2H6]hemin-re constituted cytochrome c pero×idase-F, pH 7.65; (C) [l,32H6]hemin-reconstituted cytochrome c peroxidase-F, pH 7.40.

250 relative intensity decrease confirms its assignment. The resonance assignments are in the order 5 > 1 > 8 > 3 reading from low field moving upfield. Fig. 3 provides similar data for cytochrome c peroxidase-F. Fig. 3 A - C shows the effects of reconstitution with the 1,5- and 1,3-methyl-deuterated heroins. The resonance pattern for cytochrome c peroxidase-F extends over a narrower range and the methyl group ordering is 5 > 8 > 1 >3.

A:

CCP

lOFO ' ' '910 . . . .

B. Single-proton resonance assignments Due to the greater broadness of cytochrome c peroxidase-F resonances and their high degree of overlap, single-proton assignments were made only for the native protein, the resonance lines of which are narrower and the degree of overlap less. Three hemins with selectively deuterated single-proton peripheral substituents were available for this study (See Methods). Work with isolated hemins as models for heme proteins led us to expect that, of the available deuterated groups, only the vinyl alpha protons at positions 2 and 4 would potentially be detectable in the downfield region [20]. Fig. 4 shows the results of reconstituting apocytochrome c peroxidase with each of these hemins in the downfield hyperfine spectrum. Fig. 4A is the native enzyme's spectrum. 4B indicates no relative intensity changes occur upon reconstitution with [2,4-fl-2H4]hemin. 4C shows that the two single-proton resonances indicated by arrows, display intensity decreases upon reconstitution with [2,4-a-ZHa]hemin. They are assigned to alpha-vinyl protons of positions 2 and 4. Discriminating the two, as shown in Fig. 4D, indicates that when the vinyl group at position 4 is perdeuterated the resonance near 47 p p m remains absent from the spectrum and can be assigned as the 4-alpha-vinyl proton resonance. The resonance near 38 p p m is due to the 2-alpha-vinyl proton. Assignment of protons upfield from the diamagnetic region is complicated by the fact that the reconstituted protein solutions were 5-10-times more dilute than the native solution with which they were compared. This situation introduces a major complicating factor in the upfield shift region where the single-proton resonances lie close to the diamagnetic envelope. The result is loss of resolution in the region 0 to - 5 p p m upfield. This

810 . . . .

710 . . . .

610 . . . .

SIo . . . .

L{IO' ' ' ~ 0

ppr-1

Fig. 4. Single hemin proton assignments for cytochrome c peroxidase at 360 MHz, pH 7.1-7.5, 25°C. Downfield proton hyperfine shift region between 32 and 56 ppm, of cytochrome c peroxidase reconstituted with various deuterated hemins: (A) Cytochrome c peroxidase; (B) [2,4-fl-2H4]hemin cytochrome c peroxidase; (C) [2,4-a-2H2]hemin cytochrome c peroxidase; (D) [4-2H 3]hemin cytochrome c peroxidase.

is caused by wings of the residual water peak extending into the upfield hyperfine region so that single-proton resonances appear superimposed on the broad envelope. This is demonstrated in Fig. 5, which depicts the single-proton assignments in the upfield region. In the native spectrum (Fig. 5A) four peaks are resolved, although resolution of the two further downfield is somewhat lost in spectra of the dilute reconstituted forms. The reconstitu-

I /\/\\

- ~ D:- 4.~ d3

I /\/'. / \ 4\/~C:

I

0

'

'

-~

~

-

llO

,\ ~ ...........

~j

2,4.d2

I

-20

PPId

I

-30

-"lO

Fig. 5. Upfield single-proton assignments for cytochrome c peroxidase at 360 MHz, 25°C, 0.1 M KNO3. A, B, C and D are the same as described in the caption for Fig. 4.

251

tions (Fig. 5 B - D ) show that the relative intensities of the resonances at - 8 and - 1 1 ppm decrease when the four beta vinyl protons are deuterated (Fig. 5B). When only the 4-vinyl is perdeuterated (Fig. 5D) the peak at - 8 ppm maintains the lower intensity, leading to its assignment as a 4-vinyl beta proton resonance. The peak at - 11 ppm is, therefore, a 2-vinyl beta proton resonance. All resonance assignments are given in Table I. In the downfield hyperfine region this deuterium labelling has resulted in assigning six of the ten resolved resonances. The remaining four resonances (e, f, h, i in Fig. 1) consist of a pair of single protons (f, h) and a pair with integrated intensity of two protons each (e, i). This makes six protons which remain to be assigned, with the following hemin positions as their possible origins: meso protons and the propionic acid a- and flmethylene protons. Each of these three positions consists of four protons. On the basis of their proximity to the iron ion the m e s o protons are expected to display a linewidth much broader than peaks e, f, h, i. This is due to the fact that magnetic relaxation in protein forms such as these is dominated by a distance-dependent relaxation term, leading to extreme broadening for protons closer to the ferric ion [11,21]. For metmyoglobin

the m e s o protons exhibit linewidths near 2000 Hz [19] and are expected to be much broader in proteins of higher molecular weight, such as cytochrome c peroxidase and cytochrome c peroxidaseF. Similarly, high-spin hemin models predict propionic acid a-methylene protons to resonate between 30 and 50 ppm downfield, while propionic fl-methylene protons generally resonate upfield [20]. We note, however, there is one instance where one beta resonance has been observed to lie downfield [19]. Therefore, four or five of the six remaining proton resonances can be assumed to originate from these methylene positions. The remaining one (or two) resonance must be assigned to an amino acid. The origin of hyperfine shifts in ferric high-spin proteins is primarily the contact shift mechanism [11], with the result that only groups directly bound to the iron ion can experience large shifts. Consequently, another source for protons in this region of the spectrum could be the proximal histidine, which is the only amino acid covalently linked to the hemin iron ion. Because the linewidths of all protons e, f, h, i are similar, at maximum, two protons in this group could be potentially assigned to the proximal histidine diastereopic methylene pair which lie at a separation from the iron ion nearly identical to

TABLE I PROTON RESONANCE ASSIGNMENTS IN CYTOCHROME c PEROXIDASE A N D CYTOCHROME c PEROXIDASE-F Shifts in ppm at 25°C, referenced to residual HEHO, reported relative to external DSS; linewidths given in parenthesis in Hz at 360 MHz, negative sign indicates position upfield from DSS. Assignment

Cytochrome c peroxidase

Shifts:

Observed

Methyls 5 1 8 3 Vinyls 2-a 4-a 2-/3 4-fl

Hyper. a

80.7 71.5 68.4( --- 550) 59.7

77.1 67.9 64.8 56.1

38.5 47.5( --- 500) - 10.8( ~ 200) - 8.3

29.9 38.9 - 17.1 - 14.6

Cytochrome c peroxidase-F Ave. b

-66

Spread

Observed

Hyper.

21

64.7 53.8( ~ 1000) 60.8 51.3

61.1 50.2 57.2 47.7

-

34

9

-

15.8

2.5

Ave.

Spread

54

13.4

Hyperfine, or isotropic resonances obtained by referencing observed shifts to their corresponding diamagnetic positions; see Ref. 25, Table I. b Average shift position for like protons.

252 that of the propionic acid methylene alpha protons. No hemins synthetically deuterated at the propionic a- or/3-methylene positions were available for this work. The assignment of these remaining protons will remain ambiguous until future synthetic developments. C. Methyl shift pattern The pattern and the spread of methyl resonances can, in principle, be diagnostic of a lowsymmetry perturbation which is present in the protein [20,24,26]. Incorporating hemin into cytochrome c peroxidase increases the spread of methyl shifts, indicating the presence of such a rhombic perturbation. For essentially high-spin ferric heme proteins, such as cytochrome c peroxidase and cytochrome c peroxidase-F, magnetic anisotropy is small at room temperature [27-29], indicating that the observed shifts originate primarily from the contact interaction. Therefore, the pattern of observed resonances directly corresponds to a relatively unequal distribution of unpaired spin density over the four hemin pyrroles. An analysis of the observed shift pattern of cytochrome c peroxidase reveals that methyls 5 and 1, which are substituents on opposite pyrroles (III and I, respectively), possess larger shifts than methyls 8 and 3 (pyrroles IV and II), indicating that a pairwise spin density distribution pattern is established over the heme ring. Pyrroles III and I receive comparatively greater unpaired spin density than pyrroles IV and II. Elsewhere, we have shown for the low-spin forms of cytochrome c peroxidase that the observed methyl shift pattern is consistent with the rhombic perturbation being defined by the rotational orientation of the proximal histidine plane [33]. However, differentiation of possible sources [20,25,27-29] for the rhombic perturbation is not possible here. This is because both sigma and pi type iron orbitals contain upaired spin density in ferric high-spin proteins and no model currently exists upon which a conclusion can be based. However, comparison of the methyl shift pattern for c y t o c h r o m e c p e r o x i d a s e a n d metmyoglobin is instructive. In contrast to the peroxidase, it is pyrrole IV (methyl 8) which receives the most spin density in metmyoglobin. Also, in metmyoglobin pyrrole I (methyl 1) re-

H2C:HC:: ,I CH OH2 91"12 "

CCP

C,Hz

91-12 K

ell2

Mb

Fig. 6. Projection of the axial histidine imidazole plane on protohemin IX, within the intact protein. View is along the proximal Fe-N bond for metmyoglobin(Mb), as reported in Refs. 29 and 34 from crystal structure data, and cytochrome c peroxidase (CcP), as reported in Ref. 29 from crystal data. This figure illustrates the 180° rotation about the a-'y meso axis. Note that this rotation interconverts pyrroles II(CcP)/I(Mb) and III(CcP)/IV(Mb).

ceives the least spin density (smallest shift), whereas, in cytochrome c peroxidase, pyrrole II (methyl 3) receives the least. An interpretation of these data which is consistent with the crystal structure [29] is that the interchange of methyl shift ordering is due to the crystallographically defined difference in heme rotational orientation (Fig. 6). This rotational isomerism results in the interchange of pyrroles I, II and III, IV, for each protein, when the structure is viewed along a fixed axis such as the proximal histidine-iron bond (Fig. 6). Moreover, if it can be assumed that the essential features of the crystal structure for cytochrome c peroxidase are not significantly perturbed in solution, the N M R data for both peroxidase and metmyoglobin indicate that the form of the rhombic perturbation is fixed in the protein structure and is similar in each protein. D. Axial coordination state Conclusions concerning occupation of the sixth iron coordination site of native cytochrome c peroxidase can also be drawn from these resonance assignments. From work on hemin models several criteria for six vs. five coordination have emerged which may be tested on cytochrome c peroxidase in view of the fact that the crystal structure data indicate the presence of a water molecule at the sixth coordination site [20,29]. Table II summarizes the pertinent data for cytochrome c peroxidase and cytochrome c peroxidase-F in com-

253 TABLE II SHIFT PATTERNS FOR CYTOCHROME c PEROXIDASE AND CYTOCHROME c PEROXIDASE-F COMPARED TO HIGH SPIN FERRIC PORPHYRIN COMPLEXES Average shifts from Table I (this work) and Table III (Ref. 17) in ppm; negative sign indicates upfield shift. Position: Porphyrins Five-coordinate Six-coordinate Proteins Cytochrome c peroxidase Cytochrome c peroxidase-F MB + b

CH 3

vinylH'~

vinylH ~

Qa

45 63

35 38

- 8 - 12

0.04 0.14

66.5

34.4

- 15.8

0.31

54.1 73.2

0.25 0.50

a Asymmetryparameter (Ref. 17) defined as (spread of methyl shift)-(average methyl hyperfine shift). b Metmyoglobinaverage shifts and Q calculated from data in Table I, Ref. 13.

parison to the averaged data for models and the data for metmyoglobin, where water is also acknowledged to occupy the sixth iron coordination position. By two criteria of this table (Q, vinyl fl shifts) cytochrome c peroxidase can be assigned a six-coordinate, high-spin form, implying that water occupies the sixth, axial site. The reasoning is as follows. The average methyl shift and vinyl beta proton shifts observed are significantly larger than the data for five-coordinate porphyrins and compare favorably with the six-coordinate highspin porphyrin results. Moreover, the asymmetry parameter, Q, is quite large for both peroxidases. Both are the same order of magnitude as for metmyoglobin (Table II) [19,34]. All of the protein Q values are an order of magnitude larger than the value observed for the 5-coordinate porphyrin models. Further support for this conclusion comes from additional data on deuterohemin (2,4-vinyl groups substituted by single protons)-reconstituted cytochrome c peroxidase (Satterlee and Erman, unpublished results). In the high-spin form, when no potential ligand ions except water were present, all single-proton resonances have been observed to lie upfield of the methyl resonances, or to overlap with them. This is a characteristic pattern of high-

spin six-coordinate models. In five-coordinate models the 2,4 (pyrrole) single-proton resonances lie significantly downfield of the hemin methyl resonances [20]. From all of this evidence we conclude that, in solution, water serves as a sixth ligand in native cytochrome c peroxidase.

E. pH dependence of cytochrome c peroxidase-F Previously, an analysis of the p H dependence of the native cytochrome c peroxidase hyperfine spectrum indicated the presence of three protein forms [17]. The two forms present in solution, which are linked by a p K at 5.8, are slowly interconverting and it has been suggested that this interconversion might modulate the native enzyme's reactivity. In contrast, the p H dependence of the cytochrome c peroxidase-F spectrum is less dramatic. A graph of the p H dependence of the cytochrome c peroxidase-F methyl resonances is shown in Fig. 7. Methyl 5, which lies farthest downfield, exhibits the greatest p H dependence, titrating in a smooth curve with a p K of 5.5 _+ 0.2 (Table III). The remaining three methyls all show very much smaller p H effects, as reported in Table III. The p K values were determined by nonlinear leastsquares fitting of the data points to the Henderson-Hasselbalch equation, and the lines drawn in Fig. 7 were generated by the best-fit data from these calculations. Within experimental error all of the p K values are identical. From the relative magnitudes of the methyl shift titration it is clear that the titrating group must be in close proximity to methyl 5. This methyl is a substituent on pyrrole III, next to the 6-position propionic acid. The most likely interpretation of these data is that methyl 5 is reflecting the ionization of a neighboring group on the heme, or the protein. According to the crystal structure no interactions between propionic acid 6 and protein groups are significant enough to be reported, making this group freely available for titration [29]. In contrast, propionic 7 (pyrrole IV) is implicated in interactions with either a threonine or arginine-48 [29]. Therefore, the most reasonable interpretation of the methyl 5 behavior is that it is reflecting ionization of its neighbor, propionic acid 6. Although this observed p K exhibits a value close to one which has been implicated in this

254

65

5-CH 3

6O

1-CH3

~ 55-

3-CH3

r

5

I

i

6

7

Fig. 7. pH titration data of the assigned methyl resonances of cytochrome c peroxidase-F. Only methyl 5 exhibits significant pH dependence. Spectra were obtained at 22°C, 0.1 M KNO3, at 470 MHz. Lines through the experimental points were calculated for a single ionization with a pK of 5.5, as described in the text.

enzyme's reactivity [17,35,36], we are hesitant to attribute the regulatory pK to titration of a propionic acid for two reasons. First, it has recently been shown that cytochrome c peroxidase which

TABLE III pH DEPENDENCEOF CYTOCHROMEc PEROXIDASE-F METHYL SHIFTS AND APPARENTpK VALUES Observed shifts are in ppm from DSS. Magnitude is the difference in high-pH and low-pH shift, in ppm. Apparent pK values are from nonlinear least-squares fit to the HendersonHasselbalch equation. CH 3

assignment:

Observed shift High pH Low pH Magnitude Apparent pK

5

8

l

3

64.5 59.8 4.7 5.5 +0.2

60.8 59.8 0.9 5.6 +0.2

53.8 54.5 0.6 5.6 +0.3

51.3 50.6 0.8 5.8 _+0.5

has been reconstituted with protohemin IX-dimethylester retains the pK 5.5 modulation of its reactivity with hydrogen peroxide [36]. Second, the ultraviolet-visible spectrum of cytochrome c peroxidase-F is pH-independent between pH 4.5 and 7.5 [37], whereas all other cytochrome c peroxidase forms that exhibited pH-dependent kinetics also exhibit pH-dependent (static) spectroscopic properties. Indeed, heme-linked ionizations with pK values between 5.2 and 5.8 have been found for the native enzyme by several spectroscopies [17,35,381. We are, therefore, led to these conclusions. (1) Unlike the native protein, cytochrome c peroxidase-F does not exhibit multiple conformations in solution. (2) The observed pH dependence for cytochrome c peroxidase-F is probably due to the ionization of propionic acid 6. (3) Evidently the heroin electronic structure, as sensed by ultraviolet-visible spectroscopy, is unaffected by this ionization. (4) Methyl 5's pH behavior, consequently, is a reflection of peripheral environmental changes as a consequence of acid ionization. In summary: assignments of methyl groups for cytochrome ¢ peroxidase and cytochrome c peroxidase-F have been carried out employing specifically deuterated hemins which were reconstituted into the apoenzyme. Similarly, single-proton resonances have also been assigned for the native protein. It is concluded from these results that cytochrome c peroxidase is high-spin and six-coordinate, with water occupying the sixth site. The results presented here confirm many of the hemecrevice structural features delineated for cytochrome c peroxidase by crystallography. This indicates that, in contrast to original data presented for other b-type heine proteins, there exists a high degree of correlation for heme-pocket features between the crystal and solution.

Acknowledgements We gratefully acknowledge support for this research from various sources: the American Heart Association (J.D.S.) and the Sandia-UNM cooperative research program (J.D.S.); the National Institutes of Health GM18648 and BRSG (J.E.E.); HL16807, GM22262 (G.N.L.) and HL22252 (K.M.S.). We also acknowledge the Purdue Uni-

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versity Biochemical Magnetic Resonance Laboratory, through which part of this research was supported by National Institutes of Health, Division of Research Resources grant RR01077, and the University of California, Davis, NMR Facility. We are grateful to Dr. J. Timothy Jackson for providing the nonlinear least-squares fitting program, which was written by Dr. Darrow Neves, and for the plotting program TITPLT which allowed rapid plotting of the curves in Fig. 7. We are also indebted to Dr. Stephen B. Kong for his assistance in gathering the initial spectra. References 1 Jesson, J.P. (1973) in NMR in Paramagnetic Molecules (LaMar, G.N., Horrocks, W.D. and Holm, R.H., eds.), pp. 1-52, Academic Press, New York 2 Keller, R., Groudinsky, O. and Wuthrich, K. (1976) Biochim. Biophys. Acta 427, 497-511 3 LaMar, G.N., Viscio, D.B., Gersonde, K. and Sick, H. (1978) Biochemistry 17, 361-367 4 LaMar, G.N., Budd, D.L., Viscio, D.R., Smith, K.M. and Langry, K.C. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5755-5759 5 LaMar, G.N., Smith, K.M., Gersonde, K., Sick, H. and Overkamp, M. (1980) J. Biol. Chem. 255, 66-70 6 LaMar, G.N., Overkamp, M., Sick, H. and Gersonde, K. (1978) Biochemistry 17, 352-361 7 Wright, P.E. and Appleby, C.A. (1977) FEBS Lett. 78, 61-66 8 Trewhella, J. and Wright, P.E. (1980) Biochim. Biophys. Acta 625, 202-220 9 DeRopp, J.S. (1981) Ph.D. Dissertation, University of California, Davis 10 Mayer, A., Ogawa, S., Shulman, R.G., Yamane, T., Cavaleiro, J.A.S., Rocha-Gonsalves, A.M. d'A., Kenner, G.W. and Smith, K.M. (1974) J. Mol. Biol. 86, 749-756 11 LaMar, G.N. (1979) in Biological Applications of Magnetic Resonance (Shulman, R.G., ed.), pp. 305-343, Academic Press, New York 12 Satterlee, J.D. and Erman, J.E. (1983) Biochim. Biophys. Acta 743, 149-154 13 Yonetani, T. and Ray, G.S. (1965) J. Biol. Chem. 240, 4503-4408

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