Biochimica et Biophysica Acta, 1122 (1992) 63-69 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

63

BBAPRO 34~2

Proton nuclear Overhauser effect study of the heme active site structure of Coprinus Macrorhizus peroxidase L.B. Dugad and H.M. Goff Department o f Chemistry, Unit'ersity of Iowa, Iowa City, 1.4 (USA) (Received 17 October 1991) (Revised manuscript received 24 February 1992)

Key words: Peroxidase; Mushroom peroxidase: NMR; Nuclear Overhauser effect study; Hemoprotein; ( C. macrorhizus )

Proton nuclear Overhauser effect and paramagnctic relaxation measurements have been used to define more extensively the heme active site structure of Coprinus macrorhizus peroxidase, CMP (previously known as Coprinus cblereus peroxidase), as the ferric low-spin cyanide ligated complex. The results arc compared with other well-characterized peroxidase enzymes. The NMR spectrum of CMPCN shows changes in the paramagnetically shifted resonances as a function of time, suggesting a significant heine disorder for CMP. The presence of proximal and distal histidine amino acid residues are common to the heme environments of both CMPCN and HRPCN. However, the upfield distal arginine signals of HRPCN are not evident in the 1H-NMR spectra of CMPCN.

Introduction Peroxidases are an important class of enzymes that catalyze oxidation of a wide variety of substrates in the presence of hydrogen peroxide [1,2]. Increasing interest in the peroxidases is derived from their biomedical importance as well as biotechnological applications. Although heme peroxidases are functionally as important as oxygen binding and electron-transfer proteins, the subject enzymes remain structurally less well characterized. This is evident from the fact that an X-ray crystal structure is available for only one, i.e., cytochrome c peroxidase [3,4]. Nuclear magnetic resonance spectroscopy has, however, contributed a large extent to our understanding of the active site structure of peroxidases from various sources. In spite of the inherent paramagnetism, relatively high molecular weight and the lack of crystal structure data, important structural information has been obtained for

Abbreviations: CCP, cytochrome c peroxidase; CMP, Coprinus macrorhizus peroxidase; HRP, horseradish peroxidase; LiP, lignin peroxidase; CCPCN, CMPCN, HRPCN and LiPCN, cyanide ligated complexes of CCP, CMP, HRP and LiP, respectively; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; BHA, benzohydroxamic acid. Correspondence: H.M. Goff, Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.

horseradish, lacto-, chloro-, myelo- and fungal peroxidases through application of N M R spectroscopy [5-11]. As a continuation of our initial description of the inkcap mushroom (Coprinus macrorhizus or previously Coprinus cinereus) peroxidase [9], we report here a further deiailed study using nuclear Overhauser effect, paramagnetie relaxation and substrate binding. Interest in the Coprinus enzyme as a possible source of peroxidase for clinical analyses [12] and its substrate binding properties [13] prompted us to investigate the structt:ral aspects in more detail, and to compare the results with the well-characterized horseradish ~peroxidase [5,14,15] and cytochrome c peroxidase [16]. Coprinus macrorhizus peroxidase is a glycoprotein of 4 2 kDa molecular mass with 1-5% carbohydrate content [12]. The protein contains a protoheme IX prosthetic group (Fig. 1A), it exhibits ligand binding properties similar to HRP, and the protein is acidic with p l = 3.5 [9,13]. In spite of the similarities of ligand binding between CMP and HRP, structural differences at the active site have been suggested for the two peroxidase enzymes [9,13]. The present work reveals the active site structural similarities and differences between CMP and the other two peroxidases (HRP and CCP). It is shown that important structural information can be obtained for a peroxidase enzyme of unknown primary structure by N M R spectral comparison of paramagnetically shifted resonances with those of the well-characterized enzymes H R P and CCP.

64

Materials and Methods Coprinus macrorhizus peroxidase was purchased from Chemical Dynamics and Schweizerhall and used as received. The protein was dissolved in 100% D 2 0 and 0.5 mi quantities of 3 mM protein solution were transferred to 5 mm N M R tubes. The solution pH was adjusted with 0.1 M DCI or NaOD. The reported pH values are meter readings not corrected for isotope effect. For the equilibration studies N M R samples were left at room temperature for several days and spectra were recorded at various times. The spectra in HzO solution were recorded with the Redfield selective excitation pulse sequence [17] by selection of the carrier frequency near the excitation region of interest. The low-spin cyanide complex (Fig. 1B) was prepared by addition of a 10-15-fold excess of KCN to the native protein sample. Benzohydroxamic acid and D 2 0 were purchased from Aldrich. I H-NMR spectra were recorded on a Bruker AMX600 FT NMR spectrometer operating at 600.14 MHz proton frequency. The NOE experiments were performed by collection of free induction decays with onand off-resonance irradiation in the interleaved mode. The resonance of interest was irradiated with weak decoupler power ( < 0.5 watts) and a repetition time of 0.7-0.8 s was used for the steady state NOEs. The spin-lattice relaxation times, T~, of the hyperfine shifted proton peaks were determined by the standard inver-

~CH

H

H

sion-recovery pulse sequence with a repetition rate of one per s and 90 degree observe pulse of 9/~s. The T~ values were estimated at the null point with T~ = ~-/ln 2. The N e E and T~ data were analyzed in terms of interproton and iron-proton distances, respectively, by the standard equations as described elsewhere [14,15,18,19]. Chemical shifts (in parts per million, ppm) are reported with respect to the residual water signal which was calibrated with 2,2-dimethyl-2-silapentane5-sulfonate, DSS. The ~H-NMR spectra of native CMP were obtained by a 90 KHz spectral width with 8192 data points, and a repetition rate of 40 or 80 per s. A 12 bit digitizer was used and 20000-40000 transients were collected. The FIDs were exponentially multiplied by 100 Hz line broadening to improve the signal to noise ratio. The spectra of the ferric low-spin form were obtained by a 40 or 55 KHz spectral width, 8192 data points and repetition time of 0.7 s. Generally 2000-3000 scans were accumulated and the resulting FIDs were multiplied by 20 Hz apodization.

Results The ZH-NMR spectrum of Coprinus peroxidase ferric low-spin cyanide ligated complex, CMPCN [9] is shown in Fig. 2A. This is the spectrum of a freshly prepared sample in 100% D20. The spectra recorded after storage of the same CMPCN sample at room

Oh

"

\ p

H

. ~ H ........... H C.I

H3C

CH3

H2Ca

H

;H2 ~H2

Fo

I

(

6

proximal histidin

OOH A

B

Fig. 1. (A) Schematic diagram showingthe structure of iron-protoporphyrin IX, and (B) the axial iigation of the heme iron in the low-spin ferric complexof Coprinusmacrorhizus peroxidase alongwith the distal histidine hydrogenbonded to the axial Iigand cyanide ion.

65

temperature for 1 and 2 weeks are shown in Fig. 2B and C, respectively. Several changes in the paramagnetically shifted resonances are seen as a function of time. It is noteworthy that peak b in Fig. 2A to C shows a dramatic change in its intensity. The same changes are observed in ~H 2 0 solution, thus indicating that the equilibration process does not reflect deuterium exchange and consequent isotope effects. Peak e in Fig. 2A has a shomder that is absent in Fig. 2C. Peaks i and p are better resolved in fig. 2C than in Fig. 2A. Fig. 2C shows the equilibrated protein spectrum in which peak b retains minor intensity as a shoulder to peak c. Parallel spectral changes occur in the upfield region from 0.0 to - 5 . 0 ppm. At least four paramagnetically shifted peaks of fractional proton intensity (peaks f, m, x and y, see Table I) are detected in the spectra of Fig. 2A to C that do not change intensity as a function of time. Peaks x and y show NOE between themselves (data not shown). These two peaks probably arise from a minor isoenzyme or from a modified protein. The NMR parameters for the hyperfine shifted peaks are listed in Table I. In Fig. 2D, we show a spectrum obtained with a fast repetition rate collected to observe protons with very short Tjs.

"

d

,

, .

A'L

•y

vvJ\

TABLE 1

IH-NMR parameters for the hyperfine shifted resonances of CMPCN at 298 K, pH ZO Peak label a ~ b c d e f g h a i j k ~

I m n o p q

Chemical shift (ppm) 32.5 28.9 28.6 27.8 22.8 20.9 b 20.5 19.5 19.4 19.1 15.8 15.4 14.0 h

t

13.5 12.8 12.2 11.8 0.1 -0.7 -2.7 -2.9 -3.1 - 3.25

u

-3.4

r s

v w x y

- 3.9 -4.3 -9.1 b -10.5 b

z

- 22.5

Ti¢

Assignment

(ms) 30

distal his. N-E-H

94 1.5 100

prox. his. C-8-H

I-C H 3 or 3-CH 3 5-CH.~ or 8-CH 3

72 80 87 210 65

prox. His C-/3-H? distal his. N 4 i- H prox. His C-/]-H "~

160 108 6O ! 15

distal his. C-E-H 6-H or 7-H a-prop.

70 72 120 145 120 105 100 120 120 1.5

2-H or 4-H a-vinyl 2-H or 4-H fl-vinyl 2-H or 4-H fl-vinyl

prox. his. C-~-H

a Labile proton peaks. b Peaks of fractional proton intensity. c The reported T I values have +_ 10% error.

j c

N

•

c '

' '

I

'

'

30

c

'

. . . .

'

'

I

.

2S

d

•

. . . .

.

.

~..._•

q

.

I

" '

'

'

'

I

20

J

. . . .

.

.

.

.

I'

IS

I

,'s

'

lO

'

I

-$

'

'

'

'

I

'

'

-lO

n

. . . .

,'0

......

"i';

.......

......

PPM Fig. 2. The 600 M H z i H - N M R spectra of the ferric low-spin cyanide complex of Coprinus macrorhizus peroxidase, 3 raM, in D z O , p H 7.0, at 298 K, recorded as a function of time. (A) The spectrum of a freshly prepared sample. (B) Sample in (A) stored at room temperature for 1 week. (C) Sample in (A) stored at room temperature for 2 weeks. (D) The spectrum o f sample in (C) recorded with a repetition rate of 100 per s at 303 K to detect the fast relaxing peaks d and z. in spectra (A) to (D) the vertical scale for the downfield region is twice that of the upfield region.

There are two broad resonances (peaks d and z) each of single proton intensity observed in the downfield and upfield region, respectively, at 303 K (Fig. 2D). The presence of the upfield peak z was reported earlier [9]. At 298 K, peak d is under the methyl peak c and is not resolved. In Fig. 3B to E are N O E difference spectra obtained upon irradiation of peaks c, e, o and q, respectively, along with a reference spectrum in Fig. 3A. Irradiation of peak c (Fig. 3B) gives NOEs to peaks at +0.1 ( - 2 2 % ) , - 0 . 7 ( - 13%), - 3 . 1 ( - 6 % ) , and - 3 . 9 ( - 7 % ) ppm in the upfield region. The numbers in parentheses indicate percentage NOE to that peak. Saturation of the methyl peak e (Fig. 3 0 gives NOE to peaks at 12.8 (peak o, - 10%), and - 2.9 ( - 8%) ppm. Peak o shows large N O E to a peak at 7.7 ( - 5 2 % ) ppm and small NOE to peak e (Fig. 3D). Both peak e and o show a common NOE to a peak at - 2 . 9 ppm. Peak q has two proton intensity, and at least one of the two protons shows NOE to peaks j and l, and to other peaks in the aromatic region (Fig. 3E). At least one

66 proton of peak q shows NOE to peaks at + 0.1, - 0 . 7 ppm, and these upfield peaks also show N O E from irradiation of hemc methyl peak c. Reciprocal NOE's were detected for all of the major resonances discussed here. Additional NOE relationships are shown in Fig. 4 with the reference spectrum included in Fig. 4A. Fig. 4B shows the NOE from peak g to a single peak in the aromatic region. Figs. 4C and D show the NOE difference spectra obtained upon irradiation of peaks j and 1, respectively. Large N O E is observed from peak j to 1 ( - 5 3 % ) and from peak I to j ( - 5 7 % ) . At least four common NOEs of - 2 0 % to - 3 0 % magnitude are observed upon saturation of peaks j and I (Fig. 4C and D) in the 8.3 to 11.8 ppm region. Peak i occurs as a shoulder to peak j and is only partially resolved. The estimated cross-relaxation rate, o-, is - 3 7 Hz between the protons j and I based on the NOE buildup rate (data not shown). Therefore theointerproton distance between protons j and l is --- 1.8 A and they must arise from a - C H 2 pair of protons. The downfield I H-NMR spectral region of CMPCN in 90% H 2 0 solution is shown in Fig. 5A. This spec-

-....__._

10.8,

[

! I

q

~'~.2"--s.3 9!5

"8.9

9.5

''l''''l 30

.... 25

I .... 20

I .... I .... IS 10 PPM

I .... 5

I .... 0

t''' -S

Fig. 4. (A) I H - N M R r e f e r e n c e s p e c t r u m o f 3 m M C M P C N in D 2 0 ( p H 7.0) at 298 K. (B) to (D) a r e t h e N O E difference spectra for irradiation of peaks g, j a n d I, respectively. T h e irradiated peak in e a c h difference s p e c t r u m is s h o w n by the downward arrow while the observed N O E peaks are m a r k e d with their chemical shifts o r the d e s i g n a t e d symbol.

C

A_-~

w1 ~ ~0.7

"-3.9

+0.1

%•x2 0

-2.9

-2.9

+0.1

p p M ' l

30

....

i ....

25

i ....

20

i

15

....

I ....

l0

I ....

$

I ....

0

I'''

-5

Fig. 3. (A) IH-NMR reference spectrum of 3 mM CMPCN in D20 (pH 7.0) at 298 K. (B) to (E) are the steady state NOE difference spectra obtained upon irradiation of peaks c, ¢, o and q, respectively. Each peak was irradiated for 100 ms and a recycle time of 0.7 s was used. In all difference spectra, the downward arrow shows the irradiated peak. The NOE peaks are labelled with the respective chemical shift values or the corresponding peak labels.

trum was obtained with the Redfield selective excitation pulse sequence [17] with the carrier frequency placed at 35 ppm. The labile proton peak a in Fig. 5A is not observed above p H = 9.0. The 12-20 ppm spectral region is expanded in Fig. 5B to show the other two labile proton peaks h and k obtained with the carrier frequency placed at 15 ppm. Irradiation of peak h showed no H O E to peaks in the paramagnetic region (data not shown). Therefore, peak h could not be assigned. Peak h is observed up to pH 9.5 and above this pH it completely disappears. Exchangeable protons with chemical shifts similar to peaks a and k are observed for HRPCN, CCPCN and LiPCN [5,10,11,16]. The N O E between peak n and the labile proton peak k ( - 12% NOE from each other) is shown in Fig. 5C and D. Irradiation of peak a at 31.3 ppm (Fig. 5E) shows ( - 10%) NOE to the non-exchangeable proton peak n. Irradiation of peak n shows no N O E to paramagnetically shifted peaks in 100% D 2 0 solution (not shown). Peak n has a small linewidth and is of single proton intensity with T l = 160 ms (Table I). Thus, the two exchangeable protons a and k should be close in space to the proton n. The results of benzohydroxamie acid (BHA) substrate binding to native CMP and CMPCN are shown

67 c c

F

.

~

PPM

-"~----.'

3S

T

x2

:

A

Discussion

~

30

Heme disorder?

~-"-'"--"

25

•

~"-"""

20 k

The spectral changes in Fig. 2 A - C are similar to those observed for the equilibration of orientationally disordered heme in hemoproteins [20,21]. The slow N M R spectral changes observed over a period of weeks are typical of heine disorder along the ot - y methine carbon axis (Fig. 1A) with a 180° flip of the heme. The N M R spectrum of the equilibrated CMPCN sample in Fig. 2C remains unchanged for several months. We notice that approx. 15% heine orientational disorder is retained at equilibrium. These observations suggest mobility of heme in the active site to a significant extent for CMP. The reason for this apparent heme disorder a n d / o r its structural/functional significance is not known at present.

"',sX~

15

I

n

S

k

F.

Co

Assignment of hyperfine shifted resonances Irradiation of the two heme methyl peaks c and e (Fig. 3B and C) show N O E patterns that can be ascribed to a methyl group near either a vinyl or a propionate group (Fig. 1A). The NOE data are com-

C.F.

1-CH 3 S*CH3

n I

PPM 20

I

19

I

18

I

17

I

16

I

15

I

14

I

13

|

12

Fig. 5. (A) The downfield proton NMR spectral region of 3 mM CMPCN recorded in 90% H 2 0 - 1 0 % D : O at pH 6.0 with the Redfield selective excitation pulse [17] at 308 K with the carrier frequency placed at 35 ppm. Note the most downfield shifted labile proton peak a in the spectrum. The other two exchangeable protons h and k are shown in the expanded region of trace B recorded with the carrier frequency at 15 ppm. (C) to (E) are the steady state NOE difference spectra obtained upon irradiation ot peaks n, k and a, respectively.

8-CH3 A

~

0.6 21

~6

~

I ...... '''t ....... ''I ......... I ......... I ......... I ......... I''''':'''

100

90

80

70

60

i

in Fig. 6. The spectrum of native CMP in the hypeffine shifted downfield region (Fig. 6A) is equivalent to that observed previously, and earlier assignments are listed [9]. In Fig. 6B is shown the spectrum of native CMP with 5-fold excess B H A (tool ratio). Heme methyl resonances move in an upfield direction in the presence of BHA. The 3-CH 3 and 8-CH 3 peaks experience larger upfield shifts than the degenerate 1-CH 3 and 5-CH 3 peak. The spectrum of CMPCN is shown in Fig, 6C and the result of addition of 20-fold excess BHA is shown in Fig. 6D. Spectral changes occur in the upfield spectral region from 0.0 to - 5 . 0 ppm, whereas the downfield region is unchanged. The observed chemical shift changes upon binding of B H A to CMPCN are quite small ( < 0.2 ppm) and are generally in the upfield direction.

.

50

n

40

s

q

C

• 1/2 ¢

0.2

T

~I

'

' ~01 . . . .

~I

....

o.,

0.2 J f9

~01 . . . .

°

n i

l.lJ

Uv

op

1~I . . . .

lol I

' -4

"61

I

PPM

Fig. 6. (A) The 600 MHz proton NMR downfield spectral region of 1 mM native CMP in D20 (pH 7.0) at 298 K showing hyperfine shifted heine methyl resonances as previously asfigned [9]. (B) Same as (A) but in the presence of five fold excess BHA. (C) tH-NMR hyperfine shifted spectral region of 1 mM CMPCN in DzO (pH 7.0) at 298 K. (D) same as (C) but in the presence of 20-fold excess BHA. The numbers on top of the peaks indicate the upfield changes in the chemical shifts (in ppm) observed upon addition of BHA for the corresponding peak. The spectra in (A) and (B) were obtained with repetition time 25 ms, whereas those in (C) and (D) with 700 ms.

68 pared with those of HRPCN [14,15] in which case the heine methyls close to the vinyl group show NOE to either the a-CH proton or to the fl-CH, protons depending on the orientation of the vinyl group [14] (also see Fig IA). The fl-CH, vinyl proton signals are usually observed in the upfieid region from 0.0 to - 5.0 ppm, whereas the a-CH vinyl proton is observed in the downfield region due to 1r-spin delocalization of the unpaired spin density to the vinyl group [22]. NOE is observed from the methyl peak c to two peaks at 0.1 and -0,7 ppm that are characteristic of the vinyl fl-DH 2 signals with the orientation of the vinyl group as in Fig. IA. This is further supported by the fact that saturation of peak q also shows NOE to those two peaks assigned as fl-CH 2 protons of the vinyl group (Fig. 3E). Thus, one of the protons of peak q is from an a-CH of the vinyl group. Peak c therefore must arise from either the 1-CH 3 or 3-CH a group of the heme. It is easily seen that only two of the four heme methyls are resolved from the diamagnetic region, as observed earlier for HRPCN and other hemoproteins due to the unique orientation of the proximal histidine ring with respect to the heine plane [14,22]. These heme methyl signals show a unique hyperfine shift pattern, as either the 1-CH 3 and 5-CH 3 pair or the 3-CH 3 and 8-CH 3 pair in the downfield paramagnetic chemical shift region, depending on the orientation of the proximal histidine with respect to the heme plane [14]. Thus, heme methyl peak e must arise from either the 5-CH 3 or 8-CH 3 group. The NOE pattern clearly shows that it is a methyl group near the propionate. NOE from peak e to peak o (Fig. 3C) and the reciprocal NOE from peak o to peak e are observed (Fig. 3D). Peak o has a geminal partner at 7.7 ppm. Thus, peak o and the peak at 7.7 ppm must originate from either the 6-a-CH 2 or 7-a-CH 2 pair of protons, and peak e must be from the 5-CH a or 8-CHa group. It is noted that peaks c and e also show NOE in the upfield region from - 3 . 0 to - 4 . 0 ppm. There are at least 7-8 peaks in the - 2.5 to - 4.0 ppm region which are not well resolved. These proton signals likely arise from an aliphatic amino acid residue(s) and are clustered together with similar chemical shifts and Tis (Table I). In the absence of crystal structural data, we cannot unambiguously assign these upfield peaks observed in the NOE difference spectra of Fig. 3B and C. Selective irradiation of these upfield resonances, which are near the diamagnetic region, is not possible without development of large off-resonance effects. Attempts to better resolve these peaks at higher temperature were hampered by protein denaturation. Irradiation of peak g shows NOE to only one peak at 8.6 ppm (Fig. 4B). As peak g shows no NOE connectivity to the paramagnetically shifted peaks, it cannot be assigned unambiguously. Peaks j and l show no NOE connectivity to the methyl peaks c and e, and

hence are not from either vinyl or propionate - C H 2 protons. They are likely from fl-CH 2 protons of the proximal histidine residue. Signals with characteristics similar to peak j and I have been observed in HRPCN and CCPCN [15,16]. Well resolved upfield peaks are not observed in CMPCN, as is the case for HRPCN and CCPCN. The well resolved upfield signals for these latter peroxidases are assigned to a catalytically important distal Arg residue. It is likely that any distal arginine signals for CMPCN reside in the diamagnetic region. This appears to be the case for LiPCN [10,11] as a consequence of an altered spatial disposition of the arginine residue.

Proximal histidine Fig. 2D reveals the presence of two broad, fast relaxing peaks d and z. These two peaks have been assigned to the proximal His ring C-8-H and C-~-H protons, respectively (Table I). The presence of such peaks has been detected in cyanide complexes of horseradish, cytochrome c peroxidase, lacto- and myeloperoxidase [6,8,23]. These peaks were observed earlier along with other NMR and ESR characteristics to support the presence of the proximal histidine residue in CMP [9]. The positions of both peaks d and z are more downfield shifted by 4-6 ppm as compared to those in HRPCN. The chemical shifts and other properties are consistent with the imidazolate character of the proximal histidine in CMPCN similar to HRPCN and CCPCN. Distal histidine Evidence is presented for a distal histidine residue in the heine pocket of CMP. We compare proton signals a, k and n with those assigned to the distal histidine ring protons in HRPCN [5]. Peaks a and k arise from exchangeable protons (Fig. 5A and Table I), whereas peak n is from a non-exchangeable proton. The chemical shift, NOE, and relaxation behaviour of peaks a, k and n are nearly identical to the corresponding peaks in HRPCN, and hence by analogy we propose the presence of a distal histidine in the heine pocket of CMPCN with the axial cyanide ligand hydrogen bonded to the protonated distal histidine N-e-H proton (Fig. 1B). The results of NOE data (Fig. 5B, C and D) are consistent with the distal histidine residue having irnidazolium character. This was also supported by the previous ~SN-NMR data on CMPCN with an identical chemical shift of the bound cyanide ion in both HRPCN and CMPCN [9]. These results strongly argue in favor of very similar or identical structural characteristics of the distal histidine residue with respect to the heme in both CMPCN and HRPCN. Substrate binding We now compare the binding of BHA to CMP and HRP, and their cyanide ligated complexes. The binding

69 of BHA to HRP and HRPCN induces large chemical shift changes in the hyperfine shifted heme resonances [24]. Addition of BHA to native HRP induces 3 to 4 ppm downfield shifts, whereas upfield shifts are observed for all the heme methyl resonances of CMP. For CMP the 3-CH a and 8-CH 3 peaks experience upfield shifts of up to 2.0 ppm (Fig. 6B), whereas a smaller upfield shift is observed for the I-CH 3 and 5-CH 3 degenerate peak. Thus, the structural perturbations occurring in native CMP upon BHA binding are clearly different than those observed for HRP. The binding of BHA to HRPCN induces mostly downfield shifts ranging from 0.2 to 3.0 ppm [24]. In contrast, the results for CMPCN are very different. We observe only a +0.2 ppm maximum shift among resolved signals in CMPCN (Fig. 6D). Earlier substrate binding studies suggest the binding of substrate in CMP near the 8-CH 3 heme periphery similar to HRP [13]. If a similar binding mode is considered for both HRP and CMP, then it is likely that the 8-CH 3 group is in the diamagnetic region in CMPCN. The results clearly show differences in substrate binding for CMPCN versus HRPCN. Acknowledgement This work was supported by NIH grant GM-28831. References 1 Dunford, H.B. (1982) Adv, Inorg. Biochem. 4, 41-68. 2 Dunford, H.B. and Stillman, J.S. (1976) Coord. Chem. Rev. 19, 187-251. 3 Poulos, T.L. and Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205. 4 Finzel, B.C., Poulos, T.L. and Kraut, J. (1984)J. Biol. Chem. 259, 13027-13036.

5 Thanabal, V., De Ropp, J.S. and La Mar, G.N. (1988) J. Am. Chem. Soc. 110, 3027-3035. 6 Thanabal, V. and La Mar, G.N. (1989) Biochemistry 28, 70387044. 7 Goff, H.M., Gonzalez-Vergara, E. and Bird, M.R. (1985) Biochemistry 24, 1007-1013. 8 Dugad, L.B., La Mar, G.N., Lee, H.C., lkeda-Saito, M., Booth, K.S. and Caughey, W.S. (1990) J. Biol. Chem. 265, 7173-7179. 9 Lukat, G.S., Rodgers, K.R., Jabro, M.N. and Golf, H.M. (1989) Biochemistry 28, 3338-3345. 10 Banci, L., Bertini. I., Turano, P., Tien, M. and Kirk, T.K. (1991) Pro¢. Natl. Acad. Sci. USA 88, 6956-6960. 11 De Ropp, .I.S., La Mar, G.N., Wariishi, H. and Gold, M.H. (1991) J. Biol. Chem. 266, 15001-15008. 12 Morita, Y., Yamashita, H., Mikami, B., Iwamoto, H., Aibara, S., Terada, M. and Minami, J. (1988) J. Biochem. 103, 693-699. 13 De Pillis, G.D. and Oritz de Montellano, P.R. (1989) Biochemistry 28, 7947-7952. 14 Thanabal, V., De Ropp, J.S. and La Mar, G.N. 0987) J. Am. Chem. Soc. 109, 265-272. 15 Thanabal, V., De Ropp, J.S. and La Mar, G.N. (1987) J. Am. Chem. Soc. 109, 7516-7525. 16 Satterlee, J.D. and Erman, J.E. (1991) Biochemistry 30, 43984405. 17 Redfield, A.G., Kunz, S.D. and Ralph, E.K. (1975) J. Magn. Reson. 19, 114-117. 18 Noggle, J.H. and Schirmer, R.E. (1971) The Nuclear Overhauser Effec': Chemical Applications, Academic Press, New York. 19 Neuhaus, D. and Williamson, M.P. (1988) The Nuclear Overhauser Effect in Structural and Conformational Analysis, Verlag Chemic, New York. 20 La Mar, G.N., De Ropp, J.S., Smith, K.M. and Langry, K.C. (1980) J. Am. Chem. Soc. 102, 4833-4835. 21 La Mar, G.N., Davis, N.L., Parish, D.W. and Smith, K.M. (1983) J. Mol. Biol. 168, 887-896. 22 La Mar, G.N. (1979) in Biological Applications of Magnetic Resonance (Shulman, R.G., ed.), pp. 305-343, Academic Press, New York. 23 La Mar, G.N., De P,opp, J.S., Chacko, V.P., Satterlee, J.D. and Erman, J.E. (1983) Biochim. Biophys. Acta 708, 317-325. 24 Morishima, I. and Ogawa, S. (1979)J. Biol. Chem. 254, 2814-2820.