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Deuterium exchangeable proton hyperfine resonances of low-spin cytochrome c peroxidase and the mechanism of peroxidase catalysis
Biochimica et Biophysica Acta, 743 (1983) 149-154
149
Elsevier BiomedicalPress BBA 31497
DEUTERIUM EXCHANGEABLE P R O T O N HYPERFINE R E S O N A N C E S OF L O W - S P I N C Y T O C H R O M E c PEROXIDASE AND T H E M E C H A N I S M OF PEROXIDASE CATALYSIS JAMES D. SATTERLEE a., and JAMES E. ERMAN b a Department of Chemistry, University of New Mexico, Albuquerque, N M 87131, and b Department of Chemistry, Northern Illinois University, DeKalb, I L 60178 (U.S.A.)
(Received August 3rd, 1982)
Key words: IH . N M R ; Cytochrome c peroxidase; Histidine residue," Reaction mechanism," H / 2H exchange
Deuterium exchangeable hyperfine proton N M R resonances of cytochrome c peroxidase (EC 1.11.1.5) are identified in H 2 0 solutions of the enzyme. One of these is assigned to the proximal histidine's imidazole N-H. Its shift and pH dependence indicate that an imidazolate form, which has been postulated for peroxidases, is ruled out for cytochrome c peroxidase-cyanide. A qualitative comparison of relative heine-pocket dynamics is also possible. When the bulk water resonance is irradiated with a continuous, but off acquisition, decoupler frequency the N-H resonance shows no intensity loss, indicating that saturation transfer between the proximal histidine and solvent water is either minimal, or extremely slow.
Introduction Thus far, cytochrome c peroxidase (ferrocytochrome c : hydrogen-peroxide oxidoreductase, EC 1.1 1.1.5) is the only member of the class of heme peroxidase enzymes to have its crystal structure reported [1,2]. Based on the structure Poulos and Kraut [3] have speculated about the molecular events whereby the enzyme decomposes hydrogen peroxide and they have suggested that peroxidase catalysis may be easily understood on the basis of known types of acid-base reactions. These reactions involve three critical distal amino acids in cytochrome c peroxidase's primary sequence: Arg48, His-51, Trp-52. Although these three are situated in positions to interact with heme-bound peroxide ion, none of them is readily capable of stabilizing the highly oxidized hemin iron ion which is formed during catalysis. Formally, that is considered to be Fe(IV) in both of the oxidized inter-
mediates [4,5] and, at least in cytochrome c peroxidase-I (Compound ES), a low-spin ferryl ion is implicated [6]. The possibility has been raised that stabilization of the intermediate ferryl ion may occur via the proximal histidine (His-174) [3]. In this view strong hydrogen bonding by the proximal histidine imidazole N-H (see Scheme I), or even complete
., ".'Cr
* To whom correspondenceshould be addressed. 0167-4838/83/0000-0000/$03.00 © 1983 ElsevierSciencePublishers
'P,
I
/
.2c=c
H
CH.,o ,
C=CH 2
H
CH
" fHz "~H2 CO~.
H
I
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150 deprotonation to form imidazolate ion, would result in excess negative charge localization on the histidine which, in turn, could be delocalized onto the ferryl ion. As a consequence, information about the state of protonation of the proximal histidine is important for the formulation of a correct enzyme mechanism. Under favorable conditions N M R is a technique suited for directly observing resonances such as the proximal N-H [7]. Although proton N M R spectra of cytochrome c peroxidase-I show extremely broad and very poorly resolved resonances [8,9], spectra of the low-spin native enzyme, such as cytochrome c peroxidaseCN, exhibit narrow, well-resolved hyperfine resonances [10-11]. Here, we present spectra of cytochrome c peroxidase-CN obtained in 90% H 2 0 which show four additional resonances which are not present in spectra obtained in 99.8% 2H20 solution. One of these is assigned to the proximal N-H. Together with the previously assigned proximal imidazole 2-H [11] (Scheme I), resonance positions at various pH values are determined and conclusions are drawn concerning the state of proximal imidazole protonation. Methods
Cytochrome c peroxidase was isolated and purified as previously described [12-14]. Lyophilized enzyme was dissolved in either 90% H 2 0 / 1 0 % 2H20 , or in 99.8% 2H20 (Merck), in order to form 1-2 mM solutions which were 0. l M in potassium nitrate (Sigma). Reconstitutions were carried out using hemin (protohemin IX, Sigma) according to the method of Yonetani [12] with the following modifications. The apoprotein was generated at 0 - 4 ° C with acidified butanone. 2 mM solutions of the apoenzyme in dilute potassium phosphate (Mallinkrodt, AR) buffer (0.3 M), pH 7.3, were cooled in an ice bath. The buffer solvent was either distilled, deionized H20, or 99.8% 2H20. A 10% mole excess of heroin dissolved in a minimal amount of 0.1 M sodium hydroxide was added to the apoprotein solutions and allowed to react for up to 1 h. The reconstituted protein was subsequently pressure-dialyzed in a micro-ultrafiltration apparatus (Amicon) using a phosphate- and hemin-permeable membrane (PM 10) to obtain a final enzyme solution in 0.1 M K N O 3 / E H 2 0 . Cy-
tochrome c peroxidase-CN was formed by adding a 1.1 mole ratio of KCN (Sigma) to the native enzyme solutions, pH was constantly monitored throughout this process with an Orion meter equipped with a Beckman combination pH electrode, pH titrations were carried out as previously described [14], with determinations made before and after each N M R data acquisition. The electrode was standardized prior to each use. p K values were calculated by a nonlinear leastsquares program to fit the experimental pH titration data to the Henderson-Hasselbalch equation. The program was written by Darrow E. Neves and provided for our use by J. Timothy Jackson. A Nicolet 1180 computer equipped with a Nicolet Zeta plotter was employed for this task. The solid curves drawn through the experimental points of Fig. 3 were generated by this program, along with the plotting routine TITPLT, provided by J. Timothy Jackson. N M R spectra at 360 MHz (8.45 T) were acquired at the Purdue University Biochemical Magnetic Resonance Laboratory or the Colorado State University Regional N M R Laboratory using a Nicolet Magnetics spectrometer. A recycle time of 200 ms was employed along with active temperature control and saturation (or partial saturation) of the residual water peak by decoupler irradiation which was gated off during the acquisition period. All shifts were referenced internally to the residual water peak and are reported relative to external 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) for reasons cited elsewhere [11]. Results and Discussion
Figs. 1 and 2 show comparison spectra for cytochrome c peroxidase-CN taken in H 2 0 and 2H20 solution. Fig. 1 reveals that resonance 8 is labile to deuterium exchange but only when the heine is removed from the protein. Fig. 1A is a spectrum of cytochrome c peroxidase-CN in which the apoenzyme, kept in H 2 0 solution, was reconstituted with hemin, also kept in H20. The protein was subsequently exchanged into 2H20 solution for this spectrum. The spectrum is a usual cytochrome c peroxidase-CN spectrum with eight resolved downfield hyperfine resonances [10,11] and is identical to native cytochrome c peroxidase-CN.
151
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I
- - F e - -
C
i= ~ . . . .
0
~.
PROX.HIS. 2.H
l
t ,
. . . .
-5
t ,
O
. . . .
-~0
,
....
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....
- 0
,
....
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I .
~5
.
.
.
.
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.
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.
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. . 215
.
.
.
.
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.
i . . . . 15
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A
PPM
spectra o f reconstituted cy-
tochrome c peroxidase-CN at 22°C, pH 7.2, 0.1 M potassium nitrate. (A) Reconstitution with protohemin IX carried out in H20. (B) Reconstitution carried out in 2H20. Both samples were subsequently dialyzed into 2H20 before these spectra were accumulated. Upfieid portions of these spectra display no solvent dependence and are not shown. (C) Coordination of the proximal histidine imidazole ring to the hemin iron is depicted along with the numbering convention of the imidazolewhich is employed in the text. Resonance numbers (A) and imidazole carbon numbers (C) are not related.
When the apoenzyme is maintained in 2H20 solution and the reconstitution is carried out in 2H20 , resonance 8 is missing from the spectrum (Fig. 1B). We conclude that resonance 8 is a protein proton which is exchangeable with deuterium, but is only solvent-accessible in the apoenzyme. N o solvent-dependent changes are observed in the upfield hyperfine spectrum Fig. 2 demonstrates the spectrum from an enzyme preparation handled exclusively in H20. Fig. 2, compared to 1A, reveals that there are three resolved hyperfine resonances downfield which are 2H-for-H exchangeable (resonances A - C ) . Again, no differences are observed in the upfield hyperfine region, as demonstrated by Fig. 2B compared to published spectra [8,10]. The assignments in Fig. 2 are those that we have determined with deuterium-labelled hemins [10] or by inference from hemin models [11]. One of these exchangeable protons (resonance A) is found in a position characteristic of the proximal N - H in other ferric heme proteins (Ref. 7 and G.N. LaMar, personal communication). Because of this and the similari-
~5' ....
;o ....
2s' ....
2o' ....
"s' "~P.
Fig. 2. (A) Downfieid and (B) upfield hyperfine resonances of cytochrome c peroxidase-CN in 90% H20. The three proteinassociated deuterium exchangeable resonances are labelled A-C. Heme methyl and vinyl resonances and the proximal histidine imidazole 2-H are also indicated. The numbering of resonances corresponds to Scheme I, in the text. For comparison, see Fig. 1A. Conditions are the same as in Fig. 1.
ties between the heme pocket structures of cytoc h r o m e c peroxidase, m e t - m y o g l o b i n and horseradish peroxidase, we are confident in the assignment of A as the proximal histidine N-H. In support of this, we point out that the sole sources of protons which are exchangeable on the time scale of lyophilized protein dissolution must be O or N bonded. Comparing the crystallographic and sequence data for cytochrome c peroxidase and horseradish peroxidase [2,3,15] one concludes that t h e proximal and distal histidines are consistent features. They possess N - H protons which are readily exchangeable and are situated close enough to the paramagnetic center to experience sizeable contact and dipolar (proximal) [16] or dipolar (distal) shifts [7]. In addition, for cytochrome c peroxidase (and horseradish peroxidase), the guanidinium group of Arg-48 may be a source of additional exchangeable resonances, although smaller shifts would be expected due to its greater distance from the iron ion [2,3]. To summarize the evidence for assigning resonance A as the proximal N-H, we emphasize that there are three crystallographically identified, potential candidates for exchangeable protons. The proximal and distal histidines are common to all
152
of the proteins for which comparison spectra are available. In addition, for the peroxidases Arg-48 may also be included. However, resonance A lies in a characteristic region for proximal N-H protons observed for models [16], met-myoglobin [7] and horseradish peroxidase (Ref. 18 and G.N. LaMar, personal communication). The fact that the proximal N-H resonance is observed in cytochrome c peroxidase-CN rules out deprotonation. However, three pieces of information support a strong hydrogen bonding role for the proximal N-H. Crystallography suggests that either Asp-234 or Gln-239 are positioned appropriately to act as hydrogen bond acceptors (T. Poulos, personal communication). Because these are stronger bases than polypeptide carbonyls, which are the usual proton-acceptor bases, chemical intuition supports formulation of a stronger hydrogen bond. In addition, we have shown elsewhere that the resonance pattern of non-exchangeable proximal histidine protons is suggestive of significant hydrogen bonding [11]. Further, the significant downfield shift of A relative to the proximal N-H in metmyoglobin [7] or low-spin ferric porphyrin models [16] suggests increased hydrogen bonding for cytochrome c peroxidase and parallels the downfield bias of the imidazole N-H, in bis-imidazole complexes, when hydrogen bonding is known to occur [18]. The pH dependence of these proximal resonances is also of interest because, kinetically (19), cytochrome c peroxidase exhibits a pK which regulates its activity. This raises the possibility that titration of the proximal N-H is a modulator of the enzyme's function, pH profiles for all of the exchangeable resonances and the proximal imidazole C 2H are shown in Fig. 3. Only resonances A, B and CzH demonstrate pH dependence between pH 6 and 5. All of these resonances maintain their intensity throughout the pH titration, indicating that they are not removed by ionization. A nonlinear least-squares analysis of the data results in the pK values reported in Table I. There is sizeable uncertainty associated with each of these numbers due to the fact that the titrations cannot be followed to the low-pH plateau region. In the case of resonance A, this is due to its overlap with the methyl 8 resonance. For the C2 H resonance, a major source of error is the large
30
I
29 •
•
28
27 18.5
c
I
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I
16,5
I
I
I
t
"
I
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PEAK 8
12.5 PEAK C
]
11,5
I
-19
-20
-21
I
i
i
i
i
~,0
5,0
6,0
7,0
8.0
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PH
Fig. 3. pH dependence of the deuterium exchangeable hyperfine resonances, A-C, and the upfield proximal histidine imidazole 2-H resonance. Note the different shift scales. The lines drawn through the experimental points were calculated from the Henderson-Hasselbalch equation, assuming the pK values for each resonance given in Table I.
natural linewidth which results in a large uncertainty (+0.2 ppm) in identifying the resonance maximum at individual pH values. For all of these resonances, the low-pH plateau lies in a region (pH 4.0) which is at the limit of this enzyme's TABLE I pH DEPENDENCE AND APPARENT pK VALUES FOR DEUTERIUM EXCHANGEABLE RESONANCES OF CYTOCHROME c PEROXIDASE Resonance
A B 8 C 2-H
Observed shift high pH
low pH
28.3 17.6 12.7 12.2 -21.0
27.8 17.2 12.7 12.2 -21.5
Apparent pK
4.9_+0.3 5.1 _+0.3
5.4_+0.5
153
stability range. As a result, processes associated with low-pH denaturation may also affect the observed resonances at this extreme. However, it is reasonable to conclude from these data that resonances A, B and C2H exhibit p K values which are similar to the pK identified for heme vinyl rotation (5.2 + 0.2) [20]. This p K is very near to the kinetically defined p K [ 14,19,21] which regulates the enzyme's reactivity. The pH-dependent shifts observed for cytochrome c peroxidase-CN parallel the kinetic data and heme-linked ionization results from Raman difference and visible spectroscopies for native enzyme [22]. The magnitude of the pH-induced hyperfine shift changes presented here is small compared to the large shift change associated with the vinyl group rotation [20], and indicates that none of these protons represents moieties which are themselves the ionization sites. In this respect, our conclusion about the absence of a formal imidazolate structure for the proximal histidine at neutral pH is confirmed by these titration data. The proximal imidazole protons, resonances A and C2H, maintain their intensity throughout the pH range. The observed titration behavior is consistent with the view that a titrating residue elsewhere in the protein is modulating the structural or electronic contributions to the proton hyperfine shifts of peaks A, B and C2 H. In summary, we conclude the following for cytochrome c peroxidase-CN from this data. (1) The proximal N-H is not lost by titration over the range of pH 4-8. (2) The evidence suggests a strong hydrogen-bonding role for the proximal N-H, but a formal imidazolate structure for the proximal histidine is not indicated. (3) Hyperfine shift titrations for all of the exchangeable protons between pH 5 and 6 are small, but reflect a pK which is the same as that which governs the enzyme's reactivity [14,19,21]. (4) Saturation transfer between the irradiated solvent water resonance and all exchangeable proton resonances which have been observed here is slow compared to other proteins [7], despite the fact that three of these resonances are deuterium exchangeable in the holoenzyme on the time scale necessary for dissolving the lyophilized protein (minutes). Whereas these studies give an indication of the proximal histidine protonation state in cytochrome
c peroxidase-CN, there emerges the question of the relevance of this work to the peroxidase reaction mechanism. We cannot test the state of ionization for either active intermediate (cytochrome c peroxidase-I or -II) due to the loss of spectral resolution encountered in these species. Nevertheless, because both intermediates are presumed to be low-spin six-coordinate forms, we believe that parallels may be drawn to the low-spin six-coordinate form studied here.
Acknowledgements We gratefully acknowledge the support of the National Institutes of Health G M 18648 (J.E.E.), the American Heart Association (J.D.S.) and the Research Corporation (J.D.S.). This research was also supported in part by the National Institutes of Health, Division of Research Resources, RR 01077, through an instrumentation grant to the Purdue University Biochemical Magnetic Resonance Laboratory, where part of the N M R experiments were performed. Part of the N M R work was also carried out at the Colorado State University Regional N M R Center, funded by National Science Foundation Grant, No. CHE78-18581. We would like to thank the staffs of both regional centers for their hospitality. We further thank Dr. J. Timothy Jackson for providing the computer programs and instruction in their use, and Professor Gerd N. LaMar, University of California, Davis, for allowing us to use a Nicolet 1180 data station.
References 1 Poulos, T.L., Freer, S.T., Alden, R.A., Xuong, N.H., Edwards, S.L., Hamlin, R.C. and Kraut, J. (1978) J. Biol. Chem. 253, 3730-3735 2 Poulos, T.L., Freer, S.T., Alden, R.A., Edwards, S.L., Skolglund, U.L., Takio, K., Eriksson, B., Xuong, N., Yonetani, T. and Kraut, J. (1980) J. Biol. Chem. 255, 575-580 3 Poulos, T.L. and Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205 4 Iizuka, T., Kotani, M. and Yonetani, T. (1971) J. Biol. Chem. 246, 4731-4736 5 Lan 8, G., Spartalian, K. and Yonetani, T. (1976) Biochim. Biophys. Acta 451,250-258 6 Yonetani, T. (1976) Enzymes 13, 345-361 7 Cutnell, J.D., LaMar, G.N. and Kong, S.B. (1981) J. Am. Chem. Soc. 103, 3567-3572
154 8 Satterlee, J.D. and Erman, J.E. (1981) J. Biol. Chem. 256, 1091-1093 9 Erman, J.E. and Satterlee, J.D. (1982) Symposium on the Interaction Between Iron and Proteins in Oxygen and Electron Transport (Ho, C., ed.), pp. 223-227, Elsevier, New York 10 Satterlee, J.D., Erman, J.E., LaMar, G.N., Smith, K.M. and Langry, K.C. (1982) J. Am. Chem. Soc. 105, in the press 11 LaMar, G.N., DeRopp, J.S., Chacko, V.P., Satterlee, J.D. and Erman, J.E. (1982) Biochim. Biophys. Acta 708, 317-325 12 Yonetani, T. (1967) J. Biol. Chem. 242, 5008-5012 13 Yonetani, T. and Ray, G.S. (1965) J. Biol. Chem. 240, 4503-4508 14 Satterlee, J.D. and Erman, J.E. (1980) Arch. Biochem. Biophys. 202, 608-616
15 Takano, T. (1977) J. Mol. Biol. 110, 537-568 16 Satterlee, J.D. and LaMar, G.N. (1976) J. Am. Chem. Soc. 98, 2804-2808 17 DeRopp, J.S. (1981) Ph. D. Dissertation, University of California, Davis. 18 Satterlee, J.D., LaMar, G.N. and Frye, J.S. (1976) J. Am. Chem. Soc. 98, 7275-7281 19 Loo, S., Erman, J.E. (1975) Biochemistry 14, 3467-3470 20 Satterlee, J.D. and Erman, J.E. (1983) J. Biol. Chem. 258, in the press 21 Conroy, C.W. and Erman, J.E. (1978) Biochim. Biophys. Acta 527, 370-378 22 Shelnutt, J.A., Satterlee, J.D. and Erman, J.E. (1983) J. Biol. Chem. 258, in the press
149
Elsevier BiomedicalPress BBA 31497
DEUTERIUM EXCHANGEABLE P R O T O N HYPERFINE R E S O N A N C E S OF L O W - S P I N C Y T O C H R O M E c PEROXIDASE AND T H E M E C H A N I S M OF PEROXIDASE CATALYSIS JAMES D. SATTERLEE a., and JAMES E. ERMAN b a Department of Chemistry, University of New Mexico, Albuquerque, N M 87131, and b Department of Chemistry, Northern Illinois University, DeKalb, I L 60178 (U.S.A.)
(Received August 3rd, 1982)
Key words: IH . N M R ; Cytochrome c peroxidase; Histidine residue," Reaction mechanism," H / 2H exchange
Deuterium exchangeable hyperfine proton N M R resonances of cytochrome c peroxidase (EC 1.11.1.5) are identified in H 2 0 solutions of the enzyme. One of these is assigned to the proximal histidine's imidazole N-H. Its shift and pH dependence indicate that an imidazolate form, which has been postulated for peroxidases, is ruled out for cytochrome c peroxidase-cyanide. A qualitative comparison of relative heine-pocket dynamics is also possible. When the bulk water resonance is irradiated with a continuous, but off acquisition, decoupler frequency the N-H resonance shows no intensity loss, indicating that saturation transfer between the proximal histidine and solvent water is either minimal, or extremely slow.
Introduction Thus far, cytochrome c peroxidase (ferrocytochrome c : hydrogen-peroxide oxidoreductase, EC 1.1 1.1.5) is the only member of the class of heme peroxidase enzymes to have its crystal structure reported [1,2]. Based on the structure Poulos and Kraut [3] have speculated about the molecular events whereby the enzyme decomposes hydrogen peroxide and they have suggested that peroxidase catalysis may be easily understood on the basis of known types of acid-base reactions. These reactions involve three critical distal amino acids in cytochrome c peroxidase's primary sequence: Arg48, His-51, Trp-52. Although these three are situated in positions to interact with heme-bound peroxide ion, none of them is readily capable of stabilizing the highly oxidized hemin iron ion which is formed during catalysis. Formally, that is considered to be Fe(IV) in both of the oxidized inter-
mediates [4,5] and, at least in cytochrome c peroxidase-I (Compound ES), a low-spin ferryl ion is implicated [6]. The possibility has been raised that stabilization of the intermediate ferryl ion may occur via the proximal histidine (His-174) [3]. In this view strong hydrogen bonding by the proximal histidine imidazole N-H (see Scheme I), or even complete
., ".'Cr
* To whom correspondenceshould be addressed. 0167-4838/83/0000-0000/$03.00 © 1983 ElsevierSciencePublishers
'P,
I
/
.2c=c
H
CH.,o ,
C=CH 2
H
CH
" fHz "~H2 CO~.
H
I
C.Hz ~cH~
150 deprotonation to form imidazolate ion, would result in excess negative charge localization on the histidine which, in turn, could be delocalized onto the ferryl ion. As a consequence, information about the state of protonation of the proximal histidine is important for the formulation of a correct enzyme mechanism. Under favorable conditions N M R is a technique suited for directly observing resonances such as the proximal N-H [7]. Although proton N M R spectra of cytochrome c peroxidase-I show extremely broad and very poorly resolved resonances [8,9], spectra of the low-spin native enzyme, such as cytochrome c peroxidaseCN, exhibit narrow, well-resolved hyperfine resonances [10-11]. Here, we present spectra of cytochrome c peroxidase-CN obtained in 90% H 2 0 which show four additional resonances which are not present in spectra obtained in 99.8% 2H20 solution. One of these is assigned to the proximal N-H. Together with the previously assigned proximal imidazole 2-H [11] (Scheme I), resonance positions at various pH values are determined and conclusions are drawn concerning the state of proximal imidazole protonation. Methods
Cytochrome c peroxidase was isolated and purified as previously described [12-14]. Lyophilized enzyme was dissolved in either 90% H 2 0 / 1 0 % 2H20 , or in 99.8% 2H20 (Merck), in order to form 1-2 mM solutions which were 0. l M in potassium nitrate (Sigma). Reconstitutions were carried out using hemin (protohemin IX, Sigma) according to the method of Yonetani [12] with the following modifications. The apoprotein was generated at 0 - 4 ° C with acidified butanone. 2 mM solutions of the apoenzyme in dilute potassium phosphate (Mallinkrodt, AR) buffer (0.3 M), pH 7.3, were cooled in an ice bath. The buffer solvent was either distilled, deionized H20, or 99.8% 2H20. A 10% mole excess of heroin dissolved in a minimal amount of 0.1 M sodium hydroxide was added to the apoprotein solutions and allowed to react for up to 1 h. The reconstituted protein was subsequently pressure-dialyzed in a micro-ultrafiltration apparatus (Amicon) using a phosphate- and hemin-permeable membrane (PM 10) to obtain a final enzyme solution in 0.1 M K N O 3 / E H 2 0 . Cy-
tochrome c peroxidase-CN was formed by adding a 1.1 mole ratio of KCN (Sigma) to the native enzyme solutions, pH was constantly monitored throughout this process with an Orion meter equipped with a Beckman combination pH electrode, pH titrations were carried out as previously described [14], with determinations made before and after each N M R data acquisition. The electrode was standardized prior to each use. p K values were calculated by a nonlinear leastsquares program to fit the experimental pH titration data to the Henderson-Hasselbalch equation. The program was written by Darrow E. Neves and provided for our use by J. Timothy Jackson. A Nicolet 1180 computer equipped with a Nicolet Zeta plotter was employed for this task. The solid curves drawn through the experimental points of Fig. 3 were generated by this program, along with the plotting routine TITPLT, provided by J. Timothy Jackson. N M R spectra at 360 MHz (8.45 T) were acquired at the Purdue University Biochemical Magnetic Resonance Laboratory or the Colorado State University Regional N M R Laboratory using a Nicolet Magnetics spectrometer. A recycle time of 200 ms was employed along with active temperature control and saturation (or partial saturation) of the residual water peak by decoupler irradiation which was gated off during the acquisition period. All shifts were referenced internally to the residual water peak and are reported relative to external 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) for reasons cited elsewhere [11]. Results and Discussion
Figs. 1 and 2 show comparison spectra for cytochrome c peroxidase-CN taken in H 2 0 and 2H20 solution. Fig. 1 reveals that resonance 8 is labile to deuterium exchange but only when the heine is removed from the protein. Fig. 1A is a spectrum of cytochrome c peroxidase-CN in which the apoenzyme, kept in H 2 0 solution, was reconstituted with hemin, also kept in H20. The protein was subsequently exchanged into 2H20 solution for this spectrum. The spectrum is a usual cytochrome c peroxidase-CN spectrum with eight resolved downfield hyperfine resonances [10,11] and is identical to native cytochrome c peroxidase-CN.
151
H - c ~
I
- - F e - -
C
i= ~ . . . .
0
~.
PROX.HIS. 2.H
l
t ,
. . . .
-5
t ,
O
. . . .
-~0
,
....
-l.5
B ,
....
- 0
,
....
- 5
-30
PPM
HEM[: 3-CH3 I,CH 3
I .
~5
.
.
.
.
310
.
.
.
.
. . 215
.
.
.
.
210
.
i . . . . 15
+
l0
~o' .... Fig. !. D o w n f i e l d
proton NMR
A
PPM
spectra o f reconstituted cy-
tochrome c peroxidase-CN at 22°C, pH 7.2, 0.1 M potassium nitrate. (A) Reconstitution with protohemin IX carried out in H20. (B) Reconstitution carried out in 2H20. Both samples were subsequently dialyzed into 2H20 before these spectra were accumulated. Upfieid portions of these spectra display no solvent dependence and are not shown. (C) Coordination of the proximal histidine imidazole ring to the hemin iron is depicted along with the numbering convention of the imidazolewhich is employed in the text. Resonance numbers (A) and imidazole carbon numbers (C) are not related.
When the apoenzyme is maintained in 2H20 solution and the reconstitution is carried out in 2H20 , resonance 8 is missing from the spectrum (Fig. 1B). We conclude that resonance 8 is a protein proton which is exchangeable with deuterium, but is only solvent-accessible in the apoenzyme. N o solvent-dependent changes are observed in the upfield hyperfine spectrum Fig. 2 demonstrates the spectrum from an enzyme preparation handled exclusively in H20. Fig. 2, compared to 1A, reveals that there are three resolved hyperfine resonances downfield which are 2H-for-H exchangeable (resonances A - C ) . Again, no differences are observed in the upfield hyperfine region, as demonstrated by Fig. 2B compared to published spectra [8,10]. The assignments in Fig. 2 are those that we have determined with deuterium-labelled hemins [10] or by inference from hemin models [11]. One of these exchangeable protons (resonance A) is found in a position characteristic of the proximal N - H in other ferric heme proteins (Ref. 7 and G.N. LaMar, personal communication). Because of this and the similari-
~5' ....
;o ....
2s' ....
2o' ....
"s' "~P.
Fig. 2. (A) Downfieid and (B) upfield hyperfine resonances of cytochrome c peroxidase-CN in 90% H20. The three proteinassociated deuterium exchangeable resonances are labelled A-C. Heme methyl and vinyl resonances and the proximal histidine imidazole 2-H are also indicated. The numbering of resonances corresponds to Scheme I, in the text. For comparison, see Fig. 1A. Conditions are the same as in Fig. 1.
ties between the heme pocket structures of cytoc h r o m e c peroxidase, m e t - m y o g l o b i n and horseradish peroxidase, we are confident in the assignment of A as the proximal histidine N-H. In support of this, we point out that the sole sources of protons which are exchangeable on the time scale of lyophilized protein dissolution must be O or N bonded. Comparing the crystallographic and sequence data for cytochrome c peroxidase and horseradish peroxidase [2,3,15] one concludes that t h e proximal and distal histidines are consistent features. They possess N - H protons which are readily exchangeable and are situated close enough to the paramagnetic center to experience sizeable contact and dipolar (proximal) [16] or dipolar (distal) shifts [7]. In addition, for cytochrome c peroxidase (and horseradish peroxidase), the guanidinium group of Arg-48 may be a source of additional exchangeable resonances, although smaller shifts would be expected due to its greater distance from the iron ion [2,3]. To summarize the evidence for assigning resonance A as the proximal N-H, we emphasize that there are three crystallographically identified, potential candidates for exchangeable protons. The proximal and distal histidines are common to all
152
of the proteins for which comparison spectra are available. In addition, for the peroxidases Arg-48 may also be included. However, resonance A lies in a characteristic region for proximal N-H protons observed for models [16], met-myoglobin [7] and horseradish peroxidase (Ref. 18 and G.N. LaMar, personal communication). The fact that the proximal N-H resonance is observed in cytochrome c peroxidase-CN rules out deprotonation. However, three pieces of information support a strong hydrogen bonding role for the proximal N-H. Crystallography suggests that either Asp-234 or Gln-239 are positioned appropriately to act as hydrogen bond acceptors (T. Poulos, personal communication). Because these are stronger bases than polypeptide carbonyls, which are the usual proton-acceptor bases, chemical intuition supports formulation of a stronger hydrogen bond. In addition, we have shown elsewhere that the resonance pattern of non-exchangeable proximal histidine protons is suggestive of significant hydrogen bonding [11]. Further, the significant downfield shift of A relative to the proximal N-H in metmyoglobin [7] or low-spin ferric porphyrin models [16] suggests increased hydrogen bonding for cytochrome c peroxidase and parallels the downfield bias of the imidazole N-H, in bis-imidazole complexes, when hydrogen bonding is known to occur [18]. The pH dependence of these proximal resonances is also of interest because, kinetically (19), cytochrome c peroxidase exhibits a pK which regulates its activity. This raises the possibility that titration of the proximal N-H is a modulator of the enzyme's function, pH profiles for all of the exchangeable resonances and the proximal imidazole C 2H are shown in Fig. 3. Only resonances A, B and CzH demonstrate pH dependence between pH 6 and 5. All of these resonances maintain their intensity throughout the pH titration, indicating that they are not removed by ionization. A nonlinear least-squares analysis of the data results in the pK values reported in Table I. There is sizeable uncertainty associated with each of these numbers due to the fact that the titrations cannot be followed to the low-pH plateau region. In the case of resonance A, this is due to its overlap with the methyl 8 resonance. For the C2 H resonance, a major source of error is the large
30
I
29 •
•
28
27 18.5
c
I
PEAK B 17.5
I
16,5
I
I
I
t
"
I
13.5
B.
PEAK 8
12.5 PEAK C
]
11,5
I
-19
-20
-21
I
i
i
i
i
~,0
5,0
6,0
7,0
8.0
-20
PH
Fig. 3. pH dependence of the deuterium exchangeable hyperfine resonances, A-C, and the upfield proximal histidine imidazole 2-H resonance. Note the different shift scales. The lines drawn through the experimental points were calculated from the Henderson-Hasselbalch equation, assuming the pK values for each resonance given in Table I.
natural linewidth which results in a large uncertainty (+0.2 ppm) in identifying the resonance maximum at individual pH values. For all of these resonances, the low-pH plateau lies in a region (pH 4.0) which is at the limit of this enzyme's TABLE I pH DEPENDENCE AND APPARENT pK VALUES FOR DEUTERIUM EXCHANGEABLE RESONANCES OF CYTOCHROME c PEROXIDASE Resonance
A B 8 C 2-H
Observed shift high pH
low pH
28.3 17.6 12.7 12.2 -21.0
27.8 17.2 12.7 12.2 -21.5
Apparent pK
4.9_+0.3 5.1 _+0.3
5.4_+0.5
153
stability range. As a result, processes associated with low-pH denaturation may also affect the observed resonances at this extreme. However, it is reasonable to conclude from these data that resonances A, B and C2H exhibit p K values which are similar to the pK identified for heme vinyl rotation (5.2 + 0.2) [20]. This p K is very near to the kinetically defined p K [ 14,19,21] which regulates the enzyme's reactivity. The pH-dependent shifts observed for cytochrome c peroxidase-CN parallel the kinetic data and heme-linked ionization results from Raman difference and visible spectroscopies for native enzyme [22]. The magnitude of the pH-induced hyperfine shift changes presented here is small compared to the large shift change associated with the vinyl group rotation [20], and indicates that none of these protons represents moieties which are themselves the ionization sites. In this respect, our conclusion about the absence of a formal imidazolate structure for the proximal histidine at neutral pH is confirmed by these titration data. The proximal imidazole protons, resonances A and C2H, maintain their intensity throughout the pH range. The observed titration behavior is consistent with the view that a titrating residue elsewhere in the protein is modulating the structural or electronic contributions to the proton hyperfine shifts of peaks A, B and C2 H. In summary, we conclude the following for cytochrome c peroxidase-CN from this data. (1) The proximal N-H is not lost by titration over the range of pH 4-8. (2) The evidence suggests a strong hydrogen-bonding role for the proximal N-H, but a formal imidazolate structure for the proximal histidine is not indicated. (3) Hyperfine shift titrations for all of the exchangeable protons between pH 5 and 6 are small, but reflect a pK which is the same as that which governs the enzyme's reactivity [14,19,21]. (4) Saturation transfer between the irradiated solvent water resonance and all exchangeable proton resonances which have been observed here is slow compared to other proteins [7], despite the fact that three of these resonances are deuterium exchangeable in the holoenzyme on the time scale necessary for dissolving the lyophilized protein (minutes). Whereas these studies give an indication of the proximal histidine protonation state in cytochrome
c peroxidase-CN, there emerges the question of the relevance of this work to the peroxidase reaction mechanism. We cannot test the state of ionization for either active intermediate (cytochrome c peroxidase-I or -II) due to the loss of spectral resolution encountered in these species. Nevertheless, because both intermediates are presumed to be low-spin six-coordinate forms, we believe that parallels may be drawn to the low-spin six-coordinate form studied here.
Acknowledgements We gratefully acknowledge the support of the National Institutes of Health G M 18648 (J.E.E.), the American Heart Association (J.D.S.) and the Research Corporation (J.D.S.). This research was also supported in part by the National Institutes of Health, Division of Research Resources, RR 01077, through an instrumentation grant to the Purdue University Biochemical Magnetic Resonance Laboratory, where part of the N M R experiments were performed. Part of the N M R work was also carried out at the Colorado State University Regional N M R Center, funded by National Science Foundation Grant, No. CHE78-18581. We would like to thank the staffs of both regional centers for their hospitality. We further thank Dr. J. Timothy Jackson for providing the computer programs and instruction in their use, and Professor Gerd N. LaMar, University of California, Davis, for allowing us to use a Nicolet 1180 data station.
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