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TH2 JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256. No. 13. Issue of July 10. pp. 6903-6912. 198) Printed in U.S.A. Substituted Hemins as Probes for Structure-Function Relationships in Horseradish Peroxidase* (Received for publication, May 16, 1980, and in revised form. January 19, 1981) Robert K. DiNello:{: and David H. Dolphin§ From the Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Y6 , 6903 y * These studies were supported by grants from the National Institutes of Health (AM 17989) and the Canadian National Science and Engineering Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. :\: This work was performed while R. K. D. was a travelling scholar of the Department of Biological Chemistry, Harvard Medical School. § Author to whom correspondence should be addressed. g Horseradish peroxidase was the flISt heme protein to be reconstituted in an active form from the apoprotein and free hemin. Since Theorell's classic work in 1941 (1), several investigators have examined the interaction of substituted hemins with apo horseradish peroxidase (2-13). This work has established that the hemin substituents in the 6 and 7 positions are important for efficient binding to apoperoxidase and generation of a highly active substituted enzyme. In spite of these studies, however, the following three aspects of the relationship between hemin structure and peroxidase function remained unclear: I} the relationship between the electronic structure of Compound I of horseradish peroxidase and its function in the one electron oxidation of various substrates; 2) whether the hemin 2- and 4-substituents interact with the peroxidase polypeptide chain or are exposed to the solvent; 3) whether both a certain chain length and free carboxyl groups on the 6- and 7-substituents are essential for rapid generation of a highly active enzyme. In recent years, evidence has accumulated that Compounds I of horseradish peroxidase and catalase (which are formed by two electron oxidations of the Fe(III) enzymes) contain Fe(IV) porphyrin 'IT-cation radicals (14-16). Morishima (17) recently challenged this concept on the basis of nmr experiments, but subsequent nmr (18) and Mossbauer studies (19) substantiate our initial porphyrin "IT-cation radical formulation. Such metalloporphyrin "IT-cation radicals may take one of two electronic ground states each of which possesses a characteristic electronic spectrum (14, 15, 20, 21). Catalase Compound I possesses a visible spectrum typical of a 2 A1u ground state "ITcation radical and reacts with the two-electron donor, hydrogen peroxide (14). Compound I of horseradish peroxidase possesses a 2A').'U "IT-cation radical visible spectrum (14) and reacts with one-electron donors. It was therefore of interest to know whether changing the ground state of the peroxidase 'IT-cation radical would change its reactivity toward hydrogen peroxide. It is probable that the difference in ground state between peroxidase and catalase Compounds I is due to differences in axial ligation of the protohemin (1) iron atom. Such differences in "IT-cation radical ground state due to axial ligation have also been observed in model systems (14, 15). Changing the axial ligation provided by the protein is not, at this time, a feasible endeavour, with the exception of photooxidation of the coordinated histidine (22). However, studies on metalloporphyrin 'IT-cation radicals have shown that the ground state may also be changed by changing the porphyrin peripheral side chains (15, 21). Accordingly, we set out to change the ground state of the horseradish peroxidase Compound I 'ITcation radical by changing the 2 and 4 side chains ofthe hemin to observe the effect on the reactivity of Compound I toward its substrate hydrogen peroxide. With regard to the interaction of the peroxidase apoprotein with 2,4-substituted hemins, other workers had concluded that these substituents were unimportant for hemin binding (8, 10) and peroxidase activity and were therefore exposed to the solvent (1O). With hydrophobic vinyl groups, however, such a situation would seem thermodynamically quite unfavourable. Accordingly, the interaction of the hemin substituents in the 2- and 4-positions with apoperoxidase was reexamined using two pairs of isoelectronic heroins, 2-formyl-4- j Low temperature visible spectra of Compounds I from peroxidases reconstituted with protohemin, 2formyl-4-vinyldeuterohemin, 2-vinyl-4-formyldeuterohemin, 2,4-dimethyldeuterohemin, and 2,4-diacetyldeuterohemin reveal that these Fe(IV) porphyrin 'IT-cation radicals take the ZA,u or peroxidase-type electronic ground state. Compound I of deuterohemin horseradish peroxidase, however, takes the 2A 1u or catalase type 'ITcation radical electronic ground state. Since deuterohemin horseradish peroxidase possesses no catalase activity, the structure of the peroxidase apoprotein (other than those factors which might influence the Compound I 'IT-cation radical ground state) is concluded to play the major role in determining the reactivity of Compound I toward hydrogen donors. Studies on peroxidases substituted with the hemins 2-formyl-4-vinyldeuterohemin, 2-vinyl-4-formyldeuterohemin, 2,4-dimethyldeuterohemin, and mesohemin reveal that isoelectronic hemins differentially interact with the peroxidase apoprotein. The hemin 2- and 4substituents are therefore concluded to interact sterically with the horseradish peroxidase apoprotein. While a variety of 2- and 4-substituted hemins were observed to bind rapidly with apo horseradish permodase to form active substituted enzymes, small changes in the substituents in the 6- and 7.positions had drastic effects on the rates of binding to apoperoxidase and the activities of the reconstituted enzymes. Even addition of a single methylene to form butyrate instead of propionate side chains drastically altered the rate of binding of the hemin and the activity of the substituted enzyme. It therefore appears that while the 2-, 4-, 6-, and 7-substituents of the hemins in horseradish peroxidase all interaet with the protein, the polypeptide chain possesses more conformational flexibility in the area which binds the 2- and 4-substituents. 6904 Substituted Hemins and Horseradish Peroxidase Function (6) RI= R2= -CH2-COOH (7) Rl=R2=-C 'N H2 #0 (8) the rapid generation of higbly active substituted peroxidases. Preliminary accounts of this work have appeared (24, 25). EXPERIMENTAL PROCEDURES Rj =Rz = -CH=CH z R j:: -CHO Rz:' -CH=CH z 2 3 R j;: CH==CH z 4 R 1 " Rz =- CH3 5 9 R j =R z =-CHzCH 3 10 R1=Rz =-g-CH3 110 R, :: I\~-CHO R1=Rz=-H ~l-)Q>-OH R2 =H H3C H OH lib RI:H ~1-)Q>-OH Rz: H3C H OH 12 15 = R1=R z -CHO HO ~1~ H3C H 16 17 Rz=H CH 3 Materials Horseradish peroxidase type VI (R. '" 3.0-3.2), a mixture of isozymes Band C, o-dianisidine, mesidine, protohemin (type III, equine), and a crystalline suspension of beef liver ;::atalase (2x crystallized) were all obtained from Sigma. Hematoporphyrin dihydrochloride was obtained from Nutritional Biochemicals. N,N-dimethylacetamide was obtained from Fisher and was redistilled from anhydrous copper sulfate. Hydrogen peroxide (30% aqueous solution) was obtained from Mallinckrodt and its concentration was measured by titration with potassium permanganate previously standardized to sodium oxalate. All other chemicals were reagent or the best commercially available grade. DEAE-cellulose (Whatman DE-52) and CM-cellulose (Whatman CM-52) were obtained from H. Reeve Angell. Cheng Chin polyamide thin layer plates were pUrchased from Gallard Schlessinger. Machery and Nagel polyamide CC6 «0.07 mm) was obtained from Brinkmann and silica gel for adsorbtion chromatography (catalog no. 402747) from lCN Pharmaceuticals. Silica gel thin (250 fLm) and thick (2000 fLm) layer plates were purchased from Analtech. Deuterohemin (9) and diacetyldeuterohemin (10) were prepared according to traditional procedures (26) and (27) and purified by chromatography on polyamide as described by DiNello and Chang (28). Mesoporphyrin-free acid was prepared as described by Caughey et al. (29) and isolated as the dihydrochloride as described by Corwin and Erdman (30). Mesohemin (5) was prepared by the ferrous sulfateacetic add method described in Falk (31) and was purified on polyamide (28). All of the above hemins were crystallized as the pentacoordinate chloride complexes as described by Caughey et al. (29).2- (and 4}-l'-(4"-Resorcinyl)ethyldeuterohemin (11. a and b) was prepared as described by DiNello and Chang (28). Protoporphyrin-free acid was prepared from hematoporphyrin dihydrochloride by the method of DiNello and Chang (28). Protoporphyrin di-tertiary butyl ester was prepared via the acid chloride by the method of DiN ello and Dolphin (38). Protoporphyrin di-tertiary butyl ester was converted to a mixture of 2.formyl.4-vinyldeuteroporphyrin DTBE,' 2-vinyl,4-formyldeu. 1 The abbreviations used are: DTBE, di-tertiary butyl ester; apo HRP, horseradish apoperoxidase. Downloaded from www.jbc.org at University of British Columbia on October 6, 2008 vinyldeuterohemin (2) and 2-vinyl-4-formyldeuterohemin (3) and 2.4-dimethyldeuterohemin (4) and mesohemin (5). In such pairs of hemins. the electronic structure and associated properties of the isolated hemins are identical (23). Any difference in the properties of the two hemin-substituted peroxidases must therefore be due to steric interaction of the hemin 2- and 4-substituents with the peroxidase apoprotein. Finally, the set ofisoelectronic hemins, protohemin (1) and the dibutyric acid hemin (6) were used to confum and further clarify literature reports that the hemin 6- and 7-substituents were important in both hemin binding and generation of a higbly active substituted enzyme (7-9). Comparison of the properties of these hemin-substituted peroxidases elucidated the effect of a small increase in chain length of the 6- and 7substituents while maintaining free carboxyl groups. Attempted combination of other 6- and 7-substituted hemins such as the diamide (7) and the dialcohol (8) with the peroxidase apoprotein showed that functionalities which possess some of the hydrogen bonding capabilities of the free carboxyl group are unable to duplicate the effect of free carboxyls in 6905 Substituted Hemins and Horseradish Peroxidase Function ( 13) 14b TABLE I Extinction coefficients of the Soret band of substituted hemins in the pyridine hemochrome assay Hemin Protoheroin (1) 2-Formyl-4-vinyldeuterohemin (2) 2-Vinyl-4-formyldeuterohemin (3) 2,4-Dimethyldeuterohemin (4) Mesohemin (5) Dibutyric acid hemin (6) Protohemin diamide (7) Dialcohol hemin (8) Deuterohemin (9) 2,4-Diacetyldeuterohemin (10) 2- and 4-1'-(4" -Resorcinyl)ethyldeuterohemin (Ua, Ub) 2,4-Diformyldeuterohemin (12) (X- and j1-Nitrodeuterohemin (14a, 14b) 2- and 4-1'p-Cresylethyldeuterohemin (15, £mM Source 191.5 125.3 126.3 131.5 139.5 203.6 191.5 191.5 134.6 115.4 169.6 Ref. 31 128.3 84.4 - a - d Ref. 59 16) 2,4-Di-I' -p-cresylethyldeuterohemin (17) a a - a -c - aa b d Measured using a weighed sample of the chloro Fe(IIl) complex. Based on the iron content of a pure noncrystalline sample. Assumed to be identical to that of protohemin. d Volumetric measurements used to calculate concentrations of solutions of these heroins. a b C Deuteroporphyrin dimethyl ester was synthesized and converted to a mixture of nitrodeuteroporphyrins as described by Caughey et al. (28). The porphyrins were hydrolyzed in 18% HCI and metalated using the ferrous sulfate-acetic acid method (31). (X- and j1-Nitrodeuterohemin (14) were purified by chromatography on silica gel thick layer plates as described above for the formylvinylhemins. The p-cresylethylhemins (15-17) were prepared as described by DiNello and Dolphin.2 Table I gives the extinction coefficients used to calculate hemin concentration using the pyridine hemochrome assay (39). Methods Assays-o-Dianisidine peroxidase assays and catalase assays were carried out as described in the Worthington Enzyme Manual. Because catalase catalyzes a second-order reaction, the time necessary for the 2 R. K. DiNello and D. Dolphin, unpublished observations. Downloaded from www.jbc.org at University of British Columbia on October 6, 2008 140 teroporphyrin DTBE and 2,4-diformyldeuteroporphyrin DTBE by permanganate oxidation using the method of Caughey et al. (29). The diformylporphyrin and a mixture of the formylvinylporphyrins from 3 g of starting material were purified by chromatography on silica gel (grade IV, 1 kg) using chloroform ether (100:1) to remove the starting material and chloroform ether (50:1) to separate formylvinyl- and diformylporphyrins. Diformyldeuteroporphyrin DTBE was deesterified in dry methylene chloride saturated with anhydrous HCl for 8 h. The solvent was removed in a vacuum and the free acid crystallized from pyridineacetic acid. The iron complex of the free acid (12) was synthesized by the method of Adler et al. (32) using FeClz·H2 0 as the iron salt, chromatographed on polyamide as described by DiNello and Chang for deuterohemin (28) and crystallized from pyridine/chloroform/ acetic acid/HCI (29). The pure isomers, 2-formyl-4-vinyldeuteroporphyrin DTBE and 2vinyl-4-formyldeuteroporphyrin DTBE were prepared from the corresponding photoprotoporphyrin isomers. The mixed isomeric photoprotoporphyrin di-tertiary butyl esters were prepared according to the method of Inhoffen et al. (33) except that the photolysis was performed in methylene chloride containing 10% pyridine (lliter/g of starting material)(34-37). The photoprotoporphyrin isomers were converted to the corresponding formylvinylporphyrin isomers in 80% yield as described by DiNello and Dolphin (38). The pure isomeric formylvinylporphyrins were deesterified as described for diformyldeuteroporphyrin DTBE. It is especially advantageous to use tertiary butyl esters in situations such as this because they can be removed in the absence of water. Hydration of vinyl groups has long been a problem during hydrolysis of methyl esters (31). The formylvinylporphyrin-free acids were metalated according to the ferrous sulfate method described in Falk (31) and 2-formyl-4vinyldeuterohemin (2) and 2-vinyl-4-formyldeuterohemin (3) purified on silica gel thick layer plates (one thick layer plate (20 x 20 cm) for each 50 mg of starting material) with benzene/methanol/fonnic acid (110:30:1, v Iv) as developing solvent. The heroins thus obtained ran as single spots on silica gel thin layers (benzene/methanol/formic acid, 110:30:1) and polyamide thin layers (benzene/methanol/formic acid, 110:30:1, and methanol/acetic acid, 100:2). They were crystallized as described by Caughey (29). A totally synthetic sample of 2,4-dimethyldeuteroporphyrin dimethyl ester was a gift of John B. Paine III (The University of British Columbia). It was hydrolyzed in 20% aqueous HCI for 24 h at room temperature. The free acid was metalated by the ferrous sulfateacetic acid method described by Falk (31) and the hemin was purified by chromatography on silica gel thick layers as described above for the formylvinylhemins. Protoporphyrin dinitrile (13) was a gift of C. K. Chang. It was converted to the dimethyl ester in methanol containing 2% water, which was saturated with HCI gas for 48 h at room temperature. The dimethyl ester was hydrolyzed in 18% HCI at room temperature and metalated (ferrous sulfate-acetic acid method) (31). The hemin with butyrate side chains in the 6 and 7 positions (6) was then purified by chromatography on polyamide using methanol/acetic acid (100:2) as eluent. 6906 SUbstituted Hemins and Horseradish Peroxidase Function E",M 16 E,.M 90 18 1'0 14 16 60 '2 10 40 30 E",M E",M I1J 'oS eo 12 4 8 20 10 400 500 600 0 100'>'("",) ltlOX(MI) 0 400 500 600 1OOIoml FIG. 1 (left). Low temperature spectra of protohemin-rooonstituted HRP (--) and its Compound I ( ....... ). FIG. 2 (center). Low temperature spectra of Z.formyl-4-vinyldeuterohemin-reconstituted HRP (--) and its Compound I ( .... 3 (right). ). FIG. Low temperature spectra of 2-vinyl-4.formyldeuterohemin-rooonstituted HRP ( ....... ) and its Compound I (-). FIG. 4. Low temperature spectra of 2,4.dimethyldeuterohemin-reconstituted HRP (--) and its Compound I ( ...... ). hemin under the standard conditions. The dialcohol (8) was incubated with peroxidase in 5 mM potassium acetate, pH 4.4, containing 10% by volume acetone, again for solubility reasons. Reconstitution of peroxidase with protohemin-free acid in the Tris-pyridine buffer system gave a highly active enzyme (see Table IV). Other workers have demonstrated reconstitution in acetone-buffer mixtures to give active peroxidases (43). Low temperature spectra were recorded in the following manner. N,N-dimethylacetarnide (1.68 m1) and distilled water (0,48 m1) were combined in a low temperature optical cell and cooled to -8 to -10°C. Hemin-substituted horseradish peroxidase (to give a final concentration of -9 JlM) in 10 mM potassium phosphate, pH 6.0 (0.24 m1), was then added, the solution thoroughly mixed, and the low temperature cell carefully placed in the low temperature Dewar charged with liquid propane. After allowing the temperature of the cell to equilibrate to -42°C, the assembly was placed in the modified cell compartment of a Cary 17 spectrophotometer. After recording the spectrum of the Fe (III) enzyme, hydrogen peroxide (1.2-M eq in 24 ,.u of N,N-dimethylacetamide) was added, the solution mixed, and the compound I spectrum recorded. RESULTS Low Temperature Optical Spectra af Hemin-substituted Peraxidases-Low temperature oxidations of protohemin (1), 2-formyl-4-vinyl-deuterohemin- (2}, 2-viny14-formyldeuterohemin- (3), and 2,4-dimethyldeuterohemin- (4) reconstituted peroxidases reveal that Compounds I of these hemin-substituted enzymes all possess the same metalloporphyrin 7l-cation radical ground state (see Figs. 1-4). The broad, rather invariant absorbance in the region from 530-650 nm is characteristic of the 2A2u ground state (14, 15, 2l). Diacetyldeuterohemin (10) peroxidase Compound I apparently also takes the 2A2u ground state (see Fig. 5), The increased absorption of this Compound I in the region from 530-650 nm over that of protohemin peroxidase Compound I is due to the presence of -10% Compound II, the one electron reduction product of Compound I, which absorbs strongly in this region. Deuterohemin (9) horseradish peroxidase Compound I, however, is revealed to possess a visible spectrum character- Downloaded from www.jbc.org at University of British Columbia on October 6, 2008 concentration to fall from 10.3 to 9.2 mM was used to calculate specific activity. Assays for low temperature catalase activity were carried out in the following manner. Em:.yme (l00 p.g of either a hemin-substituted peroxidase or catalase) and hydrogen peroxide (38 /-Imol) were incubated at -42°C in a mixture of 30% 3.3 roM potassium phosphate buffer, pH 6.0/70% N,N-dimethylacetamide, final volume, 1 mi. After 1 h, a 0.1-m1 aliquot of this solution was added to 1.9 mI of 0.1 M sodium acetate, pH 4.9, containing 30 /-IIDol of mesidine. The oxidation of any hydrogen peroxide was complete within 1 min at 0 ° C. Hydrogen peroxide remaining after incubation at -42°C was quantitated by the method of Paul and Avi-Dor (40) using a stoichiometry of 3 mol of hydrogen peroxide consumed for every mole of the oxidized product of mesidine produced and an EroM of 1.27 for the mesidine oxidation product. When samples of hemin-substituted peroxidases were assayed for low temperature catalase activity, the substituted peroxidase was sufficient to catalyze the oxidation of mesidine by residual hydrogen peroxide. When catalase samples were assayed, 100 p.g of horseradish peroxidase were added to the assay mixture before incubation with mesidine. In the case of catalase, low temperature activity was indicated by voluminous formation of gas bubbles within 5 min of commencement of incubation at -42°C. That destruction of peroxide was complete before warming to room temperature was verified by adding 100 p.g of horseradish peroxidase and 15 p.mol of mesidine to a solution of catalase (100 Jlg) and hydrogen peroxide (38 /-IIDoi) at -42°C. In the presence of catalase, no oxidation of meaidine (as evidenced by development of the characteristic purple color of the mesidine oxidation product) within 1 h is detected. Mesidine is oxidized by peroxidase at -42°C in the absence of catalase. Preparation of Apo Horseradish Peroxidase and Reconstitution of Apo Horseradish Peroxidase with Modified Hemins-Apo horseradish peroxidase was prepared essentially according to the method of Teale (41) as modified by Yonetani (42). Horseradish apoperoxidase was combined with substituted hemius essentially according to the method of Tamura et al. (8). Samples of apoperoxidase (5-10 mg/m1 in 10 mM Tris-HCl, pH 8.0) were combined with an equal volume of 20 mM Tris-HCI, pH 8.0, containing a 1.2-fold excess of the desired hemin. After 90 min at 0 °C, the samples were passed through a DEAE-cellulose column equilibrated in 10 mM Tris-HCl, pH 8.0, to remove excess hemin not bound at the peroxidase active site. The substituted peroxidase was then chromatographed on carboxymethyl cellulose as described by Tamura et al. (8). Tamura et al. (8) eliminated the DEAE-cellulose column and directly chromatographed their substituted peroxidases on CM-cellulose as above. We found that in cases where hemius bound slowly and incompletely at the active site within the desired time limit, direct chromatography on carboxylmethyl cellulose failed to completely separate active heminsubstituted peroxidase from inactive species in which the hemin had apparently bound nonspecifica1ly. This was especially true if an excess of hemin was used to hasten binding. In these cases, the active heminsubstituted peroxidase could easily be purified by DEAE-cellulose chromatography. When the hemin combined with apoperoxidase did not possess free carboxyl groups, the DEAE-cellulose column was omitted as such hemius do not bind to DEAE-cellulose. Proto hemin diamide (7) was bound to apoperoxidase in 10% pyridine in 15 mM Tris-HCl, pH 8.0, because of the low solubility of this 6907 Substituted Hemins and Horseradish Peroxidase Function E",M Em'" Em M Em'" 80 ItS 70 14 100 20 10 II 10 HI t;... 10.." 14 ro 60 eo 10 ~ • 40 30 6 "-' ...........~...... \ 20 \ 10 400 500 600 • ."-~-... 1OO1.!..ml 20 10 400 500 eoo 100AImll 4OO!lOO 600 500 600 700 A(nml FIG. 9. Low temperature spectrum of Mesohemin-reconstituted HRP (--) and its Compound I ( •••• ). mide-buffer from -42 to 0 "C resulted in a change from the spectrum shown in Fig. 6 to spectrum A of Fig. 8. Spectrum B was obtained after 5 min at 0 "C. Cooling the solution which FIG. 8. Spectra of deuterohemin-reconstituted HRP. A, spec- gave spectrum B of Fig. 8 to -42 "C did not restore the trum of deuterohemin-reconstituted HRP compound I formed at spectrum to that shown in Fig. 6 indicating a thermal equilib-42 ·C and warmed to 0 ·C for 1 min. B, spectrum of the sample rium was not responsible for the observed changes. Further described in A after 5 min at 0 ·C. C, room temperature Compound incubation of deuterohemin peroxidase Compound r at 0 "C I spectrum of deuterohemin-reconstituted HRP recorded by Makino resulted in gradual conversion to Compound II (spectrum D and Yamazaki (9). D, spectrum of compound II of deuterohemin HRP formed by adding IO-fold excess of luminol to com- of Fig. 8). In view of the similarity of spectrum A of Fig. 8 to published spectra of deuterohemin peroxidase Compound I pound I at -42 cc. (spectrum C of Fig. 8) (8,9), we conclude that the samples of deuterohemin peroxidase Compound I used to record the istic of the 2A lu or catalase-type metalloporphyrin 1T-cation published spectra were contaminated with -5% Compound II. radical ground state (Figs. 6 and 7). The long wavelength peak This hypothesis is consistent with a reported half-life of 15 and less intense shorter wavelength peak are characteristic of min at 20 "C for deuterohemin peroxidase Compound I (8) 2A lu ground state 1T-cation radicals (14, 15, 21). In the same and the time required to obtain II. visible spectrum. region of from 530-650 nm, the absorption of 2A2u ground state All substituted peroxidases studied in this work show Com1T-cation radicals is rather invariant (Figs. 1-5) (14, 15, 21). pound I-visible spectra which unambiguously fit either the The long wavelength visible spectrum has been established as 2 Al " or the 2 A2u 1T-cation radical ground state with the excepdiagnostic of the cation radical ground state (14). The 2,4- tion of meso hemin horseradish peroxidase (Fig. 9). This Comdiformyldeuterohemin (12) and the a- and p-nitrodeutero- pound I shows a long wavelength peak and a shorter wavehemin (14a. 14b) enzymes were also studied. These suhsti- length peak of about equal intensity. A broad absorbance tuted peroxidases gave Compounds I which were too unstable between 530 and 650 urn is, however, also observed, with the to study optically. peaks superimposed on what resembles a 2 A2u ground state Since published room temperature spectra of deuterohemin 1T-cation radical spectrum. This broad absorbance is especially (9) peroxidase Compound I are more similar to those shown noticeable between 580 and 530 urn where tbe absorbance of by 2A lu ground state 1T-cation radicals (8) and (9), the spectrum 2 A1u 1T-cation radicals is either rapidly decreasing or near a of this Compound I was studied further. Warming of a solution minimum (compare Fig. 9 with Figs. 7 and 8 and Refs. 14, 15, of deuterohemin peroxidase Compound I in dimethylaceta- and 21). Compound I of mesohemin (5) peroxidase thus gives Downloaded from www.jbc.org at University of British Columbia on October 6, 2008 FIG. 5 (left). Low temperature spectra of diacetyldeuterobemin-reconstituted HRP (--) and its Compound I ( • ••• ). FIG. 6 (center). Low temperature spectra of deuterohemin-reconstituted HRP (--) and its Compound I ( • ••• ). FIG. 7 (right). Low temperature spectrum of deuterohemin reconstituted-HRP Compound I ( .... ) and room temperature spectrum of catalase Compound I (--). Substituted Hemins and Horseradish Peroxidase Function 6908 TABLE II Catalase activity of reconstituted peroxidases at 25 and -42 ° C. Enzyme Activity at 25 ° Activity at -42 °c p.mol H 2 0, remain· ing after I·h incubation at -42 ° C units/mg Protohemin HRpa Deuterohemin HRP Mesohemin HRP Catalase a o 38 ± 38 ± 38 ± 0.0 ± o o 36,000 ± 500 1.0 1.0 1.0 1.0 HRP, horseradish peroxidase. TABLE III Relative specific activities of hemin· substituted horseradish peroxidases in the o-dianisidine assay Not all samples were from the same reconstitution. A reconstitution with protohemin was always performed to standardize specific activities. Also, see Ref. 51. Peroxidase Specific activity 3879 ± 141 (n = !O) 3574 ± 284 (n = 10) 3642 ± 166 (n = 10) 1161 ± 57 (n = 10) 113 100 85 99 78 41 26 96 64 25 32 7 (50 mM NaCl) <0.5 (100 mM NaCI) 7 (50 mM NaCI) <0.5 (100 mM NaCl) 79 0.5 or less .8 .6 .4 .2 o time (hr) I 2 3 4 5 6 FIG. 10. Binding of protohemin to apo HRP in the presence (0) and absence (e) of 1 eq of isomer 2 of the di-p-cresylethylhemin (17). Experiment 1, equal volumes (0.8 mI) of apoperoxidase (2.11 x 10-4 M apo HRP in 10 roM Tris-HCl, pH 8.0), protohemin (2.11 x 10-4 M), and 20 roM Tris-HCl, pH 8.0, were mixed at 0 °C. At the times indicated, 0.3-mI samples were withdrawn, chromatographed on DEAE-cellulose as described under "Experimental Procedures," and diluted to 2.4 mi. The optical density of the peroxidase Soret maximum at 401 nm was used as a measure of Heme binding (e). Experiment 2, the incubation was the same as above except 0.8 mI of 2.11 x 10-4 M di-p-cresylethyl hemin isomer 2 (17) in 20 mM Tris-HCl was substituted for the Tris buffer (0). 00397 1.4 1----100% bound---------------t 1.2 1.0 .8 .6 .4 .2~ o time (hr) 5 10 15 20 25 FIG. 11. Binding of 2- (and 4)-1'-(4" resorcinyl)ethyldeuterohemin to apo HRP. Experiment 1, apo HRP (1.4 X 10-4 M, 1.2 mI) in 10 roM Tris-HCl, pH 8.0, and the hemin (1.4 x 10-4 M, 1.2 mI) were mixed and incubated at O°C. At the times indicated, O.4-mI samples were withdrawn, chromatographed on DEAE-cellulose as described under "Materials and Methods," diluted to 2.4 mI, and the optical density at 397 nm, the Soret maximum of the substituted peroxidase, measured (e). Experiment 2, a single sample of apoperoxidase was incubated as above, but with 10 eq of the hemin, after 25 h, it was treated as above (0). , a spectrum with characteristics of both ground states. Low Temperature and Room Temperature Enzymic Activity of Substituted Peroxidases-Although deuterohemin (9) horseradish peroxidase Compound I possesses a visible spectrum typical of a 2A\u ground state metalloporphyrin 'IT-cation radical and Compound I of mesohemin (5) peroxidase shows some features of the 2A!u or catalase type spectrum, neither possesses any catalase activity (Table II) in assays performed at 30 and -42°C. Catalase shows activity at both temperatures (Table II). Incubation of protohemin (1), mesohemin (5), and deuterohemin (10) peroxidases at -42°C with peroxide and the peroxidase substrate, however, results in the generation of the purple color characteristic of the mesidine oxidation product. Since the conditions used to record the low temperature spectra involved a solvent system not normally encountered by horseradish peroxidase, it was of interest to know what catalytic competence was exhibited by the substituted peroxidases under low temperature conditions and whether the enzyme was damaged by exposure to the solvent system at low temperature. Protohemin- (1) reconstituted peroxidase, when incubated for 1 h at -42°C in the dimethylacetamidebuffer mixture, was observed to have the same specific activity as samples which were not exposed to the low temperature solvent mixture (Table IV). In addition, all samples of substituted peroxidases used in the low temperature studies form Compounds I rapidly and completely at -42°C. The one electron reduction product, Compound II, can be obtained within 20 min by addition of a lO-fold excess of luminol at 00401 1.0 y 2- and 4-I'-(4"Resorcinyl)ethyl deuterohemin Apoperoxidase Peroxidases Native protohemin Protohemin-reconstituted Protohemin-reconstituted (incubated at -42°C) Dibutyric acid hemin-reconstituted g Dialcoholhemin(8) TABLE IV Activities of protohemin and dibutyric acid hemin· reconstituted peroxidases in the o·dianisidine assay Apoperoxidase was reconstituted and purified as described under "Methods." A sample was assayed directly by the o-dianisidine method. Another sample (0.13 mg) of the reconstituted peroxidase was incubated for 1 h in 1.0 mI of the low temperature solvent system described under "Methods" and then its specific activity was assayed. The dibutyric acid hemin was incubated with the apoprotein for 48 h. j Native protohemin Protohemin reconstituted Protohemin reconstituted in 10% pyridine/IO roM Tris-HCl (pH 8.0) 2-Formyl-4-vinyldeuterohemin 2-Vinyl-4-formyldeuterohemin 2,4-Diformyldeuterohemin Deuterohemin 2,4-Dimethyldeuterohemin Mesohemin ex- and p-Nitrodeuterohemin Dibutyric acid hemin (6) Protohemn diamide Protohemin-reconstituted peroxidase % activity -42°C. All the enzymes for which Compound I spectra were obtained were shown to undergo the full catalytic cycle at -42 °C. Binding of Hemins with Bulky 2- and 4-Substituents to Substituted Hemins and Horseradish Peroxidase Function Apo Horseradish Peroxidase-Tamura et al. (8) and Ohlsson and Paul (10) concluded that hemin 2- and 4-substituents are not important for binding to apoperoxidase and activity of the substituted enzyme. This conclusion leads to the prediction that bulky 2- and 4-substituents will not impair binding of hemins to apo horseradish peroxidase. Our studies indicated, however, that this is not the case. The di-p-cresylethylhemin (17) possesses two asymmetric carbon atoms. Four configurational isomers are thus possible. consisting of two pairs of enantiomers. Neither pair is observed to form an active substituted peroxidase by direct assay of mixtures of the hemins and apoperoxidase for peroxidase activity. DEAE-cellulose removes all hemins from such mixtures. (Hemins studied in this work which generated active substituted peroxidases became unavailable to DEAE-cellulose.) Finally, when isomer 2 of the di-p-cresylathyl hemin is included in mixtures of protohemin and apoperoxidase, no effect on the rate of binding of protohemin is detected (Fig. 10). These data indicate the di-pcresylethyl hemins do not bind at the peroxidase active site. KO% ~nd.. -...-.. -. .• ~".~..~-----------------__- , 0.80 0.60 0.40 0.20 ,..'" .................... ! o time (min) 50 100 1.50 200 250 FIG. 12. Binding of 2-formyl-4-vinyldeuterohemin ( ..... ) and 2-vinyl-4-formyldeuterohemin (--) to apo HRP. At t = 0, hemin (final concentration, 1.30 X 10-4 M) and apoenzyme (final concentration, 1.16 X 10- 4 M) in 0.015 M TrilI-HCl were combined. At the times indicated in the figure, samples were removed and chromatographed on DEAE-cellulose to remove unbound hemin. The amount of bound hemin was assayed by measuring the optical density at 412 nm, the Soret maxima of the substituted proteins. The OD412 100% bound line refers to the 2-formyl, 4-vinyldeuterohemin-substituted enzyme. TABLE V Specific activities of substituted horseradish peroxidases in the o· dianisidine assay Peroxidases were reconstituted as described under "Methods." 3904 ± 117 (n = 10) 3454 ± 192 (n = 10) 2208 ± 191 (n = 10) Native protohemin Protohemin-reconstituted Mesohemin-reconstituted Dimethyldeuterohemin-reconstituted Spirographis hemin-reconstituted Isopirographis hemin-reconstituted 3313 ± 136 (n = 10) 3425 ± 174 (n = 20) 2704 ± 168 (n 20) ~M Similar results were obtained with the mono-p-cresylethyl hemins (15) and (16). The binding of a mixture of the 2- and 4-resorcinylethyl hemins (l1a) and (llb) to apoperoxidase was also investigated. Each structural isomer consists of two optical isomers. The binding of four hemins to apoperoxidase was thus assayed in these studies. As can be seen, the results are consistent with a maximum of 25% of the added hemin binding to apoperoxidase (Fig. 11). The resultant-substituted peroxidase proves highly active after DEAE-cellulose and CM-cellulose chromatography (Table III). Peroxidases Substituted with Isoelectronic Hemins Modified in the 2- and 4·Positions-The above results are consistent with the hemin 2- and 4-substituents being buried in the interior of horseradish peroxidase because they can be made large enough to adversely affect binding. No conclusions can be made, however, about the interaction of small 2- and 4substituents with the peroxidase polypeptide chain. To answer this question, we examined the binding of 2-formyl-4-vinyldeuterohemin (2) and 2-vinyl-4-formyldeuterohemin (3) to horseradish peroxidase. These hemins are shown to bind at different rates to apoperoxidase (Fig. 12), the enzymes substituted with these hemins have different specific activities (Table V) and the spectra of the ferrous and ferrous carbon monoxide enzymes possess significantly different wavelength maxima (Figs. 13 and 14). Since 2-formyl-4-vinyl-deuterohemin and 2-vinyl-4-formyldeuterohemin possess identical electronic properties outside their substituted peroxidases (23), the observed differences can only be due to steric interaction of the 2· and 4-substituents with the peroxidase polypeptide chain, Another pair of isoelectronic hemins, mesohemin and 2,4dimethyldeuterohemin, also exhibited different properties when combined with apoperoxidase. These substituted peroxidases differ considerably in their compound I spectra (Figs. 4 and 9) and in their specific activities (Table V). Peroxidases Substituted with Hemins Modified in the 6· and 7-Positions-The isoelectronic hemins, protohemin (1) and the dibutyric acid hemin (6), bind to apoperoxidase at vastly different rates (see Fig. 16) and possess different specific activities (see Table III). In addition to terminal carboxyl groups, the length of the hemin 6- and 7-side chains is thus important for rapid and efficient binding to apoperoxidase to form an active enzyme. Hemins with side chains in the 6 and 7 positions of identical length to those of protohemin, but with blocked carboxyl groups such as protohemin diamide (7) and the dialcohol hemin (8) possess binding properties even more aberrant than those of the dibutyric acid hemin. Less than 10% of each of these hemins was eluted from eM-cellulose by 50 roM NaCI in 5 roM potassium acetate, pH 4.4, after combination with apoperoxidase in a 1:1 molar ratio. Additional protohemin r - - - - - - - - - - - -.... Eir,M 24 ~M ~M 100 20 100 16 80 12 20 16 12 8 40 4 20 'I'OO.Mm) 6909 a 4 0 700).(nm) FIG. 13 (left). Spectra of Fe(D) 2formyl-4-vinyldeuterohemin reconstituted HRP ( - ) and Fe(D) 2-vinyl-4-formyldeuterohemin-reconstituted HRP ( ...... ). FIG. 14 (right). Spectra of Fe(U) CO 2-formyl-4-vinyldeuteroheminreconstituted HRP (--) and Fe(II) CO 2-vinyl. 4-formyldeuteroheminreconstituted HRP ( ...... ). 6910 Substituted Hemins and Horseradish Peroxidase Function 0°401 1.2 however, do not possess free carboxyl groups and do not bind to DEAE-cellulose. Binding of Excess Protohemin to Horseradish Peroxidase-It was possible to show binding of excess protohemin to apoperoxidase. Removal of this hemin had no effect on total enzyme activity (Table' VI). Whether specific binding sites exist for additional protohemin molecules or if such binding is nonspecific cannot be said at this time . 100 'Yo bound 1.0 .6 .6 .4 DISCUSSION .2 ° lime lhr) 1O 20 50 FIG. 15. Binding of protohemin (0) and the dibutyric acid hemin (6) (e) to apo HRP. Equal volumes of hemin solution (1.4 x 10- 4 M) in 20 mM Tris-HCI, pH 8.0, and apo HRP (1.4 x 10-4 M) in 10 mM Tris-HCI, pH 8.0, were mixed and incubated at ac. At the times indicated, O.4-ml samples were withdrawn and chromatographed on DEAE-cellulose to remove excess hemin as described under "Materials and Methods." The samples were then diluted to 2.4 ml and the optical density at 401 nm (the Soret maxima of the substituted peroxidases) was measured. ° Total activity units/mg units 1683 ± 83 3088 ± 144 3367 ± 166 3498 ± 175 diamide (7) and the hemin dialcohol were eluted by 100 mM NaC!. These hemin-enzyme complexes possess optical spectra which resemble that of free protohemin (Fig. 16) and exhibit very low specific activities (Table III). The low activity and atypical spectra of the dialcohol- and diamide-substituted enzymes eluted by 50 mM and 100 mM NaCI from CM-cellulose indicate that a substantial amount of the hemins may not be bound at the active site. Similar results were obtained with peroxidase substituted with the resorcinylethyl hemins (lla, llb) and even protohemin (1) (see below) when DEAE-cellulose chromatography was omitted in their preparation. The dialcohol and diamide hemins, , Before DEAE-cellulose After DEAE-cellulose Specific activity based on protohemin content y Stage of purification g TABLE VI Binding of excess protohemin to Apo horseradish peroxidase Apo horseradish peroxidase (0.7 ml) (2.68 x 10-4 M) and 5 eq of protohemin were incubated as described under "Methods." The mixture was chromatographed on CM-cellulose and then dialyzed against 10 mM Tris-HCI, pH 8.0. One sample (1.2-ml) was chromatographed on DEAE-cellulose and diluted to 2.0 ml. A second 1.2 mi was diluted to 2.0 ml without DEAE-cellulose chromatography. j FIG. 16. Spectra of proto hemin-reconstituted HRP (--) and the dialcohol hemin- (8) reconstituted HRP ( •••• ) eluted from CM-cellulose by 100 roM NaCl. Both in 10 mM Tris-HeI, pH 8.0. It proves possible through hemin substitution to change the ground state of the horseradish peroxidase Compound I 7/'cation radical. Compound I of native protohemin (1) peroxidase takes the 2A2u ground state whereas that of deuterohemin (9) peroxidase takes the 2A 1u or catalase-type ground state. In spite of the remarkable similarity between the visible spectra of Compounds I of deuterohemin horseradish peroxidase and catalase, the former possesses no catalase activity at room temperature or at -42°C. The structure of the peroxidase apoprotein and not the electronic structure of the Compound I 'IT-cation radical is thus concluded to play the major role in determining that the enzyme reacts rapidly with one-electron donors and not with hydrogen peroxide. An extensive and sometimes contradictory literature exists on how to stabilize horseradish peroxidase Compound I in order to obtain accurate optical spectra. Various workers have opined that the stability of compounds I is determined by the purity of the protein preparation (44) or the purity of the water used in making solutions of the protein (45). The existence of an endogenous electron donor within the enzyme was long advocated by workers in the field (46,47) and has recently been revived by Makino et at. (48) who obtain biphasic curves for the spontaneous decomposition of Compound I to Compound II. At various times, preincubation of peroxidase with hydrogen peroxide (49) and oxidation at low temperatures in aqueous-organic mixtures (50) have been used as means of obtaining stable compound I spectra. We found low temperature oxidation the best method of obtaining stable Compound I spectra of substituted horseradish peroxidases. Even samples of the native enzyme of comparatively low purity (R z between 2.6 and 2.8) gave Compound I spectra stable in excess of 30 min at -42 DC. Diacetyldeuterohemin (10) peroxidase, whose Compound I was reported to be too unstable to record its spectrum (9), gave a spectrum contaminated by only -10% Compound II in our system. Earlier conclusions that the hemin 2- and 4-substituents do not interact with the peroxidase polypeptide chain (8, 10) have been shown incorrect by the present work. Differences in the properties of peroxidases substituted with 2-formyl-4vinyldeuterohemin (2) and 2-vinyl-4-formyldeuterohemin (3) can only be due to the interaction of these small substituents with the peroxidase apoprotein. Outside the protein, these hemins are isoelectronic, that is, they possess identical properties which depend upon their electronic structure such as spectra of various ligated Fe(Il) derivatives (20) and pK3 values (20, 52) (pK3 is the pK. of the third porphyrin pyrrole nitrogen). Our studies on the Compound I spectra and specific activities of mesohemin (5) and dimethyldeuterohemin (4) horseradish peroxidases support the assertion that small side chains in the hemin 2- and 4-positions may interact with the peroxidase apoprotein. These hemins are again isoelectronic, but their peroxidases show differences in the above mentioned properties. When the hemin 2 and 4 side chains are made very large, as in the p-cresylethyl hemins (15, 16, and 17), the hemin may no longer be able to generate an active substituted peroxidase. It therefore appears that the 2- and 4-substituents of the 6911 Substituted Hemins and Horseradish Peroxidase Function hemin are bound in pockets of limited size in horseradish peroxidase. The interaction with apoperoxidase of the 4 isomers present in the mixture of 2- (and 4)-1'-(4"-resorcinyl)ethyldeuterohemins (lla, llb) may indicate that the geometric constraints on hemin-binding by peroxidase become extreme when the 2- or 4-substituent becomes large. Only 25% of the hemins present in the above mixture bind to apoperoxidase. This observation is consistent with only one of the four isomers binding to form an active substituted enzyme. No additional binding is observed, however, when apoperoxidase is incubated with a 1O-fold excess of the mixture of resorcinylethyl hemins (Fig. 11). This observation may be explained in the following way. Binding of hemes and hemins to apo heme proteins has, in several cases (42, 53, 54) been observed to occur in two stages: 1) rapid equilibrium formation of an intermediate complex, and 2) slow irreversible isomerization of the intermediate complex to the active heme protein. In the case of the resorcinylethyl hemins, all four isomers might be able to form the intermediate complex, but only one, the active protein. If exchange between free hemin and hemin bound in the intermediate complex is very slow, then the isomer which is capable offorming the active enzyme may not be able to displace the other three isomers from the intermediate complex. The hemins which bind only to form the intermediate complex must still be available to DEAE-cellulose, however. Confirmation that only one hemin of the four is bound at the active site would, of course, require structural analysis of that hemin. The results of our studies on peroxidases substituted with pairs of isoelectronic 2,4-substituted hemins and hemins possessing large 2- and 4-substituents are supported by studies of Ohlsson et al. (55). These workers showed that changing the 2 and 4 substituents of the hemin can cause substantial changes in the protein region of the horseradish peroxidase CD spectrum. Because a great number of functional groups of a variety of sizes may be accommodated by the binding sites for the 2and 4-hemin substituents, it may be said that these sites exhibit a high degree of conformational flexibility. Our studies and those of others (7-9) have shown, however, that a considerably smaller degree of conformational flexibility is exhibited by the pockets which bind the 6- and 7-substituents of the hemin. With respect to esterification with methanol, previous studies (7-9) have shown that at least one free carboxyl group is a requirement for rapid generation of an active substituted peroxidase. Our studies show that for rapid and efficient hemin binding, chain length of the 6- and 7-substituents is also important. Even though it possesses two free carboxyl groups, the dibutyric acid hemin (6) binds only very slowly to apoperoxidase. Its activity also differs from that of its isoelectronic counterpart, protohemin (1). Protohemin diamide (7) and the dialcohol hemin (8), which are also isoelectronic with proto hemin, bind very slowly to apoperoxidase, if at all. Substituents which possess some of the hydrogen bonding capabilities of the free carboxyl group are thus unable to duplicate the binding properties of the free carboxyl. It is tempting to speculate that the 6- and 7-hemin side chains are bound in narrow pockets and that the carboxyl groups are ionized and form tight ion pairs with positively charged groups on the protein. It is also tempting to speculate that because the 2-, 4-, 6-, and 7-substituents of the hemin interact with the protein, the hemin is buried in the protein. The function of the protein would then be to protect the strong oxidants present in Compounds I and II and allow only certain one electron reductants to transmit electrons to the oxidized hemin. One final point is of interest in considering active site binding of hemins in horseradish peroxidase. So far it has been assumed that the 2- and 4-substituents of the hemin always interact with the same binding site in apoperoxidase. If this is the case, the binding sites for the 1- and 3-substituents accommodate only methyl groups. Only one hemin studied here, dimethyldeuterohemin (4), possesses C 2v symmetry which means that the hemin possesses a 2-fold axis of symmetry. Rotation of this hemin by 180 0 would keep methyl groups in the horseradish peroxidase binding sites for the land 3-substituents. Rotation of mesohemin (5) by 180 however, would put the 2- and 4-substituents which are ethyl groups in the binding sites for the 1- and 3-substituents and the 1- and 3-methyl substituents in the binding sites for the protohemin (1) 2- and 4-substituents. The 2- and 4-substituents of a substituted hemin may thus interact with the same sites which bind the vinyl groups of protohemin or those which bind the methyl groups of protohemin. In rare cases, a portion of the hemin might bind with its 2- and 4-substituents in the sites which bind the vinyls of protohemin and a portion of the hemin with its 2- and 4-substituents in the sites which bind the methyl groups of protohemin. Interaction of the 2and 4-hemin substituents with two sites each instead of just one site, depending on the size and nature of the substituent, does not affect the conclusion that the 2- and 4-substituents interact with the peroxidase polypeptide chain. Such binding site flexibility, especially where the same hemin could bind with its 2- and 4-substituents, either in the vinyl binding sites of protohemin or the methyl binding sites of protohemin, might, however, account for the unusual compound I spectrum of mesohemin horseradish peroxidase (Fig. 10). This spectrum shows characteristics of the 2 A 1u and 2A2u ground states. 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