Download Document 8923686

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. Evidence for rotation of the hemin by 180 0 in the binding site has
been advanced in the case of deuterohemin substituted heme
proteins (56-58), and more recently in the case of horseradish
peroxidase (60).
0
,
REFERENCES
1. Theorell, H. (1941) Ark. Kemi. Mineral. Geo!. 14B, No. 20,1-3
2. Gjessing, E. C., and Sumner, J. B. (1942) Arch. Biochem. 1, 1-8
3. Theorell, H., Bergstrom, S., and Akeson, A. (1943) Ark. Kemi.
Mineral Geol. 16A, No. 18, 1-8
4. Paul, K G. (1959) Acta Chem. Scand. Ser. BOrg. Chem. Biochem.
13, 1239-1240
5. Paul, K G. (1959) Acta Chem. Scand. Ser. BOrg. Chem. Biochem.
13, 1240-1242
6. Chance, B., and Paul, K-G. (1960) Acta Chem. Scand. Ser. B
Org. Chem. Biochem. 14, 1711-1716.
7. Maehly, A. C. (1961) Nature (Lond.) 192, 630-632
8. Tamura, M., Asakura, T., and Yonetani, T. (1972) Biochim.
Biophys. Acta 268, 292-304
9. Makino, R., and Yamazaki, I. (1972) J. Biochem. (Tokyo) 72,
655-664
10. Ohlsson, P.-I., and Paul, K-G. (1973) Biochim. Biophys. Acta
315, 293-305
11. Makino, R., and Yamazaki, I. (1973) Arch. Biochem. Biophys.
157,356-368
12. Makino, R., and Yamazaki, I. (1974) Arch. Biochem. Biophys.
165,485-493
13. Yamada, H., Makino, R., and Yamazaki, I. (1974) Arch. Biochem.
Biophys.169,344-353
14. Dolphin, D., Forman, A., Borg, D. C., Fajer, J., and Felton, R. H.
(1971) Proc. Nat!. Acad. Sci. U. S. A. 68, 614-618
15. Dolphin, D., Muljiani, Z., Rousseau, K, Borg, D. C., Fajer, J., and
Felton, R. H. (1973) Ann. N. Y. Acad. Sci. 206, 177-200
16. Felton, R. H., Romans, A. Y., and Yu, N. (1976) Biochim. Biophys.
Acta 434, 82-89
17. Morishima, 1., and Ogawa, S. (1978) Biochemistry 17, 4384-4388
18. La Mar, G. N., and de Ropp, J. S. (1980) J. Am. Chem. Soc. 102,
395-397
19. Schulz, C. E., Devaney, P. W., Winkler, H., Debrunner, P. G.,
Doan, N., Chiang, R., Rutter, R., and Hager, L. P. (1979) FEBS
Lett. 103, 102-105
6912
Substituted Hemins and Horseradish Peroxidase Function
20. Dolphin, D., Felton, R H., Borg, D. C., and Fajer, J. (1970) J.
Am. Chem. Soc. 92, 743-745
21. Fajer, J., Borg, D. C., Forman, A., Dolphin, D., and Felton, R H.
(1970) J. Am. Chem. Soc. 92, 3451-3459
22. Mauk, M. R, and Girotti, A. W. (1974) Biochemistry 13, 17571763
23. Asakura, T., and Sono, M. (1974) J. Biol. Chem. 249, 7087-7093
24. DiNello, R K, and Dolphin, D. (1978) Biochem. Biophys. Res.
Commun. 80, 698-703
25. DiNello, R K, and Dolphin, D. (1979) Biochem. Biophys. Res.
Commun. 86, 190-198
26. Schumm, O. (1928) Z. Physiol. Chem. 178, 1-18
27. Fischer, H., and Zeile, K (1929) Annalen 468, 98-116
28. DiNello, R K, and Chang, C. K (1979) in The Porphyrins
(Dolphin, D., ed) Vol. 1, Part A, Chap. 7, pp. 289-339, Academic
Press, New York
29. Caughey, W. S., Alben, J. 0., Fujimoto, W. Y., and York, J. L.
(1966) J. Org. Chem. 31, 2631-2639
30. Corwin, A. H., and Erdman, J. G. (1946) J. Am. Chem. Soc. 68,
2473-2478
31. Falk, J. E. (1964) Porphyrins and Metalloporphyrins, Elsevier,
Amsterdam
32. Adler, A., Longo, F., Kampas, F., and Kim, J. (1970) J. lnorg.
Nucl. Chem. 32,2443-2445
33. Inhoffen, H. H., Brockmann, H., and Bliesener, K M. (1969)
Annalen 730,173-185
34. Clezy, P. S., and Fookes, C. J. R (1974) Aust. J. Chem. 27, 371382
35. Hamilton, A. D. (1976) M.Sc. thesis, University of British Columbia
36. DiNello, R K. (1977) Ph.D. thesis, Harvard University
37. Sivasothy, R (1978) M.Sc. thesis, University of British Columbia
38. DiNello, R K., and Dolphin, D. (1980) J. Org. Chem. 45, 51965204
39. Paul, K. G., Theorell, H. and Akeson, A. (1953) Acta Chem.
Scand. 7, 1284-1287
40. Paul, K.-G., and Avi-Dor, Y. (1954) Acta Chem. Scand. 8, 649-
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
657
Teale, F. W. J. (1959) Biochim. Biophys. Acta 35, 543
Yonetani, T. (1967) J. Biol. Chem. 242, 5008-5013
Paul, K G. (1958) Acta Chem. Scand. 12,1611-1621
Hollenberg, P. F., Rand-Meir, T., and Hager, L. P. (1974) J. Bioi.
Chem. 249, 5816-5825
Hewson, W. D., and Dunford, H. B. (1976) J. Biol. Chem. 251,
5816-5825
Nichols, P. (1966) in Hemes and Hemoproteins (Chance, B.,
Estabrook, R E., and Yonetani, T., eds) pp. 307-318, Academic
Press, New York
Paul, K G. (1963) in The Enzymes (Boyer, P. D., ed) 2nd edition,
Vol. 8 Chap. 7, pp. 227-274, Academic Press, New York
Makino, R, Yamada, H., and Yamazaki, I. (1976) Arch. Biochem.
Biophys.173,66-70
Schonbaum, G. R, and Lo, S. (1972) J. Bioi. Chem. 247, 33533360
Douzou, P., Sireix, R, and Travers, F. (1970) Proc. Natl. Acad.
Sci. U. S. A. 66, 787-792
Shannon, L. M., Kay, E., and Lew, J. Y. (1966) J. Bioi. Chem.
241,2166-2172
Caughey, W. S., Fujimoto, W. Y., and Johnson, B. (1966) Biochemistry 5, 3830-3843
Rossi-Fanelli, A., Antonini, E., and Caputo, A. (1964) Adv. Protein
Chem. 19, 73-222
Phelps, C., and Antonini, E. (1969) Biochem. J. 114, 719-724
Ohlsson, P.-I., Paul, K-G., and Sjoholm, I. (1977) J. Bioi. Chem.
252, 8222-8228
La Mar, G., Budd, D. L., Viscio, D. B., Smith, K M., and Langry,
K C. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 5755-5759
La Mar, G. N., Smith, K. M., Gersonde, K, Sick, H., and Overkamp, M. (1980) J. Biol. Chem. 255,66-70
Burns, P. D. (1980) Ph.D. thesis, University of California, Davis
Seybert, D. W., Moffat, K, Gibson, Q. H., and Chang, C. K (1977)
J. Bioi. Chem. 252, 4225-4231
La Mar, G., de Ropp, J. S., Smith, K M., and Langry, K. C. (1980)
J. Am. Chem. Soc. 102, 4833-4835