Meso-unsubstituted iron corrole in hemoproteins: Remarkable differences in effects on peroxidase activities between myoglobin and horseradish peroxidase

Article abstract of DOI:10.1021/ja907428e

(Figure Presented) Myoglobin (Mb) and horseradish peroxidase (HRP) were both reconstituted with a meso-unsubstituted iron corrole and their electronic configurations and peroxidase activities were investigated. The appearance of the 540 nm band upon incorporation of the iron corrole into apoMb indicates axial coordination by the proximal histidine imidazole in the Mb heme pocket. Based on ¹H NMR measurements using the Evans method, the total magnetic susceptibility of the iron corrole reconstituted Mb was evaluated to be S = 3/2. In contrast, although a band does not appear in the vicinity of 540 nm during reconstitution of the iron corrole into the matrix of HRP, a spectrum similar to that of the iron corrole reconstituted Mb is observed upon the addition of dithionite. This observation suggests that the oxidation state of the corrole iron in the reconstituted HRP can be assigned as +4. The catalytic activities of both proteins toward guaiacol oxidation are quite different; the iron corrole reconstituted HRP decelerates H₂O₂-dependent oxidation of guaiacol, while the same reaction catalyzed by iron corrole reconstituted Mb has the opposite effect and accelerates the reaction. This finding can be attributed to the difference in the oxidation states of the corrole iron when these proteins are in the resting state.

Full text of DOI:10.1021/ja907428e

Published on Web 10/07/2009
Meso-Unsubstituted Iron Corrole in Hemoproteins: Remarkable Differences in
Effects on Peroxidase Activities between Myoglobin and Horseradish
Peroxidase
Takashi Matsuo,*,†,‡ Akihiro Hayashi,† Masato Abe,† Takaaki Matsuda,§,| Yoshio Hisaeda,§ and
Takashi Hayashi*,†
Department of Applied Chemistry, Graduate School of Engineering, Osaka UniVersity, Suita 565-0871, Japan, Graduate
School of Materials Science, Nara Institute of Science and Technology, Ikoma 630-0192, Japan, and Department of
Chemistry and Biochemistry, Graduate School of Engineering, Kyushu UniVersity, Fukuoka 819-0395, Japan
Received September 2, 2009; E-mail: tmatsuo@ms.naist.jp; thayashi@chem.eng.osaka-u.ac.jp
Corrole is a porphyrinoid macrocycle which lacks one meso
carbon relative to the more common porphyrin framework and may
be regarded as an aromatic analogue of corrin, a unit of vitamin
B12.1,2 Although corrole retains the 18π-conjugated system, it has
trianionic character because of its contracted framework. Accord-
ingly, it is thought that the ligand shape stabilizes high-valent states
of a metal ion bound within the macrocycle core.1,3 In a past
investigation of corrole chemistry, high-valent metallocorroles
equipped with electron-withdrawing groups at the three meso
positions have been well-characterized because they have accessible
synthetic routes and tend to be thermostable.4 Syntheses of modified
corrole compounds (e.g., corrolazine), applications of corroles for
light energy conversion, and development of gas sensing by
metallocorroles have also been reported.5 On the other hand,
Figure 1. UV-vis spectra of iron corrole 1 in 100 mM KPi pH ) 7.0
controversies remain regarding electronic configurations and oxida-
tion states of metallocorroles without substituent groups at the meso
positions. This is unfortunate because meso-unsubstituted iron
containing 1% MeOH (blue line, (a)) and in the heme pocket of Mb
dissolved in 100 mM KPi pH ) 7.0 (red line, (b)) at 25 °C.
corroles are attractive from the standpoint of comparing their
(S′ ) 1/2) to generate an apparently S ) 1 electron configuration,
which has been reported previously for similar compounds.7 The
chemical properties with those of the naturally occurring prosthetic
group, heme.6,7 Investigation of meso-unsubstituted metallocorroles,
hydrolyzed form 1 is proposed to be in equilibrium between the
monomer and the µ-oxo dimer under neutral aqueous media.6b
particularly iron corroles, is needed to obtain fundamental knowl-
edge in corrole chemistry.
After the addition of 1 to apomyoglobin (apoMb) in a KPi buffer
solution (pH ) 7.0), the band at 371 nm gradually shifts to 395
nm concurrently with the appearance of a strong band at 540 nm
and a small band at 720 nm (Figure 1). The change in the higher
energy band is indicative of a shift in the equilibrium to the
monomer form, followed by incorporation of 1 into the hydrophobic
heme pocket of Mb. The appearance of the bands in the visible
region indicates the axial coordination of histidine.10 The prepared
Mb was also characterized by ESI-TOF-MS (Figure S2) with a
mass number of 17 586. The pK1/2 value, the pH corresponding to
a 50% loss of a cofactor, of the reconstituted Mb is 4.8 (Figure
S3), which is higher by only 0.3 unit than that of the native Mb.11
The stability of 1-reconstituted Mb is sufficient for conventional
column chromatography, lyophilization, and the experiments for
evaluating the oxidation activities described below. Surprisingly,
the iron corrole became EPR-active after incorporation into the Mb
matrix (Figure S4), suggesting that the ring radical was autoreduced
upon imidazole cooridination.12 Therefore, the oxidation state of
the iron corrole in the reconstituted Mb can be assigned as Fe(III)
with a neutral ring (Vide infra).
We have recently reported that the incorporation of an iron
porphycene, a structural isomer of porphyrin, enhances peroxidase
activities of myoglobin (Mb) and horseradish peroxidase (HRP) and
that it is possible to evaluate the reactivities of labile high-valent
species.8 These findings indicate that the introduction of an appropriate
heme derivative is useful not only for controlling the native functions
of hemoproteins but also for gaining an understanding of the physi-
cochemical character of the synthetic cofactor. Consequently, we
decided to incorporate iron corrole 1 (the structure shown in Figure
1) into the apo-form of horse heart Mb and HRP to explore the intrinsic
characteristics of the meso-unsubstituted iron corrole.9
The precursor of 1, dimethyl ester 2, was prepared by cyclization
of the corresponding [a,c]-biladiene 3 in the presence of FeCl3 (see
Scheme S1).6a The H NMR spectrum of 2 (Figure S1) indicates
1
spectroscopic features similar to those of the previously reported
alkyl corroles.1,7 The meso protons were observed in an unusually
far downfield region (180-200 ppm), which is indicative of Fe(III)
combined with a macrocycle π-cation radical.6 This compound is
EPR-silent, suggesting that the spin of the iron with S ) 3/2 is
antiferromagnetically coupled with the macrocycle π-cation radical
In contrast, when 1 is incorporated into apoHRP, a band at 540
nm does not appear in the UV-vis spectrum (see Figure S5). Upon
addition of sodium dithionite to the HRP solution, a new band at
540 nm is observed and the spectrum becomes similar to that of
1-reconstituted Mb (Figure S5). It is known that an axial anionic
ligand (for example C6H5-) provides a large ligand field splitting
† Osaka University.
‡ Nara Institute of Science and Technology.
§ Kyushu University.
| Current address: Kurume National College of Technology, Kurume, Fukuoka
830-8555, Japan.
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10.1021/ja907428e CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
In summary, we have successfully reconstituted Mb and HRP
with an iron corrole without substituent groups at the meso positions
of the framework. To the best of our knowledge, this is the first
example of introduction of a formal ferryl cofactor into an apo-
hemoprotein. The reconstituted proteins have remarkable differences
in electronic configurations and peroxidase activities, which are
influenced by the environments of the heme pockets in these
proteins. Efforts to precisely characterize the high-valent chemical
species of the iron corrole in the protein matrix are in progress.
Acknowledgment. The authors thank Dr. Motohiro Nakano
(Osaka University) for the suggestion of the Evans NMR measure-
ments. This work was financially supported by MEXT Japan (to T.M.
and T.H.) and Global COE program at Osaka University (to T.M.).
Figure 2. Catalytic activities for guaiacol oxidation mediated by Mbs or
HRPs; (a) HRP with the native heme; (b) HRP with 1; (c) Mb with the
native heme; (d) Mb with 1; [guaiacol]0 ) 0.25 mM, [protein] ) 2 µM,
[H2O2]0 ) 5 mM; 50 mM NaPi buffer pH ) 7.0 at 20 °C; TON1s represents
the turnover number of the catalytic reaction at 1 s.
Supporting Information Available: The synthesis of 1, UV-vis
spectra of the HRP with 1, the NMR spectra, the EPR spectrum, the
preparation of the proteins with 1 and the evaluation of peroxidase
activity are described. This material is available free of charge via the
Internet at http://pubs.acs.org.
of the corrole iron. This affords an Fe(IV) corrole with a neutral
ligand,6c which is chemically equivalent to the Fe(III) corrole
π-cation radical. Because the proximal imidazole in HRP can
provide anionic imidazolate-like coordination,13 the corrole iron
in HRP would be expected to attain the +4 metal oxidation state.
When dithionite is added to 1-reconstituted HRP, the corrole iron
is expected to be reduced to a +3 metal oxidation state. The
similarity of the UV-vis spectra between the reduced HRP (Figure
S5b) and the above-mentioned Mb (Figure 1b) supports the
assignment for the oxidation state of 1 in Mb described above.14
To address the spin state of 1 in Mb, the total magnetic
susceptibility of 1-reconstituted Mb was investigated by the Evans
method.15 When tert-butanol is employed as a standard sample,
References
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1
the peak of the butyl protons in the H NMR spectrum is shifted
as a result of the paramagnetism of 1 in the interior of Mb (Figure
S6). From the variance of the downfield shift, the effective magnetic
moment, µeff, of 1 in Mb is calculated to be 3.79 µB, suggesting
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assigned as Fe(III) with the intermediate spin state.
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around 470 nm was observed in the presence of HRPs and
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oxidation (Figure S7). It was found that the order of the catalytic
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