Artificial protein-protein complexation between a reconstituted myoglobulin and cytochrome c

Article abstract of DOI:10.1021/ja9723951

Artificial prosthetic porphyrins, 1·Fe and 1·Zn, in which two isophthalamide units having four carboxylates were bound to the terminal of each pripheral propionate side chain in protoporhyrin IX, were inserted into horse heart apomyoglobin to give novel mylglobins rMb(1·Fe) and rMb(1·Zn), respectively. The resultant reconstituted myoglobins were designed to bind cationic cytochrome c on the protein surface via elecrostatic interaction. The isoelectric point for rMB(1·Fe) was determined to be 5.5, which is about 2 pH units lower than that of native myoglobin. The pI value suggests that eight carboxylates of prosthetic group are located on the surface of the myoglobin. A construction of a myoglobin-cytochrome c complex was probed by paramagnetic ¹H NMR and flash photolysis studies. The behavior of ¹H NMR paramagnetic shifts in the rMb(1·FeCN) cytochrome c complex is comparable with that in the native pairing of cytochrome c-cytochrome c peroxidase. Laser flash photolysis shows that a long-range ET from photoexcited rMb- (1·Zn) to cytochrome coccurs within the protein-protein complex. The time- dependence of the transient spectra at 460 nm identified as the triplet excited state of rMb(1·Zn) leads to rate constant of forward ET and affinity of the protein-protein complex; k(intra) = (2.2 ± 0.1) x 10³ s^-1 and K(a) = (6.5 ± 3.0) x 10⁴ M^-1 at 10 mM ionic strength and k(intra) = (2.3 ± 0.2) x 10³ s^-1 and K(a) = (1.5 + 0.6) x 10⁴ M^-1 at 20 Mm ionic strength and pH 7.0. The binding affinity for cytochrome c decreases with increasing te ionic strength, indicating that the protein-protein complex is formed by elecrostatic interaction. This work demonstrates that the artificial functional groups bound to the terminal of porphyrin in the reconstituted myoglobin can act as an effective recognition domain for a protein at the surface of the myoglobin.

Full text of DOI:10.1021/ja9723951

4910
J. Am. Chem. Soc. 1998, 120, 4910-4915
Artificial Protein-Protein Complexation between a Reconstituted
Myoglobin and Cytochrome c
Takashi Hayashi,*,† Yutaka Hitomi, and Hisanobu Ogoshi*,‡
Contribution from the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto UniVersity, Sakyo-ku, Kyoto 606-8501, Japan
ReceiVed July 17, 1997
Abstract: Artificial prosthetic porphyrins, 1‚Fe and 1‚Zn, in which two isophthalamide units having four
carboxylates were bound to the terminal of each peripheral propionate side chain in protoporphyrin IX, were
inserted into horse heart apomyoglobin to give novel myoglobins, rMb(1‚Fe) and rMb(1‚Zn), respectively.
The resultant reconstituted myoglobins were designed to bind cationic cytochrome c on the protein surface via
electrostatic interaction. The isoelectric point for rMb(1‚Fe) was determined to be 5.5, which is about 2 pH
units lower than that of native myoglobin. The pI value suggests that eight carboxylates of prosthetic group
are located on the surface of the myoglobin. A construction of a myoglobin-cytochrome c complex was
probed by paramagnetic 1H NMR and flash photolysis studies. The behavior of 1H NMR paramagnetic shifts
in the rMb(1‚FeCN)scytochrome c complex is comparable with that in the native pairing of cytochrome
cscytochrome c peroxidase. Laser flash photolysis shows that a long-range ET from photoexcited rMb-
(1‚Zn) to cytochrome c occurs within the protein-protein complex. The time-dependence of the transient
spectra at 460 nm identified as the triplet excited state of rMb(1‚Zn) leads to rate constant of forward ET and
affinity of the protein-protein complex; kintra ) (2.2 ( 0.1) × 103 s-1 and Ka ) (6.5 ( 3.0) × 104 M-1 at 10
mM ionic strength and kintra ) (2.3 ( 0.2) × 103 s-1 and Ka ) (1.5 ( 0.6) × 104 M-1 at 20 mM ionic strength
and pH 7.0. The binding affinity for cytochrome c decreases with increasing the ionic strength, indicating
that the protein-protein complex is formed by electrostatic interaction. This work demonstrates that the artificial
functional groups bound to the terminal of porphyrin in the reconstituted myoglobin can act as an effective
recognition domain for a protein at the surface of the myoglobin.
Introduction
duced singlet electron transfer (ET) in the myoglobin-methyl
viologen complex via anionic binding site on the protein surface.
Here, we report a new architecture of a stable complex
between cytochrome c and a reconstituted myoglobin, in which
the prosthetic porphyrins 1‚Fe or 1‚Zn possesses a specific
interaction domain for the basic patch of cytochrome c (Scheme
1).7,8 The present nonmutagenic approach by the combination
of chemical modification of heme and reconstitution technique
can be exploited in the design of the assembly of proteins to
evaluate the importance of protein-protein binding, such as
cytochrome c-cytochrome c peroxidase or cytochrome c-cy-
tochrome c oxidase, in the ET process.
Protein-protein recognition at a special binding domain plays
an essential role on the formation of organized system to initiate
a biological function. To clarify the binding mechanism, a
number of synthetic host-guest models have been reported,
some of them giving a valuable insight into the nature of
molecular recognition via intermolecular weak interactions.1 The
next stage in this study should be the construction of supramo-
lecular assemblies to understand the highly ordered biological
systems.2-5 We have recently reported a new myoglobin
reconstituted with a chemically modified prosthetic group having
multiple substituted carboxylates at the terminal of two propi-
onates to give a novel complex between the myoglobin and
cationic methyl viologen.6 In this system, we found photoin-
Results and Discussion
Preparation and Characterization of Reconstituted Myo-
globin. The synthetic routes to the metalloporphyrins 1‚Fe and
1‚Zn are shown in Scheme 2. Bis(p-nitrophenyl)ester 5
prepared from protoporphyrin IX (2) and p-nitrophenyl trifluo-
roacetate was coupled with 5-aminoisophthalic acid derivative
4 having four ethoxycarbonyl groups to allow the precursor 6
† Current address: Department of Chemistry and Biochemistry, Graduate
School of Engineering, Kyushu University, Higashi-ku, Fukuoka 812-8581,
Japan.
‡ Current address: Fukui National College of Technology, Sabae, Fukui
916-8507, Japan.
(1) For recent reviews, see: Hamilton, A. D., Ed.; Molecular Recognition
(Tetrahedron Symposia No. 56). Tetrahedron 1995, 51.
(2) Vo¨gtle, F. Supramolecular Chemistry; Wiley: Chichester, 1991.
(3) Balzani, V.; Scandola, F. Supramolecular Photochemistry; Ellis
Horwood: Chichester, 1991.
(4) Lehn, J.-M. Supramolecular Chemistry; VCH: Weinheim, 1995.
(5) Sessler, J. L.; Wang, B.; Springs, S. L.; Brown, C. T. In Compre-
hensiVe Supramolecular Chemistry; Murakami, Y., Ed.; Pergamon: Oxford,
1996; Vol. 2, pp 311-336.
(6) (a) Hayashi, T.; Takimura, T.; Ogoshi, H. J. Am. Chem. Soc. 1995,
117, 11606-11607. (b) Hayashi, T.; Ogoshi, H. Chem. Soc. ReV. 1997,
26, 355-364.
(7) For recent examples of artificial assembly system of hemoprotein,
cf.: (a) Zhou, J. S.; Granada, E. S. V.; Leontis, N. B.; Rodgers, M. A. J.
J. Am. Chem. Soc. 1990, 112, 5074-5080. (b) Lahiri, J.; Fate, G. D.;
Ungashe, S. B.; Groves, J. T. J. Am. Chem. Soc. 1996, 118, 2347-2358.
(c) Tsukahara, K.; Kimura, C. J. Electroanal. Chem. 1997, 438, 67-70.
(d) Hamuro, Y.; Calama, M. C.; Park, H. S.; Hamilton, A. D. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 2680-2683.
(8) The myoglobins reconstituted with artificial prosthetic groups 1‚Fe
and 1‚Zn based on native protoporphyrin (2) have improved in their stability
compared to our previous reconstituted myoglobin.6a
S0002-7863(97)02395-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/12/1998

Artificial Protein-Protein Complexation
J. Am. Chem. Soc., Vol. 120, No. 20, 1998 4911
Scheme 1
Figure 1. Electrospray-ionization mass spectrum of rMb(1‚Fe) at pH
7.0. The peaks are labeled with the protonation state, n+, with the
molecular masses determinated from the measured m/z values.
Scheme 2a
The electronic absorption spectra of rMb(1‚Fe) and rMb(1‚Zn)
were identical to those of reference myoglobins, native met-
myoglobin Mb(2‚Fe), and zinc myoglobin rMb(2‚Zn), respec-
tively. The mass spectrum of rMb(1‚Fe) by electrospray
ionization at pH ) 7.0 shows a distribution of peaks with
protonation states ranging from 13+ to 10+ (Figure 1). The
average molecular mass measured from these peaks is 18 366.7
( 2 and corresponds to the calculated mass of rMb(1‚Fe)
1
(18 343). Furthermore, the paramagnetic H NMR spectrum
of cyanometmyoglobin rMb(1‚FeCN) shows the characteristic
shifts of peripheral methyl protons in hemin and Ile99 protons
similar to those of native Mb(2‚FeCN).12 These results indicate
that the synthetic metalloporphyrins are evidently incorporated
into the normal position of heme cavity.
Isoelectric focusing was carried out for rMb(1‚Fe) and native
Mb(2‚Fe) as shown in Figure 2. The isoelectric point, pI, of
the metaquo form of rMb(1‚Fe) is 5.5, which is about 2 pH
units lower than that of native Mb(2‚Fe).13 It is clear that the
rMb(1‚Fe) changes from a neutral protein to an acidic one due
to the formation of anionic interface located on the protein
surface.
a Key: (a) SOCl2, (b) TsOH‚HN(CH2CO2Et)2/Et3N, (c) HBr/AcOH,
(d) protoporphyrin bis(p-nitrophenyl)ester (5)/pyridine, (e) FeCl2 or
Zn(OAc)2, (f) KOH/THF/MeOH.
1
Protein-Protein Complexation Monitored by H NMR
Spectroscopy. The results of 1H NMR experiments on a
mixture of rMb(1‚FeCN) and ferricytochrome c (horse heart,
Type IV) in phosphate buffer (pH 7.4, µ ) 10 mM) are
illustrated in Figure 3. In the presence of rMb(1‚FeCN), the
peripheral methyl substituents at the 3- and 8-positions of hemin
in cytochrome c clearly shifted to lower and upper fields,
respectively, whereas the chemical shift of CꢀH3 protons of
Met80 only deviated within 0.02 ppm. The observed changes
in chemical shifts of cytochrome c upon addition of rMb-
(1‚FeCN) are comparable with those in cytochrome c-cyto-
chrome c peroxidase complexation described in previous
literature.14 Thus, the result supports that the artificial interface
in 94% yield.9 Metalation of 6 and the following basic
hydrolysis proceeded smoothly to give the desired iron and zinc
porphyrins 1‚Fe and 1‚Zn possessing eight carboxylic acids at
the terminal of two peripheral propionate side chains. These
metalloporphyrins were readily inserted into horse heart apomyo-
globin by usual method to give the reconstituted metmyoglobin
rMb(1‚Fe) and zinc myoglobin rMb(1‚Zn), respectively.6a,10,11
(9) Collman, J. P.; Denisevich, P.; Konai, Y.; Marrocco, M.; Koval, C.;
Anson, F. C. J. Am. Chem. Soc. 1980, 102, 6027-6036.
(10) Teale, F. W. Biochim. Biophys. Acta 1959, 35, 543.
(11) For examples of reconstituted myoglobin, cf.: (a) Suzuki, A.; Okuda,
K.; Kawagoe, K.; Toi, H.; Aoyama, Y.; Ogoshi, H. Chem. Lett. 1985, 1169-
1172. (b) Hamachi, I.; Tajiri, Y.; Shinkai, S. J. Am. Chem. Soc. 1994, 116,
7437-7438. (c) Willner, I.; Zahavy, E.; Heleg-Shabtai, V. J. Am. Chem.
Soc. 1995, 117, 542-543. (d) Hayashi, T.; Hitomi, Y.; Suzuki, A.; Takimura,
T.; Ogoshi, H. Chem. Lett. 1995, 911-912. (e) Hayashi, T.; Takimura, T.;
Aoyama, Y.; Hitomi, Y.; Suzuki, A.; Ogoshi, H. Inorg. Chim. Acta In press.
(12) La Mar, G. N.; Davis, N. L.; Parish, D. W.; Smith, K. M. J. Mol.
Biol. 1983, 168, 887-896.
(13) Varadarajan, R.; Lambright, D. G.; Boxer, S. G. Biochemistry 1989,
28, 3771-3781.

4912 J. Am. Chem. Soc., Vol. 120, No. 20, 1998
Hayashi et al.
Figure 2. Results of isoelectric focusing with PhastGel IEF 3-9
(Pharmacia). pI along the gel was determined by measuring the position
of protein standards (pI calibration kit 3-10, Pharmacia): (lanes 1, 3,
and 5) marker proteins; (lane 2) native metmyoglobin Mb(2‚Fe); (lane
4) rMb(1‚Fe).
Figure 4. Transient absorbance changes: (a) decay of the triplet state
of rMb(1‚Zn) in the absence of cytochrome c, monitored at 460 nm,
(b) decay of the triplet state of rMb(1‚Zn) in the presence of cytochrome
c, monitored at 460 nm. Conditions: [rMb(1‚Zn)] ) 4.3 × 10-6 M,
[cytochrome c] ) 1.6 × 10-5 M in phosphate buffer (pH 7.0, µ ) 10
mM) at 20 °C. Experimental data points are represented by dots; the
solid lines are fitting curves using a single-exponential function.
Figure 3. Changes in 1H NMR chemical shifts of selective protons in
rMb(1‚FeCN) and cytochrome c at different concentration of cyto-
chrome c; [rMb(1‚Fe)]0 ) 0.49 mM in phosphate buffer containing
1.0 mM KCN (µ ) 10 mM, pH 7.4) at 20 °C. The solid lines represent
the changes in chemical shifts of cytochrome c; heme 3-CH3 (b), Met80
CꢀH3 (2), and heme 8-CH3 (9). The broken lines represent the changes
in chemical shifts of rMb(1‚FeCN); heme 1-CH3 (O), Ile99 CγH (4),
and heme 5-CH3 (0).
state in the presence of ferricytochrome c decreased with the
increase of ionic strength, indicating that the protein-protein
complex is efficiently formed by electrostatic interaction
between eight carboxylates at the surface of rMb(1‚Zn) and
the special lysine residues of cytochrome c. Furthermore, a
short-lived transient band was observed at 680 nm, which is
representative of the zinc porphyrin cation radical species as
an intermediate of ET (Figure 5).6,16 The transient band at 680
nm increased with the decay of triplet state of rMb(1‚Zn) and
subsequently decreased in a longer time scale. The formation
and decay of another charge separated species, ferrocytochrome
c, were also detected at 550 nm, which show same profile as
those shown in Figure 5, with the formation and decay of cation
radical species of rMb(1‚Zn). Thus, it is apparent that the
of rMb(1‚Fe) interacts with the several Lys residues in the
proximity of the exposed heme edge in cytochrome c. Fur-
thermore, the significant shifts of 5-CH3 of hemin and Ile99
protons in rMb(1‚FeCN) were also observed due to the complex
formation with cytochrome c.
Photoinduced ET in rMb(1‚Zn)-Cytochrome c Complex.
Flash photolysis of rMb(1‚Zn) using a pulsed Nd:YAG laser
gave an exponential decay profile at 460 nm identified as the
triplet excited state of zinc porphyrin with a rate constant of 59
( 1 s-1, which is almost same as that for rMb(2‚Zn),15 in argon-
saturated solution at pH 7.0 (Figure 4a). Upon addition of
ferricytochrome c, the triplet state of rMb(1‚Zn) was rapidly
quenched at the ionic strength of 10 mM (Figure 4b). In
contrast, the same behavior was not obtained in a mixture of
rMb(1‚Zn) and ferrocytochrome c. The decay rate of triplet
(16) Zhou, J. S.; Rodgers, M. A. J. J. Am. Chem. Soc. 1991, 113, 7728-
7734.
(17) Estimated distance between a zinc atom of rMb(1‚Zn) and an iron
atom in cytochrome c is ca. 18 Å by computer modeling which emerged
from plausible interactions between carboxylate interface of rMb(1‚Zn) and
special Lys residues of cytochrome c. The driving force for the rMb(1‚Zn)3*
f ferricytochrome c is estimated to be ca. 1.1 eV by use of the following
papers: Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Q. ReV. Biophys.
1986, 18, 261-322. Cheng, J.; Zhou, J. S.; Kostic´, N. M. Inorg. Chem.
1994, 33, 1600-1606.
(14) (a) Moench, S. J.; Chroni, S.; Lou, B.-S.; Erman, J. E.; Satterlee, J.
D. Biochemistry 1992, 31, 3661-3670. (b) Yi, Q.; Erman, J. E.; Satterlee,
J. D. Biochemistry 1993, 32, 10988-10994.
(15) Zemel, H.; Hoffman, B. M. J. Am. Chem. Soc. 1981, 103, 1192-
1201.

Artificial Protein-Protein Complexation
J. Am. Chem. Soc., Vol. 120, No. 20, 1998 4913
due to the diffusional quenching of the triplet state, and
ferrocytochrome c was not detected at 550 nm.20 The linearity
was also obtained in rMb(1‚Zn)-cytochrome c system at the
ionic strengths of 50 mM and higher. The experimental data
at the ionic strengths of 10 and 20 mM in Figure 6 were fitted
to the following equation by an iterative, nonlinear least-squares
routine to yield a rate constant of intracomplex ET and binding
affinity between rMb(1‚Zn) and ferricytochrome c:
kobs ) k0 + kintra
) k0 + kintra[M + C + 1/Ka -
{(M + C + 1/Ka)2 - 4MC}1/2]/2M (1)
f
where k0, kintra, and f represent the rate constants of triplet-state
decay and forward ET within the complex and the fraction of
complexed rMb(1‚Zn), and M and C represent the total
concentration of rMb(1‚Zn) and cytochrome c, respectively.18
From this equation, the values of kintra and Ka are determined
to be (2.2 ( 0.1) × 103 s-1 and (6.5 ( 3.0) × 104 M-1 at 10
mM ionic strength, (2.3 ( 0.2) × 103 s-1 and (1.5( 0.6) × 104
M-1 at 20 mM ionic strength, respectively. These results clearly
indicate that the intracomplex ET rate is independent of the ionic
strength, whereas the binding affinity decreases with increasing
ionic strength.21 Furthermore, the formation of cation radical
species of rMb(1‚Zn) at 680 nm is fitted by a two-exponential
function to have a rate constant and binding affinity of (2.2 (
0.1) × 103 s-1 and (6.9 ( 3.2) × 104 M-1, respectively, at 10
mM ionic strength. These values are exactly identical with those
determined from the analysis of triplet decay profile, and this
agreement supports the reliability of experimental data. Com-
pared to the kinetic data in native protein-protein pairing,18,22
the present value shows the reasonable rate constant of intra-
complex ET, and the binding affinity derived from kinetic study
reveals that rMb(1‚Zn) forms the stable complex with cyto-
chrome c.
Figure 5. Transient absorbance changes: appearance and disappear-
ance of the cation radical of rMb(1‚Zn), monitored at 680 nm.
Conditions: [rMb(1‚Zn)] ) 4.3 × 10-6 M, [cytochrome c] ) 1.6 ×
10-5 M in phosphate buffer (pH 7.0, µ ) 10 mM) at 20 °C.
Experimental data points are represented by dots; the solid line is a
fitting curve using a double-exponential function.
Conclusion
This work indicates that the reconstitution of myoglobin with
a chemically modified prosthetic group such as 1 gives a highly
anionic protein which can form a stable complex with cationic
cytochrome c on the protein surface. Furthermore, the present
system demonstrates the first example of a long-range ET
reaction within the noncovalently linked protein-protein com-
plex via synthetic binding domain bound to the prosthetic group.
Construction of a variety of artificial protein-protein complexes
via electrostatic contacts and exploration of their properties are
currently in progress.
Figure 6. Decay rate of the triplet-state rMb(1‚Zn) as a function of
cytochrome c concentration, in phosphate buffer at pH 7.0 and 20 °C.
Ionic strengths are 10 mM (b), 20 mM (4), 50 mM (0), and 100 mM
(O), respectively. Transient absorptions were monitored at 460 nm by
using the laser flash photolysis with the 532-nm output of a Q-switched
Nd:YAG laser (6-ns fwhm). Initial concentration of rMb(1‚Zn) is 4.3
× 10-6 M. The solid lines at 10 and 20 mM ionic strengths are a fitting
curve using eq 1.
Experimental Section
photoinduced ET from rMb(1‚Zn) to ferricytochrome c occurs
within the protein-protein complex linked by specific inter-
face.17
The decay curve of triplet state could be analyzed by a single-
exponential function to give a rate constant kobs (correlation
coefficient r > 0.98).18,19 At lower ionic strength, the resulting
rate constant kobs increases nonlinearly with the cytochrome c
concentration and levels off at higher concentration as shown
in Figure 6. In contrast, in the case of rMb(2‚Zn), linear
dependence of kobs on cytochrome c concentration was observed
General Procedure. 1H NMR spectra were obtained using a JEOL
A-500 spectrometer and chemical shifts are reported relative to Me4Si
or DSS at 0 ppm. Mass spectra were measured in FAB mode and ESI
(19) Tsukahara, K.; Asami, S. Chem. Lett. 1991, 1337-1340.
(20) Decay profile of triplet state of rMb(2‚Zn) in the presence of
cytochrome c gave a second-order rate constant of kq ) (8.0 ( 0.3) × 105
M-1 s-1. Aono, S.; Nemoto, S.; Ohtaka, A.; Okura, I. J. Mol. Catal. A
1995, 95, 193-196.
(21) Figure 6 also reveals that the binding affinity remarkably decreases
at higher ionic strength, although it is difficult to obtain accurate parameters
at higher ionic strength due to the insufficient curvature of the plots.
(22) For recent reviews, see: (a) Nocek, J. M.; Zhou, J. S.; Forest, S.
D.; Priyadarshy, S.; Beratan, D. N.; Onuchic, J. N.; Hoffman, B. M. Chem.
ReV. 1996, 96, 2459-2489. (b) Durham, B.; Millett, F. S. In ComprehensiVe
Supramolecular Chemistry; Suslick, K. S., Ed.; Pergamon: Oxford, 1996;
Vol. 5, pp 219-247. (c) McLendon, G.; Hake, R. Chem. ReV. 1992, 92,
481-490. references are cited therein.
(18) Hoffman and co-workers have reported the photoinduced ET within
the native protein pairing of zinc myoglobin and cytochrome b5. In this
system, a single-exponential decay was observed due to the fast dissociation
of the complex. Our system could be followed by the same kinetic model
as their case. Nocek, J. M.; Sishta, B. P.; Cameron, J. C.; Mauk, A. G.;
Hoffman, B. M. J. Am. Chem. Soc. 1997, 119, 2146-2155.

4914 J. Am. Chem. Soc., Vol. 120, No. 20, 1998
Hayashi et al.
mode with a JEOL JMS-SX102A spectrometer and a JEOL JMS-700
MStation spectrometer, respectively. UV-vis spectra were recorded
on a HITACHI U-3410 spectrophotometer and a Hewlett-Packard
8452A diode array spectrophotometer. Protoporphyrin IX was pur-
chased from Porphyrin Products, Inc. Bis(p-nitrophenyl)ester of
protoporphyrin IX (5) was prepared by previous literature method.9
5-(Benzyloxycarbonylamino)isophthalic Acid (3). Benzyloxycar-
bonyl chloride (17.1 mL, 120 mmol) and 2 N NaOH (aq) (60.0 mL,
120 mmol) were added dropwise to a stirred solution of 5-aminoisoph-
thalic acid (18.1 g, 100 mmol) in 2 N NaOH (aq) (100 mL, 200 mmol)
at 0 °C over 0.5 h. The solution was allowed to warm to room
temperature and stirred for 6 h. The reaction mixture was then washed
with Et2O (4 × 100 mL) to remove the excess of benzyloxycarbonyl
chloride, and the resulted aqueous solution was treated with a
concentrated HCl (aq), adjusting to pH 2. The precipitated material
was filtered, washed with Et2O, and dried in vacuo for several hours
to obtain 3 (24.7 g, 80%) as a white solid: mp 297-298 °C; 1H NMR
(acetone-d6) δ ) 9.14 (1H, brs), 8.52 (1H, d, J ) 1.5 Hz), 8.35 (2H,
t, J ) 1.5 Hz), 7.32-7.46 (5H, m), 5.22 (2H, s).
Bis(N,N-diethoxycarbonylmethyl)-5-(benzyloxycarbonylamino)-
isophthalamide (4). One drop of DMF was added to a solution of 3
(0.76 g, 2.4 mmol) in thionyl chloride (3 mL), and the mixture was
then refluxed for 2 h. After the solution was cooled to room
temperature and the solvent was removed, the residue was taken up in
benzene and evaporated twice. A solution of the obtained acyl chloride
and diethyl iminodiacetate p-toluenesulfonate salt (1.9 g, 5.2 mmol)23
in dry CH2Cl2 (25 mL) was cooled to 0 °C, and triethylamine (2 mL,
14 mmol) was then added dropwise to the mixture. After the reaction
mixture was stirred overnight at room temperature, the solution was
washed successively with 1 N HCl (aq), saturated NaCl (aq), and H2O
and then dried over anhydrous Na2SO4 and evaporated to dryness.
Recrystallization from MeOH gave 4 (1.2 g, 77%) as a white solid:
1H NMR (acetone-d6) δ ) 9.10 (1H, brs), 7.72 (2H, d, J ) 1.3 Hz),
7.30-7.43 (5H, m), 7.07 (1H, t, J ) 1.3 Hz), 5.19 (2H, s), 4.29 (4H,
s), 4.19 (4H, s), 4.17 (4H, q, J ) 7.0 Hz), 4.16 (4H, q, J ) 7.0 Hz),
1.25 (6H, t, J ) 7.0 Hz), 1.19 (6H, t, J ) 7.0 Hz).
Hz), 6.25 (1H, dd, J ) 17.7 Hz, J ) 1.6 Hz), 6.19 (1H, dd, J ) 17.7
Hz, J ) 1.6 Hz), 6.13 (1H, dd, J ) 11.6 Hz, J ) 1.6 Hz), 6.12 (1H,
dd, J ) 11.6 Hz, J ) 1.6 Hz), 4.42 (4H, t, J ) 6.9 Hz), 4.17 (8H, s),
4.10 (8H, q, J ) 7.1 Hz), 4.03 (8H, s), 4.01 (8H, q, J ) 7.1 Hz), 3.72
(3H, s), 3.71 (3H, s), 3.62 (3H, s), 3.60 (3H, s), 3.33 (4H, t, J ) 6.9
Hz), 1.18 (12H, q, J ) 7.1 Hz), 1.04 (12H, q, J ) 7.1 Hz); UV-vis
(CHCl3) λabs (rel intensity) 417.0 (1.0), 545.7 (0.064), 582.8 (0.074)
nm.
The zinc porphyrin was dissolved in 9 mL of a solution containing
THF (10 mL), MeOH (10 mL), and 0.2 N aqueous KOH solution (10
mL). The solution was stirred at room temperature for 8 h. Solid
CO2 was then added, the solution was evaporated to dryness, and the
residue was taken up in MeOH. The solution was filtered over Celite
and passed through Sephadex LH-20. The solvent was evaporated and
the residue was dried to obtained 1‚Zn as a pink solid in 88% yield:
1H NMR (D2O) δ ) 10.36 (1H, s), 10.22 (1H, s), 10.21 (1H, s), 10.09
(1H, s), 8.43 (1H, dd, J ) 17.4 Hz, J ) 11.6 Hz), 8.42 (1H, dd, J )
17.4 Hz, J ) 11.6 Hz), 7.09 (2H, s), 7.05 (2H, s), 7.03 (2H, s), 6.40
(1H, d, J ) 17.4 Hz), 6.36 (1H, d, J ) 17.4 Hz), 6.14 (1H, d, J ) 11.6
Hz), 6.12 (1H, d, J ) 11.6 Hz), 4.44 (4H, t, J ) 6.4 Hz), 3.71 (4H, s),
3.69 (8H, s), 3.68 (4H, s), 3.45 (3H, s), 3.44 (3H, s), 3.42 (6H, s), 3.29
(4H, t, J ) 6.4 Hz); UV-vis (10 mM KPi, pH 7.0) λmax (relative
intensity) 414.2 (1.0), 545.0 (0.072), 582.5 (0.064) nm.
Iron(III) Complex (1‚Fe). To the solution of ferrous chloride
hydrate (40 mg) in dry acetonitrile (7 mL), a solution of 6 (30 mg) in
nitrogen-purged CHCl3 (3 mL) was added dropwise with vigorous
stirring at 50 °C under a stream of nitrogen. After complete addition,
the mixture was stirred for 10 min under nitrogen before being exposed
to air. The resulting brown solution was then diluted with CH2Cl2 and
washed with an aqueous HCl solution and then with H2O. The organic
phase was evaporated off, and the residue was purified by chromatog-
raphy (SiO2, CHCl3/MeOH ) 20:1). The iron porphyrin was hydro-
lyzed and purified as above to obtain 1‚Fe as a brown solid in 75%
yield: UV-vis (10 mM KPi, pH 7.0) λmax (rel intensity) 400.4 (1.0),
496.7 (0.048), 622.5 (0.014) nm.
Preparation of Reconstituted Myoglobins. Apomyoglobin was
prepared from metmyoglobin (horse heart, Sigma) by Teale’s 2-bu-
tanone method.10 The solution of apomyoglobin was mixed with 1‚Fe
or 1‚Zn and allowed to stand over 12 h at 4 °C. The mixture was
passed through Sephadex G-25 and CM-cellulose columns and then
lyophilized.
Isoelectric Focusing Electrophoresis. Isoelectric focusing was
curried out on an automated electrophoresis system (PhastSystem,
Pharmacia) with a Precast gel (PhastGel IEF 3-9, Pharmacia). The
quantities of proteins were detected by Coomassie blue staining. pI
along the gel was determined by measuring the positions of protein
standards (pI calibration kit 3-10, Pharmacia) having known isoelectric
points.
NMR Titration. Solutions of metcyanomyoglobin and ferricyto-
chrome c were prepared in D2O phosphate buffer containing 1.0 mM
KCN (µ ) 10 mM, pH 7.4). Aliquots of cytochrome c solution were
titrated into the metcyanomyoglobin solution, and the NMR spectra
were recorded on a JEOL A500 NMR spectrometer. The temperature
was maintained at 20 °C.
Laser Flash Photolysis Studies. The nanosecond laser photolysis
studies were carried out by a Q-switched Nd:YAG laser, which
delivered 6-ns pulses at 532 nm. The incident energy was about 10
mJ. The probe source was a continuous 150-W xenon arc lamp passed
through a monochromator. Decay of the triplet state of reconstituted
zinc myoglobin was monitored at 460 nm. Appearance and disap-
pearance of the zinc porphyrin cation radical of reconstituted myoglobin
were monitored at 680 nm. Signals were detected in transmission using
a photo multiplier (Hamamatsu Photonics, R2949), and the transient
signals were digitized using Tektronix TDS 320 oscilloscope. Signals
were averaged 1000-5120 times. The data were transferred to a NEC
PC9821Ae computer for further data analysis. No smoothing artifact
affected the results of the following data analysis. The sample solution
in a 10-mm quartz cell was prepared in a glovebox. The concentration
of reconstituted zinc myoglobin was about 4.3 × 10-6 M. The
temperature was maintained at 20 °C. UV-vis spectra of a mixture
of the reconstitued myoglobin and cytochrome c were always measured
Protoporphyrin 133,173-Bis(5-amidobis(N,N-diethoxycarbonyl-
methyl)isophthalamide) (6). The removal of the benzyloxycarbonyl
protection group was carried out by treatment of 4 (130 mg, 0.2 mmol)
with 30% HBr in AcOH (3 mL) for 0.5 h. After completion of the
reaction, the solvent was removed under reduced pressure. A solution
of the residual HBr salt and 5 (40 mg, 0.05 mmol) in dry pyridine (5
mL) was stirred at 70 °C for 8 h. The solution was condensed by
evaporator, and the residue was dissolved in CH2Cl2. The organic
solution was washed successively with saturated Na2CO3 (aq), 1 N HCl
(aq), saturated Na2CO3 (aq), saturated NaCl (aq), and H2O and then
dried over anhydrous Na2SO4 and evaporated to dryness. The main
product was separated by column chromatography (SiO2, CHCl3/MeOH
) 100:1). The solvents were evaporated off, and the residue was dried
thoroughly to yield 6 (73 mg, 94%) as a reddish purple solid: 1H NMR
(DMSO-d6) δ ) 9.71 (1H, s), 9.60 (1H, s), 9.51 (1H, s), 9.49 (1H,
brs), 9.48 (1H, brs), 9.44 (1H, s), 8.14 (1H, dd, J ) 18.0 Hz, J ) 11.6
Hz), 8.03 (1H, dd, J ) 18.0 Hz, J ) 11.6 Hz), 7.68 (4H, d, J ) 1.2
Hz), 6.95 (2H, t, J ) 1.2 Hz), 6.25 (1H, d, J ) 18.0 Hz), 6.19 (1H, d,
J ) 18.0 Hz), 6.10 (1H, d, J ) 11.6 Hz), 6.04 (1H, d, J ) 11.6 Hz),
4.25 (2H, t, J ) 7.0 Hz), 4.23 (2H, t, J ) 7,0 Hz), 4.17 (8H, s), 4.10
(8H, q, J ) 7.3 Hz), 3.94 (8H, s), 3.86 (8H, t, J ) 9.8 Hz), 3.42 (3H,
s), 3.40 (3H, s), 3.38 (3H, s), 3.35 (3H, s), 3.28 (2H, t, J ) 7.0 Hz),
3.27 (2H, t, J ) 7.0 Hz), 1.17 (12H, q, J ) 7.3 Hz), 0.91 (12 H, q, J
) 7.3 Hz), -5.40 (1H, brs); HRFAB MS calcd for C82H96O22N10
1573.6777 (MW + 1), found 1573.6758; UV-vis (CHCl3) λmax (rel
intensity) 409.7 (1.0), 506.6 (0.085), 543.1 (0.067), 576.4 (0.043), 629.8
(0.028) nm.
Zinc Complex (1‚Zn). Zinc was inserted into 6 (30 mg) in
quantitative yield using zinc acetate method:24 1H NMR (DMSO-d6)
δ ) 10.31 (2H, brs), 10.24 (1H, s), 10.16 (1H, s), 10.15 (1H, s), 10.13
(1H, s), 8.50 (1H, dd, J ) 17.7 Hz, J ) 11.6 Hz), 8.47 (1H, dd, J )
17.7 Hz, J ) 11.6 Hz), 7.69 (4H, d, J ) 0.9 Hz), 6.79 (2H, t, J ) 0.9
(23) Ueda, K. Bull. Chem. Soc. Jpn. 1979, 52, 1879.
(24) Fuhrhop, J.-H.; Smith, K. M. In Porphyrins and Metalloporphyrins;
Smith, K. M., Ed.; Elsevier: Amsterdam, 1975; p 772.

Artificial Protein-Protein Complexation
J. Am. Chem. Soc., Vol. 120, No. 20, 1998 4915
before and after flash photolysis studies in order to check that no
significant photodegradation of the protein samples was occurring.
ment to the preparation of this article. This work was partially
supported by Foundation Advanced Technology Institute, Japan.
Y.H. was supported by Research Fellowships of Japan Society
for the Promotion of Science for Young Scientists.
Acknowledgment. We are grateful to Prof. I. Morishima,
Dr. K. Ishimori, Dr. S. Takahashi, and Dr. T. Uchida, Kyoto
University, for the arrangement of laser flash photolysis
equipment. We thank Mr. K. Matsuura, JEOL Ltd., for
measuring the electrospray-ionization mass spectra. T.H. thanks
Prof. Y. Hisaeda, Kyushu University, for warmful encourage-
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