Protein engineering using molecular assembly: Functional conversion of cytochrome C via noncovalent interactions

Article abstract of DOI:10.1021/ja9711775

The structure-function relationship of cytochrome c (Cyt-c) interacted with the lipid bilayer membranes was studied by various spectroscopic methods, the reaction-products analysis, and its kinetics. Ultrafiltration binding assay, UV-visible, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies showed that Cyt-c was tightly bound to the lipid bilayer membranes bearing a phosphate head group. The anisotropic and nonnatural complexation with the phosphate-lipid membranes caused a spin-state change of the heme in the active center of Cyt-c. Depending on the membrane fluidity, two classes of the structurally altered Cyt-c were prepared and they showed the greatly enhanced N-demethylase activity. Products analysis by HPLC demonstrated that the lipid membrane bound Cyt-c performs a clean enzymatic reaction similar to native hemoenzymes. Kinetics studies established that there are two different activation manners via the phosphate lipid bilayer membranes: namely, simple enhancement of the affinity for H₂O₂, or the increased catalytic efficiency (k(cat)) in addition to the enhanced affinity for H₂O₂. The membrane fluidity again significantly affected the N-demethylation kinetics. A potential of the lipid membrane assembly to functionalize native proteins and enzymes with noncovalent but specific interactions is also discussed.

Full text of DOI:10.1021/ja9711775

9096
J. Am. Chem. Soc. 1997, 119, 9096-9102
Protein Engineering Using Molecular Assembly: Functional
Conversion of Cytochrome c via Noncovalent Interactions
Itaru Hamachi,* Akio Fujita, and Toyoki Kunitake*
Contribution from the Department of Chemistry & Biochemistry (Molecular Science Engineering),
Kyushu UniVersity, Hakozaki, Fukuoka 812-81, Japan
ReceiVed April 14, 1997X
Abstract: The structure-function relationship of cytochrome c (Cyt-c) interacted with the lipid bilayer membranes
was studied by various spectroscopic methods, the reaction-products analysis, and its kinetics. Ultrafiltration binding
assay, UV-visible, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies showed
that Cyt-c was tightly bound to the lipid bilayer membranes bearing a phosphate head group. The anisotropic and
nonnatural complexation with the phosphate-lipid membranes caused a spin-state change of the heme in the active
center of Cyt-c. Depending on the membrane fluidity, two classes of the structurally altered Cyt-c were prepared
and they showed the greatly enhanced N-demethylase activity. Products analysis by HPLC demonstrated that the
lipid membrane bound Cyt-c performs a clean enzymatic reaction similar to native hemoenzymes. Kinetics studies
established that there are two different activation manners via the phosphate lipid bilayer membranes: namely, simple
enhancement of the affinity for H2O2, or the increased catalytic efficiency (kcat) in addition to the enhanced affinity
for H2O2. The membrane fluidity again significantly affected the N-demethylation kinetics. A potential of the lipid
membrane assembly to functionalize native proteins and enzymes with noncovalent but specific interactions is also
discussed.
Introduction
proteins in basic molecular terms must be clarified. Because
the interactions between biopolymers and the molecular as-
semblies are so complicated, a suitable model system is required.
In a pioneering approach, a series of the recent resonance Raman
experiments by Hildebrandt and co-workers resulted in a unique
conformational change of cytochrome c (Cyt-c), an electron-
transporting hemoprotein, upon electrostatic binding to charged
interfaces such as phospholipid vesicles, a reversed micelle, and
heteropolytungstates.4 With respect to the intrinsic reactivity
of Cyt-c, during our study of the functional conversion of
hemoproteins by synthetic lipid bilayer assemblies, we identified
the enhanced peroxidase and N-demethylase activities of Cyt-c
bound to a lipid bilayer membrane.5
In the present article, we provide additional data on the
N-demethylase activity of Cyt-c induced by synthetic lipid
bilayer membranes bearing a phosphate head group (phosphate
lipid bilayer) and more importantly, discuss the relationship
between the lipid membranes-induced activity of Cyt-c and the
structural changes. We propose a method for noncovalent but
efficient protein engineering with supramolecular self-assembly
systems.
Hybridization of naturally occurring enzymes and proteins
with artificial molecules is one of the most promising methods
for the development of protein-based biomaterials. Among the
diverse artificial molecules, employment of self-organized
molecular assemblies is regarded as a unique approach. The
potential application for biosensors of enzyme-electrode com-
posites that use a self-assembled monolayer or multilayer films
has led to vigorous investigation of these artificial molecules.1
Enzymes encapsulated in reversed micelles or polymer ag-
gregates are applied as useful biocatalysts in some organic
synthetic (enantio- or regioselective) reactions, as well as the
separation processes of native enzymes.2 Protein-hydrophobic
polysaccharide aggregates have been proposed as a supramo-
lecular drug delivery system.3
In order to expand the potential of the molecular self-assembly
to functionalize native proteins as novel materials, how self-
assemblies affect the structure and activity of native enzymes/
* Author to whom correspondence should be addressed. E-mail:
itarutcmcmbox.nc.kyushu-u.ac.jp.
X Abstract published in AdVance ACS Abstracts, September 15, 1997.
(1) (a) Turner, A. P. F., Karube, M., Wilson, G. S., Eds. Biosensors:
Fundamentals and Applications; Oxford University Press: Oxford, 1987.
(b) Yasuda, Y.; Sugino, H.; Toyotama, H.; Hirata, Y.; Hara, M.; Miyake,
J. Bioelectrochem. Bioenerg. 1994, 36, 135. (c) Cullison, J. K.; Hawkridge,
F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877. (d) Rusling,
J.; Nassar, A. E. F. J. Am. Chem. Soc. 1993, 115, 11891. (f) Nassar, A. E.
F.; Bobbitt, J. M.; Stuart, J. D.; Rusling, J. J. Am. Chem. Soc. 1995, 117,
10986. (g) Ulman, A. An Introsuction to Ultrathin Organic Films: from
L-B to Self-Assembly; Academic Press: San Diego, 1991. (h) Katz, E.;
Schlereth, D. D.; Sxhmidt, H. L.; Olsthoorn, A. J. J. J. Electroanal. Chem.
1994, 368, 165.
(2) (a) Shield, J. W.; Ferguson, H. D.; Bommarius, A. S.; Hatton, T. A.
Ind. Eng. Chem. Fundam. 1986, 25, 603. (b) Mosbach, K. Methods Enzymol.
1988, 137, 583. (c) Klibanov, A. M. Anal. Biochem. 1979, 93, 1. (d) Naka,
K,; Kubo, Y.; Ohki, A.; Maeda, S. Polym. J. 1994, 26, 243.
(3) (a) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. Macromolecules 1994,
27, 7654. (b) Akiyoshi, K.; Nishikawa, T.; Shichibe, S.; Sunamoto, J. Chem.
Lett. 1995, 707. (c) Akiyoshi, K.; Nagai, K.; Nishikawa, T.; Sunamoto, J.
Chem. Lett. 1995, 1727. (d) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. J.
Am. Chem. Soc. 1996, 118, 6110.
Results
In order to investigate the structure and activity changes of
Cyt-c induced by lipid bilayer membranes, we used a variety
of synthetic lipids as shown in Chart 1.
Affinity of Cyt-c for Various Synthetic Lipid Bilayer
Membranes. Binding of Cyt-c to various lipid bilayer mem-
(4) (a) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710.
(b) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6722. (c)
Hildebrandt, P.; Heimburg, T.; Marsh, D. Eur. Biophys. J. 1990, 18, 193.
(d) Heimburg, T.; Hildebrandt, P.; Marsh, P. Biochemistry 1991, 30, 9084.
(e) Muga, A.; Mantsch, H. H.; Surewicz, W. K. Biochemistry 1991, 30,
7219. (f) Hildebrandt, P. In Cytochrome c-A multidisciplinary approach;
Scott. R. A., Mauk, A. G., Eds.; University Science Book: Sausalito, CA,
1996; p 285.
(5) (a) Hamachi, I.; Fujita, A.; Kunitake, T. J. Am. Chem. Soc. 1994,
116, 8811. (b) Fujita, A.; Senzu, H.; Kunitake, T.; Hamachi, I. Chem. Lett.
1994, 1219.
S0002-7863(97)01177-3 CCC: $14.00 © 1997 American Chemical Society

Protein Engineering Using Molecular Assembly
J. Am. Chem. Soc., Vol. 119, No. 39, 1997 9097
Chart 1
Figure 1. Binding assay of Cyt-c with the mixed lipid bilayer
membrane comprising of the phosphate-lipid 1a and DMPC 3: Cyt-c
5 µM, 1a + 3 ) 1mM, in 25 mM phosphate buffer (pH 7.0).
Lipid Bilayer-Induced Structural Changes in Cyt-c.
Structural changes in Cyt-c bound to the lipid bilayer membranes
were examined using UV-visible absorption, electron para-
magnetic resonance (EPR), and circular dichroism (CD) spec-
troscopy. Table 1 compares the UV-visible spectrum of native
Cyt-c with those of the membrane-bound Cyt-c both in the
oxidized (Fe(III)) and reduced (Fe(II)) forms. A sharp Soret
band at 408 nm, a broad Q band at 560 nm and a very weak
charge-transfer (LMCT) band from the sulfur atom of Met 80
(the axial ligand) to heme iron(III) at 695 nm are observed for
the oxidized form of native Cyt-c.9 Slight shift of the Soret
band (406 nm) and complete disappearance of the LMCT band
are apparent in the spectrum of 1a-bound Cyt-c. The Soret band
shows a distinct red shift (from 416 to 426 nm) with smaller
extinction coefficient (ꢀ ) 85 mM-1 cm-1) for the reduced form
of 1a-bound Cyt-c.10 Two characteristic Q bands of native Cyt-c
at 550 and 520 nm became one broad band (550 nm) in the
1a-bound Cyt-c spectrum. These spectral changes reveal the
complete cleavage of the coordination bond of Met 80 to
iron(III) upon binding to the phosphate lipid bilayer membrane
1a.
On the other hand, 1d-bound Cyt-c displays different spectral
behavior. For the oxidized state, in the 1d-bound Cyt-c
spectrum, although the LMCT band remained at 70% of the
native Cyt-c intensity, the other peaks closely resembled those
obtained for the case of 1a-bound Cyt-c. Two Q bands (550
and 520 nm) and a sharp Soret band (415 nm) similar to the
native bands were clearly retained in the reduced form. These
spectral characteristics suggest that 1d-bound Cyt-c exists in
equilibrium between a high-spin (i.e., broken Met-80 iron bond)
and a low-spin (i.e., coordinated Met 80-iron bond) state. The
1:2 ratio (the high-spin species:the low-spin species) is roughly
estimated from the intensity of LMCT band. No changes occur
upon addition of ammonium lipid 2a or zwitterionic 3.
The spin-state alteration of the active site of Cyt-c was directly
monitored using EPR at 20 K. Figure 2 shows the EPR spectra
of frozen solutions in the presence and absence of the lipid
bilayer membranes. Without lipid membranes, two peaks at g
) 3.0 and 2.3 appear as reported in the literature, indicating a
ferric low-spin state (Figure 2a).11 The signals due to the low
spin are remarkably reduced for the 1a-bound Cyt-c (Figure
2b). Instead, a strong peak (g ) 5.9) and a weak peak (g )
branes was evaluated using the ultrafiltration binding assay.6
The fraction of Cyt-c bound to the phosphate lipid bilayer 1a,
1b, 1c, and 1d, an ammonium-lipid bilayer, 2a, and a
zwitterionic DMPC 3, was 96, 98, 90, 95, 6, and 10%,
respectively. Clearly, the N-demethylase bilayers tightly bound
Cyt-c under neutral pH, because of the high isoelectric point of
Cyt-c (pI ) 10).7 Next, the binding of Cyt-c with a mixed lipid
bilayer membrane of 1a and DMPC 3 was evaluated. When
the lipid bilayer membrane consisted of less than 10% 1a, only
10% Cyt-c bound. As the percentage of 1a in the mixed lipid
bilayer membrane increased, the amount of membrane-bound
Cyt-c sigmoidally increased (shown in Figure 1).8 These results
indicate that Cyt-c is selectively bound to an anionic phosphate
lipid bilayer membrane.
(6) (a) Hamachi, I.; Nakamura, K.; Fujita, A.; Kunitake, T. J. Am. Chem.
Soc. 1993, 115, 4966. (b) Hamachi, I.; Higuchi, S.; Nakamura, K.; Fujimura,
H.; Kunitake, T. Chem. Lett. 1994, 1219.
(7) Barlow, G. H.; Margoliash, E. J. J. Biol. Chem. 1966, 241, 1473.
(8) In the mixed lipid membrane of the lower content of 1a (less than
10 %), it is reasonable to consider that 1a is homogeneously dispersed in
the DMPC lipid. With increase of the 1a ratio, the lipid 1a easily aggregates
to form a cluster in the mixed membrane. The sigmoidal curve for the
binding affinity study suggests that such a cluster structure of 1a is needed
for the Cyt-c binding. The N-demethylase activity was also found to be
sigmoidal in the presence of the mixed lipid bilayer membrane.
(9) (a) Sreenathan, B. R.; Taylor, C. P. S. Biochem. Biophys. Res.
Commun. 1971, 42, 1122. (b) Schechter, E.; Saludjian, P. Biopolymers 1967,
5, 788. (c) Kaminsky, L. S.; Ivanetich, K. M.; King, T. E. Biochemistry
1974, 13, 4866.
(10) The molecular extinction coefficient of native Cyt-c (reduced form)
was reported to be 129.1 /mM cm. Margoliash, E.; Frohwirt, N. J. Biochem.
1959, 71, 570.
(11) Ikeda-Saito, M.; Iizuka, T. Biochim. Biophys. Acta 1975, 393, 335.

9098 J. Am. Chem. Soc., Vol. 119, No. 39, 1997
Table 1. Absorption Spectra of Cyt-c Derivatives
Hamachi et al.
ferric state (λmax (ꢀmM))
ferrous state (λmax (ꢀmM))
Q band, nm
SfFe charge
transfer (695 nm)
Q band
(R/â), nm
Soret band,
nm
Soret band,
nm
hemoprotein
native Cyt-c
bilayer 1a-bound Cyt-c
bilayer 1b-bound Cyt-c
bilayer 1c-bound Cyt-c
bilayer 1d-bound Cyt-c
bilayer 1e-bound Cyt-c
myoglobin
R
â
yes
no
no
528
525
523
530
525
528
502
409 (106.1)
406
407
407
407
407
409
550 (29.5)
520
415 (129.1)
426 (98)
418 (102.5)
419 (91)
415 (107)
428 (88)
434
550 (broad; 11.6)
550 (broad; 14.5)
550 (broad; 12.9)
520
550 (broad; 10.3)
556 (broad)
no
yesa
no
550 (19.7)
a Mixture of high-spin and low-spin states.
Figure 3. CD spectra of the N-demethylase bilayer membranes-bound
Cyt-c: (a) Soret band region and (b) the amide bond region.
When Cyt-c is bound to 1a (- ‚ -, Cyt-c/1a), the CD spectrum
changes markedly and exhibits a positive (423 nm) peak, a
strong negative (412 nm) peak, and a weak positive (393 nm)
peaks. A similar but intensified CD pattern is observed for Cyt-
c/1e (‚‚‚). In contrast, the CD spectrum of Cyt-c/1d is very
different; the positive peak at 423 nm disappears, and instead a
sharp negative (413 nm) band and a broad positive (398 nm)
band are observed. Thus, the heme environment of 1d-bound
Cyt-c appears to be different from 1a-bound Cyt-c. It should
be noted that these spectra are distinct from that of urea-
denatured Cyt-c in which a broad and less structured CD signal
at 410nm (positive) is observed.15 In addition, the CD spectrum
of the R-helix region for Cyt-c/1e is almost identical to that of
native Cyt-c.16 Clearly, the N-demethylase induced structural
changes of Cyt-c are not due to a simple denaturation.
Anisotropic Binding of Cyt-c to the Phosphate-Lipid
Bilayer Membranes. The molecular orientation of Cyt-c on
the lipid bilayer membrane was examined using the angular
dependence of EPR measurement of the immobilized lipid film
containing Cyt-c at the temperature of liquid helium (4 K).17
EPR spectra of the film of the Cyt-c/1a composite were
measured at different angles (φ) of the normal of the film plane
against the direction of the applied magnetic field (Figure 4).
The EPR signals appeared to change depending on the angle.
When the film plane was disposed parallel to the magnetic field
(φ ) 90°), an intense signal at g ) 5.9 (g component of the g
parameters) and a very weak signal at g ) 2.0 (g| component)
Figure 2. EPR spectra of frozen solutions containing Cyt-c in the
absence and presence of lipid bilayer membranes at 20 K: Cyt-c 210
µM, 8.3 mM of lipid 10 mM of the phosphate buffer (pH 7.0). (a)
without lipid, (b) with 1a, (c) with 1d, (d) with 2a. EPR conditions:
in a quartz EPR tube (diameter 2 mm), JEOL JES-2X X-band EPR
spectrophotometer equipped with a microwave liquid helium cryostat
with 100 kHz magnetic field modulation, 5 mW microwave power,
0.79 mT modulation amp.
2.0) which are ascribed to the ferric high-spin state emerge.12
In sharp contrast, the EPR spectrum of 1d-bound Cyt-c is almost
identical to the ferric low-spin state (Figure 2c) and the high-
spin component is not observed at 20 K.13 EPR signals of Cyt-c
in the presence of ammonium 2a (Figure 2d) and zwitterionic
3 (data not shown) lipid bilayers do not differ from those of
native Cyt-c.
Figure 3 shows the CD spectra of Cyt-c in the presence of
various phosphate-lipid bilayer membranes. A negative (419
nm) and a positive (410 nm) peak due to a split Cotton effect
are observed for native Cyt-c (represented as - - - in Figure 3).14
(15) Myer, Y.P.; MacDonald, L. M.; Verma, B. C.; Pande, A. Biochem-
istry 1980, 19, 199.
(16) Unfortunately, a strong CD band due to the chiral amide residue of
the lipids 1a and 1d prevented us from estimating the R-helix region by
CD spectroscopy.
(17) This technique has been utilized for determining the orientation of
membrane-bound hemoproteins and iron-sulfur proteins in biomembranes.
Blasie, J. K.; Erecinska, M.; Samuels, S.; Leigh, J. S. Biochim. Biophys.
Acta 1978, 501, 33.
(12) The observed g values and the signal pattern are closely similar to
those of myoglobin which has His and H2O coordinated to the heme iron.
Yonetani, T.; Schleyer, H. J. Biol. Chem. 1967, 242, 3926.
(13) For 1d-bound Cyt-c, the low spin state became predominant at 20
K, instead of the spin-state equilibrium at room temperature.
(14) Myer, Y. P. Methods Enzymol. 1978, 54, 249.

Protein Engineering Using Molecular Assembly
J. Am. Chem. Soc., Vol. 119, No. 39, 1997 9099
Figure 5. The initial rates of N-demethylase activity of Cyt-c in the
absence and presence of various lipid bilayer membranes: Cyt-c 5 µM,
NNDA 2 mM, H2O2 1 mM, lipid 0.25 mM in 25 mM phosphate buffer
(pH 7.0) at 30 °C.
is available to react with small molecules, just like the heme
center of peroxidase or monooxygenase in the family of
hemoenzymes. N-Demethylation is one of the typical oxidation
reactions catalyzed by such hemoenzymes. Thus we evaluated
the catalytic activity of Cyt-c in the presence of the various
lipid bilayer membranes using a N-demethylation reaction of
N,N-dimethylaniline (eq 1). The initial reaction rates were
determined by detecting the amount of formaldehyde produced21
and are summarized in Figure 5. The addition of 1a accelerated
the initial reaction rate (14 nmol/mL min) by 10-fold, relative
to that of native Cyt-c (i.e., without lipid bilayers, the reaction
rate is 1.3 nmol/(mL min)). This rate enhancement produced
typical saturation behavior with respect to the 1a concentration
(data not shown). Other N-demethylase bilayer membranes also
enhanced the reaction rate (11 nmol/(mL min) for 1b, 27 nmol/
(mL min) for 1c and 30 nmol/(mL min) for 1d). In contrast,
the reaction rates in the presence of the ammonium-lipid
bilayers were almost identical to that of native Cyt-c (2.0 nmol/
(mL min) for 2a and 1.6 nmol/(mL min) for 2b). The reaction
was moderately suppressed by the addition of zwitterionic lipid
3 (0.8 nmol/(mL min)). An anionic micelle, sodium dodecyl
sulfate (SDS), did not influence the rate (2.0 nmol/(mL min)).
These results suggest that the phosphate-lipid bilayer mem-
branes enhance the N-demethylase activity of Cyt-c.
Analysis of the N-Demethylation Reaction. Figure 6a
shows a HPLC (high-performance liquid chromatography) chart
obtained during the demethylation reaction catalyzed by the 1a-
bound Cyt-c. We observed only two peaks; the peak due to
the starting material (N,N-dimethylaniline (NNDA), retention
time tR ) 5.4 min) and the product (N-methylaniline, tR ) 3.8
min) in addition to the peaks of the solvent (trichloroacetic acid)
and the internal standard (N-ethylaniline). No other peaks due
to byproducts were detected, indicating that the present dem-
ethylation is a clean oxidation reaction with no considerable
side reactions. Figure 6b displays the time courses of N-
methylaniline and formaldehyde produced, along with that of
the consumed H2O2. The amount of N-methylaniline produced
as determined by HPLC is in good agreement with that of
formaldehyde produced as determined by the Nash method. The
stoichiometry of consumed H2O2 to product is 2:1. This ratio
is different from the native peroxidase-catalyzed N-demeth-
Figure 4. Angular dependence of EPR spectra of Cyt-c immobilized
in a cast film of 1a: Cyt-c/1a ) 1/200 (mol/mol) at 5 K. EPR conditions
are same as Figure 2.
were observed. As the angle decreases to 0°, the g signal is
weakened and g| is intensified. The present angular dependence
of both g components implies that the heme plane in Cyt-c is
oriented almost parallel relative to the lipid multilayer film plane
(i.e., parallel to the lipid bilayer 1a surface).18 Similar orienta-
tion of Cyt-c has been reported in anionic Langmuir-Blodgett
films and self-assembled monolayers on gold.19 This orientation
of Cyt-c has been ascribed to the electrostatic interaction
between the cationic domain of Cyt-c and the anionic membrane
surfaces. Notably, the present regular orientation indicates that
the phosphate lipid bilayer 1a anisotropically acts upon the
specific domain of Cyt-c.20
Lipid Bilayer-Enhanced N-Demethylase Activity of Cyt-
c. The above findings with respect to the structural changes in
membrane-bound Cyt-c suggest that the axial site of the heme
(18) The direction of g| tensor is assigned parallel to the normal axis of
the heme plane and the direction of g components are in the heme plane.
The g signal is intensified when the direction of g tensor is parallel to the
direction of the applied magnetic field. The simulation was conducted
according to the same manner reported by us. (a) Hamachi, I.; Noda, S.;
Kunitake, T. J. Am. Chem. Soc. 1991, 113, 9625. (b) Ishikawa, Y.; Kunitake,
T. J. Am. Chem. Soc. 1991, 113, 621.
(19) (a) Pachence, J. M.; Blasie, J. K. Biophys. J. 1987, 52, 735. (b)
Pachence, J. M.; Fischetti, R. F.; Blasie, J. K. Biophys. J. 1989, 56, 327.
(c) Pachence, J. M.; Amador. S. M.; Mamoara, G.; Vanderkooi, J.; Dutton,
P. L.; Blasie, J. K. Biophys. J. 1990, 58, 379.
(20) Since the phosphate lipid 1d did not form a stable cast film, we
could not evaluate the orientation of Cyt-c against the membrane comprising
of 1d.
(21) Werringloer, J. Methods Enzymol. 1978, 52, 297.

9100 J. Am. Chem. Soc., Vol. 119, No. 39, 1997
Hamachi et al.
Figure 6. Assay of the products formed during the N-demethylation reaction catalyzed by 1a-bound Cyt-c: (a) HPLC elution profile of the
reaction mixture at the reaction time of 6 min and (b) time courses of the amounts of the produced formaldehyde and N-methylaniline, and of the
consumed H2O2.
ylation (stoichiometry of 1:1).22 Therefore, the oxidation
efficiency (the ratio of the product based on the oxidant; i.e.,
[product]/ [H2O2]) is 0.5 for the present system and 1 for the
native peroxidase system. This reduction in efficiency suggests
that the present reaction system contains some nonproductive
side reactions that waste H2O2.23 Similar behavior was observed
for the other N-demethylase bilayer bound Cyt-c samples.
The hemoenzyme-catalyzed N-demethylation has been re-
ported to proceed via two major steps, in which the hemoenzyme
reacts with H2O2 to form an active intermediate (a hypervalent
iron-oxo species was confirmed in some cases), followed by
the oxidation of substrates (NNDA in this case).24 All attempts
to detect the active intermediates of Cyt-c failed in our system
due to the instability. Instead, rapid decomposition of the heme
unit of Cyt-c occurred in the absence of NNDA and the
decomposition rate was simply suppressed by the addition of
NNDA. The heme degradation rate in the absence of NNDA
was dependent on the type of lipid bilayer membrane. In the
presence of 1a, the initial rate (2.5 µM/min) was greatly
accelerated, relative to that in the absence of lipids (0.27 µM/
min), or relative to 2a (0.26 µM/min) or to 3 (0.25 µM/min).
Interestingly, the rate in the presence of 1d (11.0 µM/min) was
4 times greater than that of 1a.25
because of the tightly coordinated axial ligands of His 18 and
Met 80.26 The fast autooxidation (1.6 µM/min) of Cyt-c
occurred upon binding to 1a. Binding to phosphate lipid 1d
(4.4 µM/min) was again more effective than 1a. Conceivably,
1a-bound Cyt-c or 1d-bound Cyt-c acquired N-demethylase
activity, instead of losing native electron-transfer activity under
aerobic conditions. The ammonium lipid 2a and DMPC 3 did
not facilitate the autooxidation reaction.
Kinetics of N-Demethylase Activity of Membrane-Bound
Cyt-c. The hemoprotein-catalyzed N-demethylation by H2O2
is regarded as a typical two-substrate reaction in general. The
initial rate was found not to be sensitive to the concentration of
NNDA, but dependent on the concentration of H2O2 in a
saturation manner. Therefore, the rate-determining step of the
overall reaction was assigned to the generation step of the active
intermediate of Cyt-c with H2O2. Under these conditions,
double-reciprocal plots of the initial rates against the H2O2
concentration yielded satisfactorily linear relationships as shown
in Figure 8. The parameters obtained in the Michaelis-Menten
scheme were as follows: kcat ) 9 min-1, Km ) 2 mM in the
presence of 1a, kcat ) 87 min-1, Km ) 5 mM in the presence of
1d-bound Cyt-c, kcat ) 10 min-1, Km ) 28 mM in the presence
of Cyt-c with 2a, and kcat ) 12 min-1, Km ) 40 mM for Cyt-c
without lipids. The following points should be noted: (i) Km
for the phosphate-lipids 1a and 1d are greatly lower than the
value in the absence of lipids; (ii) the kcat value for 1a is almost
identical with that in the absence of lipids, whereas kcat for 1d
is 7-fold greater; (iii) the kinetic parameters for 2a are the same
as those in the absence of lipids within the margin of
experimental errors. These results indicate that phosphate lipid
1a facilitates the N-demethylase activity of Cyt-c by the
increased affinity of Cyt-c for H2O2 (1/Km), not by the catalytic
efficiency (kcat). In contrast, in the case of 1d, the enhanced
net activity is reasonably ascribed to not only the increased
The autooxidation rate of the reduced Cyt-c was also
accelerated by the addition of the phosphate lipid bilayers as
shown in Figure 7. The reduced Cyt-c without lipids was hardly
autooxidized under aerobic conditions (less than 0.01 µM/min)
(22) Kedderis, G. L.; Hollenberg, P. F. J. Biol. Chem. 1983, 258, 8129.
(23) The catalase-like reaction (2H2O2 f 2H2O + O2) might be a
candidate of such a reaction. We actually confirmed that H2O2 was
consumed by 1a-bound Cyt-c in the absence of NNDA.
(24) (a) Kedderis, G. L.; Koop, D. R.; Hollenberg, P. F. J. Biol. Chem.
1980, 255, 10174. (b) Kedderis, G. L.; Hollenberg, P. F. J. Biol. Chem.
1983, 258, 12413. (c) Miwa, G. T.; Walsh, J. S.; Kedderis, G.L.; Hollenberg,
P. F. J. Biol. Chem. 1983, 258, 14445.
(25) The order of the magnitude of the heme-degradation rate was in
good correspondence with that of the net N-demethylase activity.
(26) Bushnell, G. W.; Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1983,
214, 585.

Protein Engineering Using Molecular Assembly
J. Am. Chem. Soc., Vol. 119, No. 39, 1997 9101
Figure 7. UV-visible spectral change of the autooxidation process of the reduced Cyt-c by addition of 1a. (Inset) Time course of autooxidation
of the reduced Cyt-c in the presence of various lipid bilayer membranes: 10 µM of Cyt-c in 10 mM phosphate buffer (pH 7.0) under aerobic
conditions. The reaction was initiated by the addition of the lipid bilayer dispersion.
Figure 8. Double-reciprocal (Lineweaver-Burk) plots of the initial
Figure 9. Dependence of the initial rates of N-demethylation reaction
rate of N-demethylation by Cyt-c against H2O2 concentration in the
catalyzed by Cyt-c in the presence of the lipid membranes on Tc (phase
absence and presence of the lipid bilayer membranes (1a, 1d, 2a): Cyt-c
transition temperature): Tc - T, the difference between the phase
5 µM, NNDA 2 mM, lipid 1 mM, in the phosphate buffer(pH 7.0).
transition temperature of the corresponding lipid membranes and the
reaction temperature (38 °C).
affinity for H2O2, but also the enhanced catalytic efficiency of
the membrane-bound Cyt-c.
membrane surface. This implies that another cationic domain
which is not used in the natural system interacts with the
membrane surface. Such a nonnatural electrostatic interaction
may play a crucial role in the structural perturbation of the
membrane-bound Cyt-c.
Interestingly in the present study, these structurally perturbed
Cyt-c molecule showed enhanced N-demethylase activity.
Product analysis demonstrated that the present N-demethylation
is a clean reaction similar to the native enzyme system although
the oxidation efficiency is rather less efficient than native one.
We plotted the initial rates of N-demethylation catalyzed by
Cyt-c bound to five different phosphate lipid bilayer membranes
(1a, 1b, 1c, 1e, and 1d) against their gel-to-liquid-crystal phase-
transition temperatures (Tc ) 58, 48, 36, 30, and 18 °C,
respectively).28 As evident in Figure 9, an interesting feature
is that the Cyt-c bound to the membranes in the liquid-crystalline
Discussion
Two classes of structurally altered Cyt-c were prepared upon
binding to different phosphate lipid bilayer membranes; Cyt-c
bearing the high spin state of heme (1a-bound Cyt-c) or Cyt-c
bearing the heme in an equilibrium between the high spin and
the low spin state (1d-bound Cyt-c). This is consistent with
the results obtained by Hildebrandt and co workers.4 Cyt-c is
known to possess cationic lysine clusters.26,27 When Cyt-c
interacts with cyt-c-oxidase (CcO), a natural electron-accepting
partner, the lysine cluster surrounding the heme crevice of Cyt-c
binds to the negatively charged domain of CcO. If the same
lysine cluster is employed to attach Cyt-c to the phosphate-
lipid membrane, the heme of Cyt-c can be assumed to be
oriented perpendicular to the membrane surface. However, the
heme orientation has been determined to be parallel to the
(28) The phase-transition temperature of the lipid bilayer membranes
was determined by differential scanning calorimeter (DSC). Although Figure
9 displayed the plot of the reaction rate at 38 °C, similar dependence was
obtained at the reaction temperature of 30 °C.
(27) Scott. R. A., Mauk, A. G., Eds. Cytochrome c-A multidisciplinary
approach; University Science Book: Sausalito, CA, 1996.

9102 J. Am. Chem. Soc., Vol. 119, No. 39, 1997
Hamachi et al.
Calcd for C43H84NO9P: C, 65.35; H, 10.74; N, 1.77. Found: C, 65.61;
H, 10.76; N, 1.78. The synthesis of lipids 1b, 2a and 2b was reported
previously.18a,31 Other chemicals were commercially available.
Assay Procedure for N-Demethylase Activity. The N-demethylase
activity of Cyt-c was assayed by measuring the amount of the produced
formaldehyde using a modified Nash method.21 Powdery lipids were
suspended in 25 mM phosphate buffer (pH ) 7.0) and sonicated at the
appropriate temperatures (i.e., 5-10 °C higher than the corresponding
Tc) for 3-10 min. In the case of the phosphate lipids, equimolar amount
of NaOH was added before sonication. After the solution was incubated
at room temperature overnight,32 Cyt-c and NNDA was then added to
the solution. The mixture was incubated for several minutes in a water
bath equipped with a thermostatic controller (30 or 38 °C). The reaction
was initiated by the addition of H2O2 and terminated by the addition
of 30% trichloroacetic acid (TCA). After centrifugation of the reaction
mixture at 15000 rpm for 20 min, 1 mL of the supernatant was mixed
with Nash reagent (11.3 g of ammonium acetate, 150 uL of 2,4-
pentanedione and 225 uL of acetic acid in 25 mL of H2O) and the
solution was incubated at 40 °C for 1 h. The absorbance of the resulting
conjugate (3,5-diacetyl-1,4-dihydrolutidine) was monitored at 412 nm
(ꢀ412nm ) 7950 M-1 cm-1) on a Shimadzu UV2200 UV-visible
spectrophotometer.
Product Determination in N-Demethylation Reaction. From the
reaction mixture (20 mL scale), a 3 mL sample was taken out at 0, 2,
4, 6, 8, 10, 12 min and mixed with 0.9 mL of TCA. After
centrifugation, the supernatant was applied to HPLC: column; a
reversed-phase TOSOH, internal standard; N-ethylaniline,22 eluent;
acetonitrile/H2O (1/1, v/v)) at a flow rate of 1.0 mL/min. The amount
of the unreacted H2O2 was determined as follows:33 To the supernatant
(1 mL), 0.2 mL of ferroammonium sulfate (10 mM, freshly prepared)
and 80 µL of 2.5 M of KSCN were added. The solution was incubated
for 10 min at 25 °C, and the absorbance at 480 nm was monitored.
Binding Assay of Cyt-c to Lipid Bilayer Membranes.6 The
solution containing Cyt-c and lipid membranes was ultrafiltered using
a MOLCUT II system (UPF1 THK24, cut of molecular weight 100000;
Millipore). The concentration of unbound Cyt-c was determined by
the absorbance of the filtrate (408 nm).
state more effectively catalyze the reaction rather than the Cyt-c
bound to the gel state membrane. No Tc dependence was
observed in the case of ammonium-lipid membranes. Thus, the
membrane fluidity directly affects the intrinsic reactivity of the
membrane-bound Cyt-c, in addition to the simple surface charge.
The structural study demonstrates that for the Cyt-c bearing
the high spin-state of heme (1a-bound Cyt-c), the anisotropic
lipid-binding causes the complete dissociation of the axial Met
80. The vacant axial site of the heme center is readily accessible
to react with H2O2. This is clearly reflected by the enhanced
affinity for H2O2 . On the other hand, the Cyt-c bearing the
heme in a high-spin/low-spin equilibrium state shows different
kinetic features. We conducted a kinetic study of 1d-bound
Cyt-c as a typical example. The affinity for H2O2 was 2.5-fold
lower than that of 1a-bound Cyt-c, whereas the catalytic
efficiency was 10-fold greater. The increased kcat may have
been due to the rapid exchange between the coordinated and
uncoordinated Met 80. Dynamic swing of Met 80 in the axial
site can facilitate the generation of the active species of heme
that has reacted with H2O2. Such an increase in the dynamics
of the active center is reflected in the observed accelerated heme
degradation and autooxidation of the 1d-bound Cyt-c.
We previously reported membrane-facilitated electron transfer
and aniline hydroxylase activity of myoglobin.29 These func-
tions were ascribed to the unique capability of the lipid bilayer
membrane to two-dimensionally organize plural functional
molecules. The results of the present study established that
specific binding of a lipid bilayer membrane alter the intrinsic
activity of a native protein. Very recently, Sekimizu and co-
workers reported that the activity of Dna A, an initiation protein
of DNA replication, can be switched on by synthetic phosphate
lipid membranes.30 Based on these findings, we conclude that
lipid bilayer membranes can operate as an active effector on
protein molecules through both noncovalent and anisotropic
interactions, as well as controlling the assembly of functional
groups. Such interactions are potentially useful for protein-
based bioengineering.
EPR Measurement and Simulation.18a,b EPR measurements at 4
or 20 K, and the simulation for the orientation of the heme plane of
Cyt-c against the film surface was conducted according to the reported
method by us.
CD Measurement. CD spectra were obtained using a JASCO J-720
spectropolarimeter (sensitivity, 10 mdeg; resolution, 0.2 nm; bandwidth,
2.0 nm; response, 0.5 s; speed; 200 nm/min) at 25 °C.
Experimental Section
Materials. Horse heart cytochrome-c (Cyt-c, type IV) was pur-
chased from Sigma Chemical Co. and purified by passing through a
Sephadex G-25 column. The synthetic lipids were prepared according
to the method reported previously by us6,18a,31 and characterized by
NMR, IR and elemental analysis. 1a: Anal. Calcd for C44H84NO13P:
C, 64.38; H, 9.86; N, 1.63. Found: C, 64.19; H, 9.73; N, 1.67. 1c:
Anal. Calcd for C38H68NO10P: C, 61.84; H, 9.17; N, 1.92. Found:
C, 62.04; H, 9.17; N, 1.92. 1d: Anal. Calcd for C42H75NO12P: C,
61.81; H, 9.16; N, 1.72. Found: C, 61.97; H, 9.22; N, 1.78. 1e: Anal.
Acknowledgment. The authors thank Mrs. Atsushi Baba and
Takeshi Kawakami for the preparation of the synthetic lipids.
We are also grateful to Mr. Hideki Horiuchi for his skillful
preparation of the specially designed quartz plate for EPR
measurements. This research is partially supported by the
Foundation Advanced Technology Institute and Nissan Science
Foundation.
(29) (a) Hamachi, I.; Fujita, A.; Kunitake, T. J. Inorg. Biochem.1993,
51, 327. (b) Hamachi, I.; Fujita, A.; Kunitake, T. Chem. Lett. 1995, 657.
(30) (a) Sekimizu, K. Chem. Phys. Lipids. 1994, 73, 223. (b) Mizushima,
T.; Ishikawa, Y.; Obana, E.; Hasa, M.; Kubota, T.; Katayama, T.; Kunitake,
T.; Sekimizu, K. J. Biol. Chem. 1996, 271, 3633.
JA9711775
(32) The morphology of all of the lipid bilayer membranes was a mixture
of tube, disk, and stringlike structures.
(31) Nakashima, N.; Asakuma, S.; Kunitake, T. J. Am. Chem. Soc. 1985,
107, 509.
(33) Hildebrandt, A. G.; Roots, I.; Tjoe, M.; Heinemeyer, G. Methods
Enzymol. 1978, 52, 342.