Mimicking hemoproteins: A new synthetic metalloenzyme based on a Fe(III)-mesoporphyrin functionalized by two helical decapeptides

Article abstract of DOI:10.1002/psc.2586

A new metalloenzyme formed by a Fe(III)-mesoporphyrin IX functionalized by two helical decapeptides was synthesized to mimic function and structural features of a hemoprotein active site. Each decapeptide comprises six 2-aminoisobutyric acid residues, whi

Full text of DOI:10.1002/psc.2586

Research Article
Received: 30 July 2013
Revised: 15 October 2013
Accepted: 16 October 2013
Published online in Wiley Online Library: 19 November 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2586
Mimicking hemoproteins: a new
synthetic metalloenzyme based on a Fe
(III)-mesoporphyrin functionalized by
two helical decapeptides
Mariano Venanzi,* Sabrina Cianfanelli and Antonio Palleschi
A new metalloenzyme formed by a Fe(III)-mesoporphyrin IX functionalized by two helical decapeptides was synthesized to
mimic function and structural features of a hemoprotein active site. Each decapeptide comprises six 2-aminoisobutyric acid
residues, which constrain the peptide to attain a helical conformation, and three glutamic residues for improving the solubility
of the catalyst in aqueous solutions. The new compound shows a marked amphiphilic character, featuring a polar outer sur-
face and a hydrophobic inner cavity that hosts the reactants in a restrained environment where catalysis may occur. The cat-
alytic activity of this synthetic mini-protein was tested with respect to the oxidation of L- and D-Dopa by hydrogen peroxide,
showing moderate stereoselectivity. Structural information on the new catalyst and its adduct with the L- or D-Dopa substrate
were obtained by the combined use of spectroscopic techniques and molecular mechanics calculations. Copyright © 2013
European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: peptide catalyst; mini-enzyme; hemoprotein models; Dopa oxidation kinetics; helical oligopeptides; Aib
linked to the porphyrin scaffold or (ii) self-assemble around the
porphyrin site by exploiting noncovalent interactions. Among the
Introduction
Hemoproteins are impressive chemical machineries: A single
prosthetic group, the heme, is able to promote an amazing
variety of different biological functions, such as transport and
storage of oxygen, transfer of electrons, oxidation of organic sub-
strates, and destruction of peroxides [1].
The protein 3D structure controls the reactivity and specificity
of the prosthetic group by tuning the geometry, number and
type of metal coordination, nature of the axial ligands in the pri-
mary coordination shell, local dielectric constant, and nature of
the interactions in the secondary coordination shell [2]. The envi-
ronment hosting the metal ion in the reactive pocket determines
the specific potential needed to start a given redox reaction and
hinders the entry to the active site discriminating between com-
petitive substrates [3].
Several model systems have been developed trying to embed
the properties of a protein in a simpler molecule, with the aim to
establish structure–activity relationships and understand the ba-
sic requirements that make possible to fulfill a specific function.
Peptide-based compounds are intermediates between an intact
large protein and a small molecular catalyst, thus being of suffi-
cient complexity to retain the principal structural features of a
protein environment and simple enough to improve the compre-
hension of the factors determining the reactivity of natural
systems.
Several excellent review articles, collecting seminal results in
the field, have appeared in the literature, to which the interested
reader can refer for a detailed description of the history of peptide
models of heme proteins [4]. Shortly, they can be classified in two
broad families: models in which (i) the peptide chains are covalently
former, the field was pioneered by the introduction of
microperoxidases, heme–peptide fragments obtained by the pro-
teolytic digestion of cytochrome c [5], and the monopeptide–
deuteroheme complex of Casella and co-workers [6]. This work
culminated in the paradigmatic four-helix bundle model developed
by DeGrado and co-workers [7], which, besides fundamental acqui-
sition on the understanding of protein folding and function, leads
to a wide range of applications from catalysis [8] to biosensing
[9], paving the way for the fast-growing field of bio-inspired nano-
technology [10].
At an intermediate level of complexity between simple por-
phyrin derivatives and isolated protein fragments, Pavone and
co-workers [11] and Benson and co-workers [12] developed a
helix–heme–helix sandwich model composed of two helical
peptides covalently linked through the amino group of a Lys side
chain to the twin propionates of deuteroporphyrin IX and
mesoporphyrin II or IX, respectively.
*
Correspondence to: Mariano Venanzi, Department of Chemical Sciences and
Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1,
00133 Rome, Italy. E-mail: venanzi@uniroma2.it
Department of Chemical Sciences and Technologies, University of Rome Tor
Vergata, Rome, Italy
Abbreviations: 10Np, H-Ala-Aib-Glu(OtBu)-Aib-Glu(OtBu)-(Aib)3-Glu(OtBu)-
Aib-OCH2-CH2-Napht; mP(10N)2, mesoporphyrin IX/(Ala-Aib-Glu-Aib-Glu-(Aib)
3-Glu-Aib-OCH2-CH2-Napht)2 conjugate; FemP(10N)2, Fe(III)-mesoporphyrin IX/
(Ala-Aib-Glu-Aib-Glu-(Aib)3-Glu-Aib-OCH2-CH2-Napht)2 conjugate.
J. Pept. Sci. 2014; 20: 36–45
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

A PEPTIDE CATALYST MIMICKING HEMOPROTEINS
In their peptide models of hemoprotein, one or two His resi-
dues were suitably inserted in the peptide chain to coordinate
the central iron ion, giving rise to pentacoordinated high-spin
species in the monoadducts and hexacoordinated low-spin sys-
tems in the bis-adducts. Interestingly, the molecule showing the
highest content of pentacoordinated structures also showed
the highest activity, suggesting that a tight binding of the protein
environment in peptide–heme adducts or bis-histidine coordina-
tion in peptide-sandwiched porphyrins prevents the heme from
reacting with an exogenous ligand.
to the porphyrin macrocycle through the heme propionates [15].
This is particularly relevant in view of the choice of not inserting
His residues in the peptide chains of the newly designed catalyst.
In natural hemoproteins, histidines strongly coordinate the Fe(III)
ion, and for this reason, they were generally introduced in most
of the peptide models described so far in the literature. An issue
of concern in this contribution is the capacity of peptide-based
catalysts lacking His residues to mimic the activity of hemopro-
teins and protecting the active site from oxidative degradation,
but still preserving its functionality.
We report here on a new synthetic catalyst also based on the
peptide–heme–peptide motif, composed of a Fe(III)-mesoporphyrin
IX covalently linked to two amphiphilic helical decapeptides. The
new compound is characterized by a polar outer surface and a
hydrophobic inner cavity, which could host the reactants in a
restrained catalytic environment. The idea is to have a peptide
catalyst soluble in water, sufficiently protected against oxidative
degradation, and capable of chiral discrimination.
The molecular formula of the investigated compound, denoted
in the following as FemP(10N)2, is shown in Figure 1. It embodies
a Fe(III)-mesoporphyrin IX, the propionyl sites of which were co-
valently linked to the N-terminal Ala residues of two identical
decapeptides (10N). The presence of six α-aminoisobutyric acid
(Aib) residues constrains the two peptide chains to attain a helical
conformation, because of the steric hindrance exerted by the
gem-dimethyl substitution on the Aib Cα atom [13]. Three Glu res-
idues were also included (at positions 3, 5, and 9) in the peptide
chain for improving the solubility of the catalyst in aqueous
solutions, giving rise to the formation of an amphiphilic helix.
Mesoporphyrin IX was used instead of protoporphyrin IX, be-
cause the former, lacking the two ring-substituent vinyl groups,
is less sensitive to the attack of oxidants, thus improving the cat-
alyst resistance [12]. Furthermore, the two peptide chains were
each C-terminally derivatized as naphthyl esters to obtain struc-
tural information from fluorescence energy transfer (FRET) mea-
surements [14].
Materials and Methods
Synthesis
The synthesis of the side chain-protected decapeptide H-Ala-Aib-
Glu(OtBu)-Aib-Glu(OtBu)-(Aib)3-Glu(OtBu)-Aib-OCH2-CH2-Napht
(10Np) was performed by classical methods in solution via frag-
ment condensations following strategies previously elaborated
for Aib peptides [16]. At first, the Z-Aib-OH was esterified with
2-(2-naphthyl)ethanol by 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC·HCl) in the presence of
DMAP. N-terminal elongation of this ester to the decapeptide
derivative was performed in stepwise manner by intermediate
Nα-hydrogenolytic removal of the Z protecting group and acyla-
tion with the related Z-Glu(OtBu)-OH and Z-Aib-OH derivatives
via hydroxybenzotriazole(HOBt)/EDC·HCl and 1-hydroxy-7-
azabenzotriazole(HOAt)/EDC·HCl, respectively, and finally with
Z-Ala-OH via HOBt/EDC·HCl. In the final step, mesoporphyrin IX
and Fe(III)-mesoporphyrin IX were solubilized in CH2Cl2 and
reacted with H-Ala-Aib-Glu(OtBu)-Aib-Glu(OtBu)-Aib-Aib-Aib-
Glu(OtBu)-Aib-OCH2-CH2-Napht, respectively, which was obtained
by catalytic hydrogenation (Pd/C in CH3OH) of the corresponding
Z derivative, and N-methylmorpholine. The still protected
mesoporphyrin IX and Fe(III)-mesoporphyrin IX conjugates were
purified by flash chromatography on silica gel using CH2Cl2/
EtOH (9.2 : 0.8) and CH2Cl2/EtOH/AcOH (8.9 : 0.65 : 0.45) as eluent,
respectively. Final deprotection of the two conjugates was
achieved by exposure to TFA/CH2Cl2 (1 : 1) for 3 h. The desired
mesoporphyrin IX and Fe(III)-mesoporphyrin IX constructs were
obtained as well as characterized compounds after HPLC using
a semi-preparative reversed phase column (Supelco, Bellefonte,
Pennsylvania) and as eluent CH3CN/H2O/TFA (75 : 25 : 5) and
monitoring the absorption of the product at λ = 226 nm. Final
and intermediate products were characterized by melting point
determination, TLC, rotatory optical power, IR absorption, mass
spectrometry, and 1H-NMR. Details on the synthetic procedures
and characterization of intermediates and products are provided
in Supporting Information.
The Glu residues play a key role in mimicking the hemoprotein
activity for both structural and reactivity reasons. From the struc-
tural point of view, the Glu carboxyl groups could coordinate the
central iron atom, stabilizing a specific conformation. Further-
more, the protonation of the Glu side chains can be suitably var-
ied, modulating the local polarity of the active site environment
and, hence, its redox potential and reactivity. Recently, it was
shown that carboxyl groups can exert a distal effect on the active
site reactivity in peroxidases, opening electron transfer pathways
Methods
TLC was carried out on silica gel (60F254, Merck Italy, Vimodrone,
Italy) using five different eluents: chloroform/ethanol 9 : 1 (Rf1),
n-butanol/AcOH/H2O 3 : 1 : 1 (Rf2), toluene/ethanol 7 : 1 (Rf3),
AcOEt/petroleum ether 1 : 3 (Rf4), and AcOEt/petroleum ether 1 : 1
(Rf5). Flash chromatography was carried out using silica gel as sta-
tionary phase. HPLC purifications were carried out by a Hewlett-
Packard A7554 (Agilent Technologies, Santa Barbara, CA, USA) with
UV detection at λ = 226 nm using a semi-preparative reverse-phase
column (Supelco LC-18-DB, l = 25 cm, d = 10 mm, Bellefonte,
Pennsylvania). CD measurements were carried out by a JASCO
Figure 1. Molecular formula and acronym of the synthesized peptide–
heme–peptide compound. O-Napht denotes a 2-(1-naphthyl)ethyl ester
group by which the two decapeptides are C-terminally derivatized.
J. Pept. Sci. 2014; 20: 36–45 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

VENANZI, CIANFANELLI AND PALLESCHI
J600 spectropolarimeter (Jasco Europe, Cremella, Italy), by using
quartz cells (l = 0.1, 0.5, and 1 cm). CD spectra are reported as molar
ellipticity per residue (θ, degreecm2 dmol–1). Steady-state and
time-resolved fluorescence experiments were performed on the
same apparatus (CD900, Edinburgh Analytical Instruments, UK) by
using a Xe lamp (450 W) for spectral measurements (analog detec-
tion) or a pulsed arc lamp (30 kHz repetition rate) for time decays
measurements (single photon counting detection). The arc lamp
was filled with ultrapure hydrogen (pressure 0.4atm) for exciting
the sample in the UV (λ = 280 nm). Under these conditions, the full
width at half maximum of the excitation pulse was 1.1ns. Time
decays were analyzed through iterative reconvolution of a
multiexponential function by using standard software supplied by
Edinburgh Analytical Instruments (Edinburgh, UK). All solutions
were freshly prepared and fluxed for 20 min in ultrapure N2 to min-
imize oxygen quenching effects. Polarimetric measurements were
carried out on a Perkin-Elmer polarimeter (PE-241, Waltham, MA,
USA). Temperature (T = 25 °C) was controlled by a Haake D8
thermostat (Vreden, Germany). FAB- and ESI-mass spectra were
obtained with a Sciex API-365 mass spectrometer (Perkin-Elmer,
Waltham, MA, USA) equipped with a triple quadrupole ESI-MS.
Fourier transformed infrared (FTIR) spectra were obtained with a
PE 1720 FTIR spectrometer (Perkin-Elmer, Waltham, MA, USA) by
Results and Discussion
Spectroscopic Results
FTIR absorption
Helical peptides feature characteristic IR absorption in the
3420–3310 cmꢀ1 range, associated to the stretching of NH amide
groups, and between 1720 and 1660 cmꢀ1 (stretching of CO am-
ide groups) [19]. The onset of secondary structure, promoted by
formation of intramolecular hydrogen bonds, can be easily ob-
served by the progressive shift to lower energies of the NH and
CO stretching frequencies with increased length of the peptide
chain from two to ten residues during the synthesis of the
protected decapeptide Z-Ala-Aib-Glu(OtBu)-Aib-Glu(OtBu)-(Aib)
3-Glu(OtBu)-Aib-OCH2-CH2-Napht (10Np) (Figures S1 and S2 of
Supporting Information). The FTIR spectrum of FemP(10N)2
in CDCl3, reported in Figure 2, shows a very weak absorbance at
ν = 3435–3425 cmꢀ1, ascribable to free NH groups, and a predomi-
nant absorption band peaked at ν = 3325 cmꢀ1, assigned to
hydrogen-bonded NH’s [19]. The intramolecular character of the
hydrogen bonding interaction was confirmed by concentration-
dependent FTIR absorption measurements from 0.1 to 10 mM.
The ratio between the number of NH protons involved in hy-
drogen bond formation (nb) and the number of free NH’s (nf)
can be estimated from the areas of the two corresponding IR ab-
sorption bands and the associated molar extinction coefficients
εb and εf, that is,
1
using CaF2 cells (optical length = 0.01, 0.1, 1 cm). H-NMR experi-
ments were carried out with a Bruker AM400 NMR spectrometer
(Billerica, MA, USA). Chemical shifts were expressed as parts per
million with respect to tetramethylsilane. 2D-NMR experiments
were processed by the UXNMR code from Bruker. UV–vis absorp-
tion measurements were recorded on a thermostated JASCO
7850 (Jasco Europe, Cremella, Italy), using quartz cells (0.5 and
1 cm). Kinetic experiments were performed on the same apparatus
for nine different substrate concentrations in the range between
8.0 10ꢀ6 and 2.9 10ꢀ4 M. The enzyme concentration was kept
constant at 8.5 10ꢀ6 M, while the H2O2 oxidant concentration
was 1.5 10ꢀ4 M. Each kinetic experiment was repeated in triple
(pH = 7.5, T = 25 °C). Molecular mechanics calculations were carried
out by using homemade programs, exploiting the MM4 force field
[17,18]. The following procedure was adopted: (i) The two
peptide backbones were initially considered in an ordered
helical conformation, exploring both 310- and α-helical struc-
tures. The conformers arising from internal rotation around the
torsional angles of the side chains linking the porphyrin and
naphthyl chromophores were built up by making use of the
rotational isomeric state model. (ii) The conformational energy
was then optimized by varying all torsional angles associated
to both the peptide backbones and the side chains; (iii) the Glu
side chain carboxyl groups were considered in both neutral
and anionic (carboxylate) forms.
nb Ab εf
¼
ꢁ
Af εb
(1)
nf
The εb/εf ratio was determined by us through extensive IR mea-
surements on several conformationally constrained oligopeptides
attaining a 310-helix conformation [19]. Deconvoluting the IR spec-
trum reported in Figure 2, we obtained nb/nf = 2.7, a value interme-
diate between those expected for a pure α-helix (2.3) and a 310-
helix structure (4) [20].
1H-NMR
By TOCSY and ROESY experiments in CDCl3, we were able to
identify the amide protons of Ala1 and Aib2 and of two Glu
Assumption (i) strongly reduced the computational effort and
is based on the CD and NMR experimental evidences on the sec-
ondary structure attained by the two decapeptide chains. The po-
tential energy comprised electrostatic, nonbonding, hydrogen
bond, and torsional interactions and was calculated by using
standard bond angles and lengths for both the backbone and
the side chains, as implemented in the MM4 force field [18].
Coulombic interactions were assessed by assigning partial atomic
charges to all atoms. Energy minimization refinement was
performed by relaxing the fixed geometry for all the internal de-
grees of freedom and introducing stretching and bending terms.
Solvent effects were taken into account by assuming a distance-
dependent dielectric constant [ε(Rik)], where Rik is the distance
between the i and k atoms.
Figure 2. FTIR absorption spectra of FemP(10N)2 in CDCl3 in the NH ab-
sorption region [1 mM (dashed); 0.1 mM (continuous)].
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A PEPTIDE CATALYST MIMICKING HEMOPROTEINS
and one Aib residues of 10Np. It was not possible to identify the
other amide protons because of the extensive overlap between
the naphthyl and Z group’s resonances. Titration experiments
with DMSO-d6 allowed us to have a clear indication on the con-
formational properties of this decapeptide derivative, because
only the resonance frequencies of protons not involved in hydro-
gen bonding are affected (downshifted) by DMSO, a strong hy-
drogen bond acceptor. In Figure 3, we reported the effects of
the DMSO titration on the NMR signals of the 10Np protons
identified by 2D-NMR experiments. As can be seen, only the
Ala1 and Aib2 protons were seriously perturbed by adding DMSO,
suggesting that the protected decapeptide in CDCl3 predominantly
populates a 310-helix structure characterized by an i ← i + 3 hydro-
gen bond network.
CD
CD measurements in CH3OH confirmed that 10Np predominantly
attains a helically ordered conformation also in this solvent,
showing well-defined dichroic negative maxima at λ = 207
(π→π* amide transition) and 226 nm (n→π* amide transition).
Very similar CD spectra were obtained for mP(10N)2 and FemP
(10N)2 in the same solvent (Figure 4), the ratio between the CD
molar ellipticities at λ = 208 and 227 nm, that is,
Figure 4. CD spectra of 10Np(dashed), mP(10N)2 (continuous), and
FemP(10N)2 (dotted) in CH3OH. Peptide concentration is 0.1mM in all cases.
achiral residues in the peptide chain (CD ellipticities are normal-
ized per residue molar concentration) [22].
θð227Þ
Interestingly, the CD spectra of FemP(10N)2 in H2O/CH3OH
(9 : 1, v/v) solutions at pH 4 and pH 7.5, reported in Figure 5, re-
veal a strong pH dependence of the CD curves, the R ratio being
0.90 at pH 4 and 0.48 at pH 7.5, respectively. These results sug-
gest that an α- → 310-helix transition on going from pH 4 to pH
7.5 takes place. This conformational transition could be promoted
by either the repulsive interaction between the deprotonated Glu
carboxylates at pH 7.5 [pKa(Glu) = 4.1] or the coordination of the
Fe(III) ion to the Glu carboxyl groups of the two peptide chains.
However, the CD spectrum of the free-base compound mP
(10N)2 in H2O/CH3OH (9 : 1, v/v) at pH 7.5 shows an R value equal
to 0.81, suggesting that the Fe(III)–COOꢀ interaction is mainly re-
sponsible for the conformational transition observed in the case
of FemP(10N)2 (Supporting Information).
R ¼
(2)
θð208Þ
being 0.71, 0.75, and 0.79 for 10Np, mP(10N)2 and FemP(10N)2,
respectively. R values in the range 0.15–0.40 are considered diag-
nostic of the 310-helical conformation, while values close to 1
were found to be typical of the α-helix [21]. These results, in
agreement with the FTIR data, indicate the predominant popula-
tion of helical structures in both apolar (CDCl3) and polar (CH3OH)
solvents, probably in equilibrium between 310- and α-helical con-
formations. The close similarity between the mP(10N)2 and FemP
(10N)2 CD curves suggests that in CH3OH, only a weak interaction
between the Fe(III) central ion and the carboxylic side chain of
the Glu residues is established. The relatively low intensity of
the obtained CD spectra can be ascribed to the presence of six
Figure 3. DMSO titration in CDCl3 of the NMR signals of the protected
decapeptide 10Np protons identified by 2D-NMR experiments. (◆)Ala1;
(▲)Aib2; (▼) Aib; (●) Glu; (■)Glu.
Figure 5. CD spectra of FemP(10N)2 in CH3OH (continuous) and in H2O/
CH3OH (9 : 1, v/v) at pH 4 (dashed) and pH 7.5 (dotted). Peptide concen-
tration is 0.1 mM in all cases.
J. Pept. Sci. 2014; 20: 36–45 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

VENANZI, CIANFANELLI AND PALLESCHI
UV–vis absorption
UV–vis absorption spectroscopy may provide relevant informa-
tion on the structural features of the hemoprotein active site, be-
cause the characteristic absorption spectrum of iron porphyrins
depends on the symmetry and strength of metal coordination,
spin, and oxidation state [23]. The UV–vis absorption features in
CH3OH of both FemP(10N)2 and mP(10N)2 are reported in Table 1.
As it is well known, the insertion of a metal ion strongly perturbs
the absorption features of free-base porphyrins in both the Soret
(λ = 400 nm, S0 → S2) and the Q bands (λ = 450–650 nm, S0 → S1)
regions. The mP(10N)2 absorption spectrum shows the two UV ab-
sorption bands typical of the naphthyl chromophores (λ = 222 and
280 nm), the very intense Soret transition peaked at λ = 395nm,
and the four Q bands system in the visible region, characteristic
of free-base porphyrins. The insertion of the metal ion shifts the
Soret transition to shorter wavelengths (λmax = 392 nm), sensibly
decreases its intensity, and gives rise to a shoulder at lower wave-
lengths (λ = 345 nm, sh), typical of high-spin Fe(III). Major perturba-
tions can be observed in the visible region where the Q bands
collapse to a two-band system, because of the D4h symmetry of
Fe(III) coordination to the porphyrin N-ligands. Interestingly, in
H2O/CH3OH (9: 1)v/v at pH 7.5, the shoulder at λ = 345 nm markedly
increases, and the Q bands sensibly shift to lower energies,
suggesting a stronger coordination of Fe(III) to the Glu3 carboxylate
side chain groups. It should be noted, however, that the spectral
features of the metalated porphyrin indicate that, in this environ-
ment, the iron center is in a high-spin state, suggesting that either
a single carboxylic group is coordinating the metal ion or a weak
bis-coordination of the two Glu3 carboxyl groups takes place at
relatively large distances.
Figure 6. Fluorescence spectra of 10Np (a, dashed), mP(10N)2 (b, continu-
ous), and FemP(10N)2 (c, dotted) in CH3OH. The emission bands of mP(10N)2
and FemP(10N)2 were magnified by a factor 4 for clarity (λexc = 280 nm).
Energy transfer efficiencies can be also measured by evaluat-
ing the enhanced fluorescence emission of the acceptor moiety
caused by energy transfer from the donor group [25]. In the pres-
ent case, however, this can be carried out for the free-base por-
phyrin compound only, because of total quenching of the
porphyrin emission caused by Fe(III) in FemP(10N)2. The energy
transfer efficiency, calculated from the enhanced fluorescence
intensity of mP(10N)2 (EP = 0.85), indicates that energy transfer
is the only quenching mechanism affecting the relaxation of
the excited naphthyl chromophores in this compound.
In favorable conditions, time-resolved fluorescence experi-
ments may give detailed information on the conformations pop-
ulated by the molecules and their different contributions to the
overall quenching process. This is because FRET quenching effi-
ciency depends on the distance and orientation between the
D–A pairs and, hence, it is the characteristic of the specific confor-
mation attained by a macromolecule [26].
Fluorescence measurements
The presence of naphthalene and porphyrin chromophores, a
well-known energy transfer donor–acceptor (D–A) pair, allowed
us to apply Förster resonance energy transfer (FRET) to obtain
structural information on the conformations attained in solution
by the compounds investigated [14,24].
In Figure 6, we reported the fluorescence spectra of 10Np, mP
(10N)2, and FemP(10N)2 in CH3OH obtained by excitation at
λ = 280 nm and dominated by the emission of the naphthyl
fluorophore. From the figure, the quenching of the naphthalene
emission caused by N* → mP or N* → FemP transfer of excitation
energy can be readily observed. The FRET quenching efficiency
was obtained by measuring the naphthyl quantum yield in
FemP(10N)2 and mP(10N)2 with respect to the protected
decapeptide assumed as reference compound, that is,
Fluorescence time decays are usually described by a sum of ex-
ponential time components, that is,
ꢀ
ꢁ
n
t
IðtÞ ¼ ∑ αi exp ꢀ
(4)
τi
i¼1
ϕN½FemPð10NÞ2 or mPð10NÞ2ꢂ
EN ¼ 1 ꢀ
(3)
where αi is the weight associated to the lifetime τi. Provided that
the different conformers do not interconvert in the nanosecond
time scale, so that dynamical averaging of the instantaneous rel-
ative positions of the donor and acceptor pair cannot take place,
each decay time component can be associated to a specific
ϕNð10NpÞ
From steady-state fluorescence measurements, we obtained
EN = 0.86 and 0.91 for mP(10N)2 and FemP(10N)2, respectively.
Table 1. UV–vis absorption features of FemP(10N)2 and mP(10N)2 in CH3OH
Sample
λmax (nm) (ε, Mꢀ1 cmꢀ1
)
λ
max (nm) (ε, Mꢀ1 cmꢀ1
)
λmax (nm) (ε, Mꢀ1 cmꢀ1
)
λ
max (nm) (ε, Mꢀ1 cmꢀ1
)
mP(10N)2
Q bands
222 (70 000)
499 (17 800)
222 (70 000)
483 (13 000)
280 (6100)
530 (13 400)
280 (6100)
590 (10 000)
395 (125 000)
568 (9600)
345 (sh)
618 (6400)
FemP(10N)2
Q bands
392 (110 000)
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A PEPTIDE CATALYST MIMICKING HEMOPROTEINS
conformer. Furthermore, in the absence of ground-state interac-
tions, the experimental pre-exponential factors αi can be directly
associated to the Boltzmann-weighted populations of each con-
former [26].
The emission time decay of the naphthyl groups in FemP(10N)
2 and mP(10N)2 is described by a three-exponential function, the
decay parameters of which are reported in Table 2. From these
values, it is possible to define an average time decay, that is,
populated conformers (98–99%, from the sum of α1 and α2 in
Table 2) are characterized by a very high quenching efficiency.
Although these results are mainly determined by the large R0
values of the naphthyl–FemP pair, they strongly suggest that
FemP(10N)2 attains compact 3D structures in both the CH3OH
and H2O/CH3OH environments. In particular, analyzing the
short-time components of the naphthyl time decay in the differ-
ent compounds, it appears that the FemP(10N)2 time decay in
CH3OH is almost similar to that measured for mP(10N)2 in the
same solvent, in nice agreement with CD results. On the contrary,
the naphthyl time decay of FemP(10N)2 in H2O/CH3OH 9 : 1 (v/v)
at pH = 7.5 is accounted for by definitely shorter lifetimes
(Table 2), suggesting that the naphthyl–FemP pair is located at
short distances. Conformational energy calculations were there-
fore carried out to substantiate this finding.
∑iαiτi
∑iαi
< τ >¼
(5)
also reported in Table 2. The experimental quenching efficiencies
associated to the i-th time component of the mP(10N)2 and FemP
(10N)2 time decays can be obtained from the simple equation:
τi
τ0
Ei ¼ 1 ꢀ
(6)
Conformational Analysis of FemP(10N)2
where τ0 is the lifetime of the naphthyl fluorophore in absence
of the porphyrin quencher, measured from the 10Np time decay
in CH3OH (τ0 = 52.5 ns) and in H2O/CH3OH (9 : 1)v/v at pH 7.5
(τ0 = 36.2 ns), respectively. From the average time decay < τ >,
an average quenching efficiency, which can be directly com-
pared with the static quenching efficiency [Eqn 3], can be intro-
duced. The quenching efficiencies obtained for mP(10N)2 and
FemP(10N)2 in CH3OH and H2O/CH3OH (9 : 1)v/v are reported in
Table 3.
The conformational properties of FemP(10N)2 were investigated
by molecular mechanics calculations, exploring both 310- and
α-helical structures and considering the Glu side chain carboxyl
groups in both neutral and anionic (carboxylate) forms.
The most stable structures of FemP(10N)2 in H2O/CH3OH (9 : 1)
at pH 4 and pH 7.5 are reported in Figure 7A and 7B, respec-
v/v
tively. In the minimum energy structure at pH 4, the closest
Fe–O (COOH) distance is 3.58 Å, suggesting that only a very weak
interaction between the Fe(III) ion and the two Glu3 carboxylic
oxygen atoms takes place, in good agreement with spectroscopic
results. As a consequence, the helical peptide chains are dynam-
ically free to attain the most stable α-helix conformation. On the
other hand, at pH 7.5, electrostatic interactions favor the apical
coordination of the two Glu3 carboxylate groups to the central
metal ion causing a severe distortion of the peptide backbone
(Fe–O minimal distance = 2.59 Å). Interestingly, the Fe(III)–(Glu3)
carboxylate interaction constrains the N-terminal peptide seg-
ment to attain a β turn-like structure that closely reproduces
the onset of a 310-helix conformation. This finding parallels the
CD results, suggesting an α-helix to a partial 310-helix transition
on going from pH 4 to pH 7.5. It should be noted, however, that
the Fe(III)–O(Glu3) distances are relatively large with respect to
the distances (~2 Å) attained by the strongly coordinated iron
In structural terms, the energy transfer efficiency can be
accounted for by the Förster dipole–dipole model:
(
)
ꢀ1
ꢀ
ꢁ
6
2
3k2i
Ri
Ei ¼ 1 þ
ꢁ
(7)
R0
where Ri is the center-to-center distance of the D–A pair in the
i-th conformer and ki dimensionless geometric factor
a
determined by the D–A relative orientation in space [27]. R0 is a
pure spectroscopic parameter characteristic of the D–A pair,
representing the distance at which 50% transfer of excitation en-
ergy occurs. For the naphthyl–FemP pair, R0 = 38.0 Å in CH3OH
and 32.9 Å in H2O/CH3OH (9 : 1)v/v. The data reported in Table 3
reveal that, for both the compounds investigated, almost all the
Table 2. Time decay parameters for mP(10N)2 and FemP(10N)2 in CH3OH and 9 : 1 (v/v) H2O/CH3OH solutiona
Sample
α1
τ1/ns
α2
τ2/ns
α3
τ3/ns
< τ >/ns
mP(10N)2
0.58
0.65
0.74
1.6
1.6
0.9
0.40
0.34
0.24
4.7
4.2
3.8
0.02
0.01
0.02
35.2
34.6
36.2
3.5
2.8
2.3
FemP(10N)2 (CH3OH)
FemP(10N)2 H2O/CH3OH 9 : 1 (v/v) pH = 7.5
aT = 25 °C, λexc = 280 nm, and λem = 340 nm.
Table 3. Experimental quenching efficiencies of mP(10N)2 and FemP(10N)2 in CH3OH and H2O/ CH3O H (9 : 1)v/v
a
a
a
a
b
Sample
E1
E2
E3
< E >exp
< E >th
mP(10N)2
0.97
0.97
0.97
0.90
0.92
0.90
0.67
0.66
0.94
0.95
0.93
0.94
0.95
0.96
FemP(10N)2 (CH3OH)
FemP(10N)2 H2O/CH3OH (9 : 1)v/v, pH = 7.5
≈0
aFrom the data reported in Table 2.
bFrom molecular mechanics calculations.
J. Pept. Sci. 2014; 20: 36–45 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

VENANZI, CIANFANELLI AND PALLESCHI
Figure 7. Most populated conformer of FemP(10N)2 in H2O/CH3OH (9 : 1)v/v at pH = 4 (A) and at pH 7.5 (B) from molecular mechanics calculations. C
atoms: white circles; N: blue circles; O: red circles; Fe: green circle. Hydrogen atoms were omitted for clarity. The helical arrangement of the peptide back-
bone was schematized by a ribbon representation.
center in Fe–His or Fe–imidazole adducts [2]. This finding is in
good agreement with the UV–vis absorption spectrum of FemP
(10N)2, typical of a high-spin Fe(III) system. As clearly shown in
Figure 7B, the Fe(III)–O(Glu3) interaction constrains the peptide
segment in the proximity of the porphyrin active site, leaving
however to the oxidant the possibility to approach the porphyrin
ring and compete with the carboxylate group for Fe(III) coordina-
tion. As expected, the other Glu residues are positioned at the
outer surface of the peptide helices, completely exposed to the
solvent molecules.
center-to-center distance increases to ~30 Å. In this case, the cal-
culated FRET quenching efficiencies take values ranging from 0.6
to 0.8, definitely smaller than the experimental data. Taken
together, the results of conformational analysis and FRET experi-
ments indicate that FemP(10N)2 at pH 7.5 attains a rather com-
pact 3D structure, the access to the active site being somewhat
restricted by the folding of the peptide chains. The Fe(III) porphy-
rin moiety appears to be more protected against a direct attack
of oxidants and the peptide chains more rigidly held in the prox-
imity of the active site. For this reason, all kinetic experiments
were carried out at pH 7.5.
From the calculated structures, an average theoretical
quenching efficiency can be obtained by summing over the
quenching efficiencies associated to each of the significantly
populated conformers:
Kinetic and Conformational Studies on the Catalytic Activity
of FemP(N10)2
2
< E>th ¼ ∑jwj ∑ Ei
(8)
Kinetic results
i¼1
The catalytic activity of FemP(10N)2 has been investigated in the
oxidation of L- and D-Dopa by H2O2 in a H2O/CH3OH (9 : 1)v/v solu-
tion at pH = 7.5 (T = 25 °C). The iron-catalyzed oxidation of L- and
D-Dopa is a well-known reaction, and it is here addressed as a ref-
erence reaction to assess the catalytic properties of FemP(10N)2
(Scheme 1) [28].
Detailed mechanistic studies on the peroxidase-catalyzed oxida-
tion of Dopa revealed the formation of two active intermediates,
often referred as Cpd I (+E-FeIV = O) and Cpd II (+EH-FeIV = O)
[29,30]. A third low-reactive intermediate was found to be respon-
sible for inactivation of peroxidase [31]. Recent literature also
reported on the formation of phenoxy radicals during the oxidation
of L-Dopa in the presence of horseradish peroxidase followed by
radical dismutation [32]. Despite the complexity of the mechanism,
a Michaelis–Menten approach has been generally found most
appropriate to describe enzymatic oxidation of Dopa substrates
[33,34]. The formation of dopaquinone, an intermediate product
in the Dopa oxidation pathway, can be easily monitored by UV–vis
absorption spectroscopy [35,36]. Final oxidation products of the re-
action are dopachrome and, after polymerization, melanine [37].
where wj is the Boltzmann weight associated to the j-th con-
former, as given by
ꢂ
ꢃ
exp ꢀUj=RT
j
ꢂ
ꢃ
wj ¼
(9)
∑
exp ꢀ Uj=RT
and Ei is the efficiency associated to each of the two naphthyl–
FemP FRET pairs in the j-th conformer, characterized by a potential
energy Uj. The calculated average quenching efficiency < E >th can
be directly compared with the experimental average quenching
efficiency < E >exp (Table 3). The fine agreement between experi-
mental and calculated parameters makes reasonable to consider
the computed structures a good representation of the conforma-
tions actually populating the solution.
It should be noted that the large R0 value of the naphthyl–
FemP D–A pair makes the experimental quenching efficiencies
not very sensitive to the naphthyl–FemP distance. However,
when the Glu3–Fe interaction is relaxed and the peptides forced
to dip straight into the solvent in extended conformation, so that
the catalyst attains a totally ‘open’ structure, the naphthyl–FemP
Scheme 1. Oxidation of Dopa by hydrogen peroxide in the presence of FemP(10N)2 catalyst.
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A PEPTIDE CATALYST MIMICKING HEMOPROTEINS
Figure 8. Absorption spectra of the L-Dopa/FemP(10N)2 solution at in-
creasing time intervals: (a) 3 min, (b) 9 min, (c) 20 min, (d) 28 min, (e)
40 min, and (f) 50 min from the addition of H2O2 (t = 0). The reported spec-
tra were subtracted by the spectrum at t = 0.
Figure 9. Double reciprocal plot of initial velocity versus concentration
for the oxidation reaction of L- (○) and D-Dopa (●) by H2O2 in the presence
of the FemP(10N)2 catalyst.
The time evolution of the absorption spectrum of an L-Dopa
solution when reacted with H2O2 at micromolar concentration of
FemP(10N)2 is reported in Figure 8 (actually, the reported spectra
were subtracted by the spectrum at t = 0). The formation of
dopaquinone can be clearly observed at λmax = 304 and 475 nm.
Most importantly, the Soret absorption at λmax = 393nm did not
show any perturbation, indicating that the catalyst was not
degraded in the course of the reaction. This result was confirmed
by studying the stability of FemP(10N)2 in the presence of in-
creasing concentrations of H2O2. For a FemP(10N)2 : H2O2 1 : 10
solution and in absence of the substrate molecule, the observed
catalyst degradation after 30 min was only 9%. This finding
indicates that the peptide environment shielded effectively the
FemP active site from unwanted side reactions and degradation
processes, still allowing for the catalyst–oxidant interaction
leading to the formation of the Fe-oxo reactive intermediates.
Control measurements carried out on a Dopa/H2O2 solution in
the absence of the catalyst did not reveal the formation of
dopaquinone in detectable amount.
The stereoselective properties of FemP(10N)2 were investi-
gated by comparing the oxidation kinetics of L- and D-Dopa by
H2O2, following the time dependence of the dopaquinone
absorption at λ = 310 nm. The kinetic curves showed a typical sat-
uration trend, suggesting that a simple Michaelis–Menten treat-
ment can be successfully applied.
The double reciprocal plot of the initial velocity versus L- or
D-Dopa substrate concentration at a fixed micromolar catalyst
concentration is shown in Figure 9. The kinetic parameters of
the reaction, that is, the catalytic (kcat) and Michaelis–Menten
(KM) constants, obtained by the reported experimental data, are
listed in Table 4. Very similar catalytic rate constants were
obtained for the oxidation of the L- and D-Dopa enantiomers,
suggesting poor stereoselective properties of the FemP(10N)2
catalyst. However, the Michaelis–Menten constants differ by
more than 30%, suggesting a significant major affinity of L-Dopa
for the catalyst. As a result, the ratio between the catalytic and
Michaelis–Menten constants, that is, kcat/KM, is 5.31 105 Mꢀ1 sꢀ1
and 3.35 105 Mꢀ1 sꢀ1 for the L- and D-Dopa, respectively, denoting
weak but not negligible stereoselective properties [38]:
Table 4. Kinetic parameters for the oxidation of L- and D-Dopa by
H2O2 catalyzed by FemP(10N)2
kcat/sꢀ1
10ꢀ5·KM/M
105 kcat/KM
6.8 0.2
6.2 0.2
1.28 0.05
1.85 0.05
5.31
3.35
Conformational analysis of the (L,D)Dopa–catalyst adducts
The formation of L- and D-Dopa adducts with the new FemP(10N)
catalyst was investigated by molecular mechanics calculations.
2
In particular, the approach of the substrate to the active site
was analyzed along 100 different trajectories, starting from initial
positions equally distributed on a spherical surface having a 15 Å
radius and centered on the Fe(III) ion, and searching for the min-
imum energy structure of the L- and D-Dopa/FemP(10N)2 adducts.
The obtained minimum energy structures in H2O/CH3OH (9 : 1)v/v
at pH = 7.5 are reported in Figure 10A and 10B for the L- and
D-Dopa adducts with FemP(10N)2, respectively. It was found that
both L- and D-Dopa can reach the active site, approaching the
periphery of the porphyrin ring through its catecholic group.
UV–vis measurements confirmed the weak character of the
catalyst–substrate interaction, the Soret transition remaining
unperturbed by the formation of the L- or D-Dopa/FemP(10N)2
adducts. By taking into account the most populated computed
structures, the energetic binding of the L- and D-Dopa with
FemP(10N)2 results from a complex interplay of electrostatic
and weak interactions that ultimately leads to a modest differ-
ence in the stability of the two adducts, slightly favoring the
L-Dopa/FemP(10N)2 encounter complex [ΔU(L-Dopa/FemP(10N)
2) – ΔU(D-Dopa/FemP(10N)2) = ꢀ0.9 kcal molꢀ1]. This result quali-
tatively confirms the experimental finding of a lower Michaelis–
Menten constant for the L-Dopa/FemP(10N)2 adduct.
From the structures reported in Figure 10, it can be noted that
(i) the penetration of L-Dopa into the catalyst sphere of action
causes the partial displacement of the Glu3 residue involved in
the coordination of Fe(III) and (ii) for both L- and D-Dopa, the
approach to the FemP active site takes place through its
catecholic oxygen, relatively far from the chiral groups
kcatðL-DopaÞ kMðD-DopaÞ
ꢃ
¼ 1:6 0:3
kcatðD-DopaÞ kMðL-DopaÞ
J. Pept. Sci. 2014; 20: 36–45 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

VENANZI, CIANFANELLI AND PALLESCHI
Figure 10. Minimum energy structure of the L-Dopa (A) and D-Dopa (B) adduct with FemP(10N)2 in H2O/CH3OH (9 : 1)v/v at pH 7.5 from molecular me-
chanics calculations. C atoms: white circles; N: blue circles; O: red circles; Fe: green circle. Hydrogen atoms were omitted for clarity. The helical arrange-
ment of the peptide backbone was schematized by a ribbon representation.
discriminating between the L and D enantiomers. This finding
clearly explains for the observed moderate stereoselectivity of
the FemP(10N)2 catalyst.
This contribution emphasizes the importance to control the
conformational properties of biomimetic synthetic catalysts for
determining the efficiency and the stereochemical preference
of the catalyzed reaction.
Acknowledgements
Conclusions
The kind hospitality of the Laboratory of Peptide Synthesis headed
by Prof. Claudio Toniolo (University of Padova, Italy) is gratefully
acknowledged. We thank Prof. Basilio Pispisa (University of Rome
Tor Vergata) for stimulating discussion and critical reading of the
paper. This research was funded by the Italian Minister for Univer-
sity and Research (MIUR), PRIN 2010–2011 No. 2010FM738P.
The structural features of a de novo designed catalyst based on a
Fe(III)-mesoporphyrin IX group covalently linked to two helical
decapeptides were investigated by spectroscopic techniques
(CD, FTIR, NMR, UV–vis, steady-state, and time-resolved fluores-
cence) and theoretical conformational analysis. The most stable
structure in CH3OH was obtained when the peptide chains attain
an α-helical conformation, experiencing only a weak interaction
with the heme group. On the other hand, in H2O/CH3OH (9: 1)v/v
at pH 7.5, the synthetic mini-enzyme attains a more compact 3D
structure, characterized by the coordination of one or, possibly,
two Glu carboxylate groups to the Fe(III) central ion. This coordina-
tion is promoted by an α- → 310-helix conformational transition of
the two peptide chains, giving rise to a sterically hindered chiral
environment, protecting the active site. This effect is particularly
relevant in the absence of Hys residues in the iron-porphyrin
coordination shell and represents the most important element of
novelty of this work.
The catalytic properties of FemP(10N)2 were investigated with
respect to the oxidation of L- and D-Dopa by hydrogen peroxide.
The synthesized catalyst effectively speeds up the reaction, show-
ing optimal resistance against degradation under the experimen-
tal conditions used. Kinetic studies revealed that the catalyzed
reaction can be satisfactorily described by a simple Michaelis–
Menten mechanism. Although the stereoselective properties of
the new catalyst were found to be of moderate relevance when
considered in absolute terms, they are encouraging in compari-
son with the enantiomeric discrimination capacity of low molec-
ular weight analogs. These effects principally originate from the
rigid helical structure and the conformationally constrained char-
acter of the two decapeptide chains, determined by the high
content of Cα-tetrasubstituted residues (Aib). Molecular mechan-
ics calculations showed that the approach of both L- and D-Dopa
to the FemP active site occurs through the catecholic oxygen of
Dopa, which limits the influence of the chiral region of the
substrate on the reaction.
References
1 Metalloporphyrins in Catalytic Oxidations, Sheldon RA (ed.). Marcel
Dekker: New York, 1994.
2 Loew GH, Harris DL. Role of the heme active site and protein environ-
ment in structure, spectra, and function of the cytochrome P450s.
Chem. Rev. 2000; 100: 407–419; Meunier B, de Visser SP, Shaik S.
Mechanism of oxidation reactions catalyzed by cytochrome P450
enzymes. Chem. Rev. 2004; 104: 3947–3980.
3 Woggon W-D Metalloporphyrins as active site analogues – lessons from
enzyme and enzyme models. Acc. Chem. Res. 2005; 38: 127–136; Shaik S,
Kumar D, de Visser AP, Altun A, Thiel W. Theoretical perspective on the
structure and mechanism of cytochrome P450 enzymes. Chem. Rev.
2005, 105: 2279–2328.
4 Lombardi A, Nastri F, Pavone V. Peptide-based heme-protein models.
Chem. Rev. 2001; 101: 3165–3189; Reedy CJ, Gibney BR. Heme-protein
assemblies. Chem. Rev. 2004; 104: 617–649; Nicolis S, Casella L, Roncone
R, Dallacosta C, Monzani E. Heme-peptide complexes as peroxidase
models. C.R. Chimie 2007; 10: 380–391.
5 Low DW, Yang G, Winkler JR, Gray JB. Modification of heme peptides
by reverse proteolysis: spectroscopy of microperoxidases-10 with
C-terminal histidine, tyrosine and methionine residues. J. Am. Chem.
Soc. 1997; 119: 4094–4095.
6 Casella L, Monzani E, Fantucci P, Gullotti M, De Gioia L, Strini A,
Chillemi F. Axial imidazole distortion effects on the catalytic and binding
properties of chelated deuterohemin complexes. Inorg. Chem. 1996; 35:
439–444; Monzani E, Linati L, Casella L, De Gioia L, Favretto M, Gullotti M,
Chillemi F. Synthesis, characterization and stereoselective catalytic
oxidations of chelated deuterohaemin-glycyl-L-histidine complexes.
Inorg. Chim. Acta 1998; 273: 339–345; Franceschi F, Gullotti M, Monzani
E, Casella L, Papaefthymiou V. Cytochrome C oxidase models. A novel
dinuclear iron-copper complex derived from a covalently modified
deuteroporphyrin-L-histidine-bis(benzimidazole) ligand. Chem. Commun.
1996; 1645–1646.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 36–45

A PEPTIDE CATALYST MIMICKING HEMOPROTEINS
7 Choma CT, Lear JD, Nelson MJ, Dutton PL, Robertson DE, DeGrado WF.
Design of a heme-binding four-helix bundle. J. Am. Chem. Soc. 1994;
116: 856–865.
8 Broo KS, Nilsson H, Nilsson J, Baltzer L. Substrate recognition and sat-
uration kinetics in de novo designed histidine-based four-helix bundle
catalysts. J. Am. Chem. Soc. 1998; 120: 10287–10295.
9 Aili D, Enander K, Baltzer L, Liedberg B. Synthetic de novo designed
polypeptides for control of nanoparticle assembly and biosensing.
Biochem. Soc. Trans. 2007; 35: 532–534.
10 Ye S, Discher BM, Strzalka J, Xu T, Wu SP, Noy D, Kuzmenko I, Gog T,
Therien MJ, Dutton PL, Blasie JK. Amphiphilic four-helix bundle pep-
tides design for light-induced electron transfer across a soft interface.
Nano Lett. 2005; 5: 1658–1667.
11 Nastri F, Lombardi A, Morelli G, Maglio O, D’Auria G, Pedone C, Pavone
V. Hemoprotein models based on a covalent helix-heme-helix sand-
wich: 1. Design, synthesis and characterization. Chem. Eur. J. 1997; 3:
340–349; D’Auria G, Maglio O, Nastri F, Lombardi A, Mazzeo M, Morelli
G, Paolillo L, Pedone C, Pavone V. Hemoprotein models based on a
covalent helix-heme-helix sandwich: 2. Structural characterization of
Co(III) mimochrome I δ and λ isomers. Chem. Eur. J. 1997; 3: 350–362;
Lombardi A, Nastri F, Marasco D, Maglio O, De Sanctis G, Sinibaldi F,
Santucci R, Coletta M, Pavone V. Design of a new mimochrome with
unique topology. Chem. Eur. J. 2003; 9: 5643–5654.
12 Benson DR, Hart BR, Zhu X, Dougthy MB. Design, synthesis and circu-
lar dichroism investigation of a peptide sandwiched mesoheme.
J. Am. Chem. Soc. 1995; 117: 8502–8510; Arnold PA, Benson DR, Brink
DJ, Hendrich MP, Jas GS, Kennedy ML, Petasis DT, Wang M. Helix in-
duction and springboard strain in peptide-sandwiched mesohemes.
Inorg. Chem. 1997; 36: 5306–5315; Wang M, Kennedy ML, Hart BR,
Benson DR. The bis-p-nitrophenyl ester of iron(III) mesoporphyrin II
acetate: preparation and incorporation into a model protein. Chem.
Commun. 1997; 883–884; Williamson DA, Benson DR. Remarkable
helix stabilization via edge-to-face tryptophan-porphyrin interaction in
a peptide-sandwiched mesoheme. Chem. Commun. 1998; 961–962; Liu
D, Williamson DA, Kennedy ML, Williams TD, Morton MM, Benson DR.
Aromatic side chain-porphyrin interactions in designed hemoproteins.
J. Am. Chem. Soc. 1999; 121: 11798–11812.
20 Polese A, Formaggio F, Crisma M, Valle G, Toniolo C, Bonora GM,
Broxterman QB, Kamphuis J. Peptide helices as rigid molecular rulers:
a conformational study of isotactic homopeptides from α-methyl-α-
isopropylglycine, [L-(αMe)Val]n. Chem. Eur. J. 1996; 2: 1104–1111.
21 Toniolo C, Polese A, Formaggio F, Crisma M, Kamphuis J. Circular
dichroism spectrum of a peptide 310-helix. J. Am. Chem. Soc. 1996;
118: 2744–2745.
22 Toniolo C, Formaggio F, Tognon S, Broxterman QB, Kaptein B, Huang
R, Setnicka V, Keiderling TA, McColl IH, Hecht L, Barron LD. The com-
plete chirospectroscopic signature of the peptide 310-helix in aqueous
solution. Biopolymers 2004; 75: 32–45.
23 Eaton WA, Hofrichter J. Polarized absorption and linear dichroism
spectroscopy of hemoglobin. Methods Enzymol. 1981; 76: 175–261.
24 Pispisa B, Palleschi A, Venanzi M, Zanotti G. Conformational statistics and
energetic analysis of sequential peptides undergoing intramolecular
transfer of excitation energy. J. Phys. Chem. 1996; 100: 6835–6844.
25 Toniolo C, Formaggio F, Crisma M, Mazaylerat J-P, Wakselman M, George
C, Deschamps JR, Flippen-Anderson JL, Pispisa B, Venanzi M, Palleschi A.
First peptide-based system of rigid donor-rigid interchromophore
spacer-rigid acceptor: a structural and photophysical study. Chem. Eur.
J. 1999; 5: 2254–2264.
26 Venanzi M, Gatto E, Bocchinfuso G, Palleschi A, Stella L, Baldini C,
Formaggio F, Toniolo C. Peptide folding dynamics: a time-resolved
study from the nanosecond to the microsecond time regime.
J. Phys. Chem. B 2006; 110: 22834–22841.
27 Pispisa B, Palleschi A, Stella L, Venanzi M, Toniolo C. A nitroxide deriv-
ative as a probe for conformational studies of short linear peptides in
solution. A spectroscopic and molecular mechanics investigation.
J. Phys. Chem. B 1998; 102: 7890–7898.
28 Pispisa B. In The Coordination Chemistry of Metalloporphyrins, Bertini I,
Drago RS, Luchinat C (eds). D. Reidel Publ. Co.: Dordrecht, The
Netherlands, 1983; 279–290.
29 Dunford HB. Heme Peroxidases. Wiley-VCH: New York, 1999.
30 Sanchez Ferrer A, Rodriguez-Lopez JN, Garcia-Canovas F, Garcia-
Carmona F. Tyrosinase: a comprehensive review of its mechanism.
Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1995; 1247: 1–11.
31 Adedrain S, Lambeir AM. Kinetics of the reaction of compound II of
horseradish peroxidase with hydrogen peroxide to form compound
III. Eur. J. Biochem. 1989; 186: 571–576.
13 Toniolo C, Crisma M, Formaggio F, Peggion C. Control of peptide confor-
mation by the Thorpe-Ingold effect (Cα-tetrasubstitution). Biopolymers
(Pept. Sci.) 2001; 60: 396–415.
14 Pispisa B, Palleschi A, Mazzuca C, Stella L, Valeri A, Venanzi M,
Formaggio F, Toniolo C, Broxterman QB. The versatility of combining
FRET measurements and molecular mechanics results for determining
the structural features of ordered peptides in solution. J. Fluoresc.
2002; 12: 213–217.
32 Munoz-Munoz JL, Garcia-Molina F, Varon R, Tudela J, Garcia-Canovas
F, Rodrigues-Lopez JN. Generation of hydrogen peroxide in the
melanin biosynthesis pathway. Biochim. Biophys. Acta 2009; 1794:
1017–1029.
33 Adams PA, de L Milton RC, Byfield MP, Pratt JM. Oxygen activation and
ligand binding by pure heme-octapeptide microperoxidase-8 (MP-8).
J. Inorg. Biochem. 1988; 34: 167–175.
15 Guallar V. Heme electron transfer in peroxidases: the propionate
e-pathway. J. Phys. Chem. B 2008; 112: 13460–13464.
34 Kubo I, Chen Q-X, Nihei KI, Calderon JS, Cespedes CL. Tyrosinase in-
hibitors from galls of Rhus javanica leaves and their effects on insects.
Z. Naturforsch. 2003; 58c: 703–718.
35 Martin RB. Zwitterion formation upon deprotonation in L-3,4-
dihydroxyphenylalanine and other phenolic amines. J. Phys. Chem.
1971; 75: 2657–2661.
36 Barteri M, Pispisa B. Stereospecific oxidation of ascorbic acid by chiral
association complexes between polypeptides and iron(III) chelate
ions. Biopolymers 1982; 21: 1093–1106.
16 Pispisa B, Stella L, Venanzi M, Palleschi A, Viappiani C, Polese A,
Formaggio F, Toniolo C. Quenching mechanisms in bichromophoric
310-helical Aib-based peptides, modulated by chain length depen-
dent topologies. Macromolecules 2000; 33: 906–915.
17 Pispisa B, Venanzi M, Stella L, Palleschi A, Zanotti G. Photophysical and
structural features of covalently bound peptide-protoporphyrin-
peptide compounds carrying naphthalene chromophores. J. Phys.
Chem. B 1999; 103: 8172–8179.
18 Allinger NL, Chen K, Lii J-H. An improved force field (MM4) for satu-
rated hydrocarbons. J. Comp. Chem. 1996; 17, 642–668; Nevens N,
Chen K, Allinger K. Molecular mechanics (MM4) calculations on al-
kenes. J. Comp. Chem. 1996; 17, 669–694; Nevins N, Lii J-H, Allinger
NL. Molecular mechanics (MM4) calculations on conjugated hydrocar-
bons. J. Comp. Chem. 1996; 17, 695–729; Nevins N, Allinger NL. Molec-
ular mechanics (MM4) vibrational frequency calculations for alkenes
and conjugated hydrocarbons. J. Comp. Chem. 1996; 17, 730–746.
19 Pispisa B, Palleschi A, Stella L, Venanzi M, Mazzuca C, Formaggio F,
Toniolo C, Broxterman QB. Effects of helical distortions on the optical
properties of amide NH infrared absorption in short peptides in solu-
tion. J. Phys. Chem. B 2002; 106: 5733–5738.
37 In Bioinorganic Catalysis, Reedijk J (ed.). Marcel Dekker: New York,
1993.
38 In Metalloporphyrins Catalyzed Oxidation, Montanari F, Casella L,
Kluwer Academic Publ.: Dordrecht, The Netherlands, 1994.
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