Peptide-bound dinitrosyliron complexes (DNICs) and neutral/reduced-form Roussin's red esters (RREs/rRREs): Understanding nitrosylation of [Fe-S] clusters leading to the formation of DNICs and RREs using a de novo design strategy

Article abstract of DOI:10.1021/ic201529e

This manuscript describes the interaction of low-molecular-weight DNICs with short peptides designed to explore the stability and structure of DNIC-peptide/RRE-peptide constructs. Although characterization of protein-bound and low-molecular-weight DNICs i

Full text of DOI:10.1021/ic201529e

ARTICLE
pubs.acs.org/IC
Peptide-Bound Dinitrosyliron Complexes (DNICs) and Neutral/
Reduced-Form Roussin’s Red Esters (RREs/rRREs): Understanding
Nitrosylation of [FeꢀS] Clusters Leading to the Formation of DNICs
and RREs Using a De Novo Design Strategy
Zong-Sian Lin,† Feng-Chun Lo,† Chih-Hsiang Li,† Chih-Hao Chen,† Wei-Ning Huang,‡ I-Jui Hsu,§
Jyh-Fu Lee,^ Jia-Cherng Horng,† and Wen-Feng Liaw*,†
†Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan
‡Department of Biotechnology, Yuanpei University, Hsinchu 30015, Taiwan
§Department of Molecular Science and Engineering, National Taipei University of Technology, Taipei 10608, Taiwan
^National Synchrotron Radiation Research Center (NSRRC), Hsinchu 30076, Taiwan
S Supporting Information
b
ABSTRACT: This manuscript describes the interaction of low-
molecular-weight DNICs with short peptides designed to
explore the stability and structure of DNICꢀpeptide/RREꢀ
peptide constructs. Although characterization of protein-bound
and low-molecular-weight DNICs is possible via EPR, XAS, and
NRVS, this study demonstrates that the combination of aqu-
eous IR νNO and UVꢀvis spectra can serve as an efficient tool to
characterize and discriminate peptide-bound DNICs and RREs.
The de novo chelate-cysteine-containing peptides KC(A)nCK-
bound (n = 1ꢀ4) dinitrosyliron complexes KC(A)nCK-DNIC
(CnA-DNIC) and monodentate-cysteine-containing peptides
KCAAK-/KCAAHK-bound Roussin’s red esters (RREs) KCAAK-RRE/KCAAHK-RRE were synthesized and characterized by
aqueous IR, UVꢀvis, EPR, CD, XAS, and ESI-MS. In contrast to the inertness of chelate-cysteine-containing peptide-bound DNICs
toward KCAAK/KCAAHK, transformation of KCAAK-RRE/KCAAHK-RRE into CnA-DNIC triggered by CnA and reversible
transformation between CnA-DNIC and CnA-RRE via {Fe(NO)2}9-{Fe(NO)2}10 reduced-form peptide-bound RREs demon-
strate that the {Fe(NO)2}9 motif displays a preference for chelate-cysteine-containing peptides over monodentate-cysteine-
containing peptides. Also, this study may signify that nitrosylation of [FeꢀS] proteins generating protein-bound RREs, reduced
protein-bound RREs, or protein-bound DNICs are modulated by both the oxidation state of iron and the chelating effect of the
bound proteins of [FeꢀS] clusters.
’ INTRODUCTION
nucleic acid and the construction of structural analogues of the active
sites of metalloproteins, have been designed and synthesized.5 As has
been known, de novo designed peptides can not only be considered as
a bridge between metalloenzymes and organic model compounds but
also may reveal both types of functions.6 In particular, de novo
designed peptides can regulate the metalꢀligand-bonding interactions
and tune the intrinsic properties of the metal centers.7 For synthetic
chemistry, one approach for mimicking the active site of reactive
metalloenzymes to a thorough comprehension of their structural and
functional roles in biology is to expand from traditional small-molecule
biocoordination chemistry to the design and synthesis of peptide-based
analogues, which were named “bridged biological assemblies”.8 The
rational design of the stable de novo designed peptide-bound metal-
ion active sites has shown remarkable success for the past decades.8
Dinitrosyliron complexes (DNICs), the intrinsic NO-derived
species existing in various NO-overproducing tissues,1 and
S-nitrosothiol are known as the two possible forms for storage
and delivery of NO in a biological system.2 Nitric oxide (NO)
regulates multiple physiological reactions such as vasodilation,
neuronal transmission, inflammation, immune system response,
and cancer remedy.3 NO in vivo can be stabilized and stored in
the form of DNICs with proteins (protein-bound DNICs) and is
probably released from cells in the form of low-molecular-weight
DNICs (LMW-DNICs).4 As has been known, characterization
of both protein-bound DNICs and LMW-DNICs in vitro is
possible via their distinctive electron paramagnetic resonance
(EPR) signals at g = 2.03.1ꢀ4
A large number of metallopeptides, motivated by studies of electron
transfer, the effect of metal-ion binding on protein folding/assembly
and stability, catalytic function, electrochemistry, interactions with
Received: July 19, 2011
Published: September 22, 2011
r
2011 American Chemical Society
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The peptide-based [4Feꢀ4S] synthetic analogue was synthe-
sized to study the electron-transfer event and the midpoint reduc-
tion potentials of the [4Feꢀ4S] clusters regulated by proton-
coupled cofactor.9 In metallobiochemistry, the X-ray crystal
structures of de novo apoprotein and metalated peptide, re-
garded as a basis to comprehend interaction between the metal
ions and protein, were reported.10 In addition, in the field of
materials, de novo designed light-emissive peptide-bound por-
phyrin complexes have been synthesized based on the computa-
tional design.11
NO has been demonstrated to react with the [FeꢀS] clusters
of several proteins for regulating the physiological function of
ironꢀsulfur protein.112 The anaerobic reaction of high-potential
iron protein (HiPIP) or NsrR, required for the upregulation of
ResDE-dependent genes, with NO showed the generation of
protein-bound DNICs.4,13 Ding and co-workers showed that the
ferredoxin [2Feꢀ2S] clusters were converted into protein-bound
DNICs when Escherichia coli cells were treated with NO.14
Lippard and co-workers demonstrated that the [2Feꢀ2S] clus-
ters were nitrosylated to yield protein-bound Roussin’s red esters
(RREs) upon exposure of a Rieske-type ToMOC protein to
NO.15 Also, nitrosylation of WhiB-like protein containing a
[4Feꢀ4S] cluster resulting in the formation of a protein-bound
RRE was reported.16
S-Nitrosation, coupling of NO and a cysteine sulfur, has been
identified as a post-translational modification of proteins to
convey part of the ubiquitous influence of NO on thiol-depen-
dent cellular-signaling transduction.2 It is noticed that the NO
motif of Cys-SNO in proteins may originate from GSNO (GS =
glutathione), protein-bound SNO, NO-donor drugs, and HNO2
derived from acidification of nitrite and DNICs.2
In chemistry, the {Fe(NO)2} motif coordinated by different
ligands ([SPh]ꢀ, [ꢀSC4H3S]ꢀ, [C3H3N2]ꢀ, [OPh]ꢀ, and [NO2]ꢀ)
displaying distinct electronic properties that are cooperatively
regulated by the noninnocent NO ligands was concluded in the
previous study.17 The binding affinity of the coordinated ligands
([SPh]ꢀ, [ꢀSC4H3S]ꢀ, [C3H3N2]ꢀ, [OPh]ꢀ, and [NO2]ꢀ)
toward the {Fe(NO)2}9 motif was also delineated. Of impor-
tance, the facile interconversion among {Fe(NO)2}9 DNICs,
[(NO)2Fe(μ-SEt)2Fe(NO)2] RREs, and the {Fe(NO)2}9
{Fe(NO)2}10 reduced RREs (rRREs) [(NO)2Fe(μ-SEt)2Fe-
(NO)2]ꢀ was demonstrated.18 In addition, the redox shuttling
between the {Fe(NO)2}9 and {Fe(NO)2}10 DNICs modulating
the nitrite binding modes and then triggering nitrite activation to
generate NO were identified.19 Understanding the fundamental
chemistry of biomimetic DNICs may provide new insights into the
physiological process of NO storage and liberation. Efforts to look
for an efficient tool to characterize and discriminate the existence of
protein-bound DNICs/RREs/rRREs in a biological system led to
the design and synthesis of peptide-bound DNICs/RREs. In this
study, an anionic water-soluble DNIC, [Fe(SPhNO2COOH)2-
(NO)2]ꢀ (1), and a series of short peptides containing cysteine(s)
with the sequences KCACK (C1A), KCAACK (C2A), KCAAACK
(C3A), KCAAAACK (C4A), KCAAK, and KCAAHK, respec-
tively, were synthesized to study the binding affinity of chelate/
monodenate-cysteine-containing peptides toward the {Fe(NO)2}9
motif. The UVꢀvis spectra and the aqueous IR νNO stretching
frequencies/patterns of the peptide-bound DNICs/RREs/rRREs
were demonstrated to be an efficient tool to characterize and
discriminate the existence of peptide-bound DNICs/RREs/rRREs.
In particular, a study of the transformation among peptide-bound
DNICs, RREs, and rRREs may provide a way to understanding the
Figure 1. ORTEP drawing and labeling scheme of 1 in a [Na+-18-crown-
6-ether] salt with thermal ellipsoids drawn at 50% probability. Selected bond
distances (Å) and angles (deg): Fe1ꢀS1 2.2845(16), Fe1ꢀS2 2.2879(16),
Fe1ꢀN1 1.688(5), Fe1ꢀN2 1.681(5), N1ꢀO1 1.173(6), N2ꢀO2 1.176(6),
Na1ꢀO6 2.239(4); S1ꢀFe1ꢀS2 112.33(6), N1ꢀFe1ꢀN2 116.8(2).
nitrosylation products/pathways of [FeꢀS] proteins, regulated by
both the oxidation state of iron and the chelating effect of the bound
proteins of [FeꢀS] clusters in a biological system.
’ RESULTS
Synthesis of [Na-18-Crown-6-ether][Fe(SPhNO2COOH)2-
(NO)2] (1), a Water-Soluble DNIC. Upon the addition of 2
equiv of 5,50-dithiobis(2-nitrobenzoic acid) into the CH3CN
solution of complex [Fe2(μ-S)2(NO)4]2ꢀ at ambient tempera-
ture, a reaction ensued over the course of 30 min to yield the
water-soluble {Fe(NO)2}9 DNIC 1, characterized by IR, UVꢀvis,
and single-crystal X-ray crystallography (Figure 1). The conver-
sion of [Fe2(μ-S)2(NO)4]2ꢀ to complex 1 was monitored by IR.
The shift of IR νNO stretching frequencies from (1667 s, 1647 s cmꢀ1)
to [1759 s, 1712 s cmꢀ1 (νNO), 1732 s cmꢀ1 (νCO, CH3CN)]
implicatedthe formation of complex 1. The oxidative additionof 5,
50-dithiobis(2-nitrobenzoic acid) to [Fe2(μ-S)2(NO)4]2ꢀ accom-
panied by reductive elimination of S8 may rationalize the forma-
tion of complex 1.
Syntheses of Peptides KCAAK, KCAAHK, KCACK (C1A),
KCAACK (C2A), KCAAACK (C3A), and KCAAAACK (C4A). The
differences of the binding affinity of ligands (thiolate, imidazo-
late, phenoxide, and nitrite) toward the {Fe(NO)2}9 motif,
probed by ligand-substitution reactions, were demonstrated in
the previous study.17a In order to understand the role(s) that the
chelating effect of thiobiomolecules or proteins may play in
creating/stabilizing the protein-bound DNICs/RREs and ratio-
nalizing the conversion of LMW-DNIC into protein-bound
DNICs, the cysteinate-containing scaffold peptides (KCAAK,
KCAAHK, C1A, C2A, C3A, and C4A) expected to bind to
{Fe(NO)2} were synthesized. Alanine was selected as a spacer to
separate two binding cysteinates, and the positively charged
lysine was adopted to balance the negative charge of the DNIC
core. The peptides containing 5ꢀ8 residues (KCAAK, C1A,
C2A, C3A, and C4A) were synthesized by solid-phase methods
using standard Fmoc protocols, purified by semipreparative high-
performance liquid chromatography (HPLC) and identified by
electrospray ionization mass spectrometry [ESI-MS; Figure S1 in
the Supporting Information (SI)].20
Syntheses of De Novo Peptide-Bound DNICs CnA-DNIC
(n = 1ꢀ4). Transformation of the water-soluble DNIC 1 into
peptide-bound DNICs CnA-DNIC (n = 1ꢀ4) accompanied
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Table 1. Aqueous IR νNO Stretching Frequencies, Electronic Absorption Bands, EPR Signals at 77 K, and Fe K-Edge XAS Preedge
Energies for Peptide-Bound DNICs CnA-DNIC (n = 1ꢀ4) and RREs KCAAK-RRE/KCAAHK-RRE
IR νNO (cmꢀ1
)
UVꢀvis (nm)
EPR (77 K)
preedge energy (eV)
C1A-DNIC
C2A-DNIC
C3A-DNIC
C4A-DNIC
KCAAK-RRE
KCAAHK-RRE
rC1A-RRE
rC2A-RRE
rC3A-RRE
rC4A-RRE
C1A-RRE
1722, 1767
1722, 1768
1720, 1767
1721, 1766
309, 413
g1 = 2.040, g2 = 2.032, g3 = 2.014
g1 = 2.039, g2 = 2.029, g3 = 2.014
g1 = 2.040, g2 = 2.030, g3 = 2.014
g1 = 2.040, g2 = 2.029, g3 = 2.014
7113.4
7113.7
7113.7
7113.7
7113.7
313, 409
315, 407
313, 407
1764, 1788, 1820
1764, 1788, 1820
312, 366, 448
310, 364, 448
310, 369, 464, 613, 952
306, 368, 467, 646, 957
312, 367, 464, 639, 939
310, 370, 462, 637, 950
312, 366, 448
312, 364, 448
314, 366, 448
312, 364, 448
400
g^ = 2.006, g = 1.970
g1 = 2.012, g2 = 1.999, g3 = 1.988
g1 = 2.012, g2 = 1.999, g3 = 1.989
g1 = 2.012, g2 = 2.000, g3 = 1.988
1763, 1788, 1821
1764, 1788, 1822
1763, 1788, 1822
1764, 1788, 1822
C2A-RRE
C3A-RRE
C4A-RRE
complex 1
g1 = 2.036, g2 = 2.029, g3 = 2.022
[Fe(Cys)2(NO)2]ꢀ
1727, 1772
310, 405
by the release of 3-carboxy-4-nitrobenzenethiolate ([SPhNO2-
COOH]ꢀ (a strong absorption band at 410 nm)) was observed
when a phosphate buffer solution (pH 8.5) of complex 1 was
treated with de novo peptides (C1A, C2A, C3A, and C4A,
respectively) in a 1:1 molar ratio for 1 h at ambient temperature.
All CnA-DNIC were purified by the gel-filtration chromatogra-
phy (Bio-Gel P-2 Gel, Bio Rad). The CnA-DNIC were char-
acterized by IR, UVꢀvis, EPR, and ESI-MS (Figure S2 in the SI
and Table 1). The Fourier transform infrared (FTIR) spectra of
peptide-bound DNICs CnA-DNIC in aqueous solution are
depicted in Figures 2a and S3 in the SI. Compared to the
thiolate-containing DNICs [Fe(SEt)2(NO)2]ꢀ displaying
IR νNO (1674, 1715 cmꢀ1) in THF,21 the aqueous IR νNO
spectra of peptide-bound DNICs CnA-DNIC exhibit the diag-
nostic stretching frequencies 1722, 1767 cmꢀ1 (C1A-DNIC),
1722, 1768 cmꢀ1 (C2A-DNIC), 1720, 1767 cmꢀ1 (C3A-
DNIC), and 1721, 1766 cmꢀ1 (C4A-DNIC), respectively
(Table 1). In contrast to complex [Fe(SPh)2(NO)2]ꢀ domi-
nated by intense absorption bands 479 and 798 nm in THF,17c
the electronic spectra of peptide-bound DNICs CnA-DNIC
display different patterns and absorptions in aqueous solution:
309 sh, 413 nm (C1A-DNIC), 313 sh, 409 nm (C2A-DNIC),
315 sh, 407 nm (C3A-DNIC), and 313 sh, 407 nm (C4A-
DNIC) (Figures 3b and S4a,b in the SI). The UVꢀvis spectra of
CnA-DNIC observed in this work are in very good accord with
those published by Fontecave and co-workers using cysteamine
as the thiol ligand.17e
EPR spectra of the peptide-bound DNICs CnA-DNIC are
depicted in Figures 4a,b and S5bꢀd and S6bꢀd in the SI. At 77 K,
C1A-DNIC displays a rhombic EPR signal at g1 = 2.040, g2 =
2.032, and g3 = 2.014 (g1 = 2.039, g2 = 2.029, and g3 = 2.014 for
C2A-DNIC, g1 = 2.040, g2 = 2.030, and g3= 2.014 for C3A-
DNIC, and g1 = 2.040, g2 = 2.029, and g3 = 2.014 for C4A-DNIC;
Table 1). Intriguingly, the EPR spectrum of C2A-DNIC exhibits
a well-resolved seven-line pattern with a g value of 2.029 (coupl-
ing constants AN(NO) = 2.45 G and AH(Cys) = 1.90 G) at 298 K.
Compared to those of C1A-DNIC, C3A-DNIC, and C4A-
DNIC, C2A-DNIC displays a seven-line EPR signal ascribed
to the higher contribution of cysteine-containing peptide to the
singly occupied molecular orbital of C2A-DNIC.22 This could be
further supported by the intense circular dichroism (CD) signals
in the near-UV and visible regions (from 260 to 600 nm;
Figure 5). In addition, the far-UV (from 200 to 260 nm) CD
spectra indicate that these peptide-bound DNICs CnA-DNIC do
not have significant secondary structures (Figure S7 in the SI).
Syntheses of De Novo Peptide-Bound RREs KCAAK-RRE
and CnA-RRE (n = 1ꢀ4). Upon the addition of 1 equiv of
Fe(CO)2(NO)2 to the aqueous solution of de novo monoden-
tate-cysteine-containing peptide KCAAK at ambient tempera-
ture, the electronic absorption bands [312, 366, 448 sh nm
(H2O); Figure S4c in the SI] and IR νNO stretching frequencies
[1764 s, 1788 s, 1820 w cmꢀ1 (H2O); Figure 2b] suggested
formation of the peptide-bound RRE KCAAK-RRE containing
two monodentate-cysteine-containing KCAAK peptides as brid-
ging ligands (Figure S8a in the SI). Of importance, attempts to
synthesize the de novo monodentate-cysteine-containing pep-
tide-bound DNIC (KCAAK)2-DNIC were unsuccessful by
adding 2 equiv of KCAAK into a phosphate buffer (20 mM,
pH = 8.5) of KCAAK-RRE at ambient temperature.17e In a
similar fashion, the monodentate-cysteine-containing peptide-
bound RRE KCAAHK-RRE, characterized by aqueous solution
IR, UVꢀvis, and ESI-MS (Figure S8b in the SI and Table 1), was
synthesized by the reaction of peptide KCAAHK and Fe(CO)2-
(NO)2 at ambient temperature. The electronic absorption bands
[310, 364, 448 sh nm (H2O); Figure S4c in the SI] and IR νNO
stretching frequencies [1764 s, 1788 s, 1820 w cmꢀ1 (H2O);
Figure 2b] demonstrated the formation of monodentate-
cysteine-containing peptide-bound RRE KCAAHK-RRE. The
absence of formation of KCAAHK-DNIC with the cysteine-
histidine-chelating mode is consistent with the binding affinity of
coordinated ligands toward the {Fe(NO)2}9 motif observed in
the biomimetic model study ([SPh]ꢀ > [ꢀSC4H3S]ꢀ > [C3H3-
N2]ꢀ > [OPh]ꢀ > [NO2]ꢀ).17a The unique ability of the
[Fe(NO)2] motif to bind chelate-cysteine-containing peptides
CnA and monodentate-cysteine-containing peptide KCAAK
provides the opportunity to probe the binding preference of
the [Fe(NO)2] motif to CnA and KCAAK peptides. As shown in
Scheme 1, upon the addition of 2 equiv of C1A to the aqueous
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Figure 2. Aqueous IR νNO spectra (H2O) of (a) (I) C1A-DNIC and (II) [Fe(Cys)2(NO)2]ꢀ and (b) (III) KCAAK-RRE, (IV) KCAAHK-RRE, and
(V) C1A-RRE.
solution of KCAAK-RRE and stirring at ambient temperature for
1/2 h, the IR νNO shift from [1764, 1788, 1820 cmꢀ1 (H2O)] to
[1722, 1767 cmꢀ1 (H2O)] suggested formation of the chelate-
cysteine-containing peptide-bound DNIC C1A-DNIC. How-
ever, the addition of 2 equiv of KCAAK to CnA-DNIC in
aqueous solution reveals no detectable changes in IR νNO
(Scheme 1).
In contrast to the inertness of CnA-DNIC toward KCAAK,
the chelating peptides CnA triggering the transformation of
KCAAK-RRE into peptide-bound DNICs CnA-DNIC impli-
cates that the [Fe(NO)2] motif displays a preference for CnA
over KCAAK (Scheme 1). The differences in the [Fe(NO)2]
affinities for chelate-cysteine-containing peptide CnA and
monodentate-cysteine-containing peptide KCAAK explain that
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Figure 3. UVꢀvis spectra of (a) complex 1, (b) C1A-DNIC, (c) rC1A-RRE, and (d) C1A-RRE in H2O.
inclusion of the chelate-cysteine-containing peptide CnA may
produce the more thermally stable {Fe(NO)2}9 DNICs (Scheme 1).
This result rationalizes the failure of isolation of the monoden-
tate-cysteine-containing peptide-bound DNIC and opens an
avenue for the synthesis of chelate-cysteine-containing peptide-
bound RREs.
(Figure S10 in the SI). In comparison with RRE [Fe(SEt)-
(NO)2]2 showing IR νNO 1749 s, 1774 s, 1809 w cmꢀ1 in THF,23
aqueous IR νNO spectra of peptide-bound RREs CnA-RRE
exhibit the diagnostic stretching frequencies 1763 s, 1788 s,
1821 w cmꢀ1 (C1A-RRE), 1764 s, 1788 s, 1822 w cmꢀ1 (C2A-
RRE), 1763 s, 1788 s, 1822 w cmꢀ1 (C3A- RRE), and 1764 s,
1788 s, 1822 w cmꢀ1 (C4A-RRE) in H2O, respectively
(Figures 2b and S11 in the SI). It is noticed that the separation
(ΔνNO = ∼45 cmꢀ1) of νNO stretching frequencies of the
peptide-bound DNICs CnA-DNIC is different from that
(ΔνNO = ∼25 cmꢀ1) of peptide-bound RREs CnA-RRE
(Table 1).
In particular, conversion of chelate-cysteine-containing pep-
tide-bound RREs CnA-RRE to chelate-cysteine-containing pep-
tide-bound DNICs CnA-DNIC was displayed when 1 equiv of
peptide CnA was added to the phosphate buffer (20 mM, pH
8.5) solution of CnA-RRE at ambient temperature (Scheme 2c).
These results illustrate that the transformation pathway of CnA-
DNIC into CnA-RRE exclusively occurs via the intermediate
rCnA-RRE. In contrast, CnA peptides trigger the direct conver-
sion of CnA-RRE to CnA-DNIC in aqueous solution at ambient
temperature, as shown in Scheme 2. This study demonstrates
that the {Fe(NO)2}9 motif shows a strong preference for the
formation of chelate-cysteine-containing peptide-bound DNICs over
the formation of chelate-cysteine-containing peptide-bound RREs.
Of importance, we noticed that the combination of UVꢀvis
spectra of the peptide-bound RRE CnA-RRE (or KCAAK-RRE)
and peptide-bound DNIC CnA-DNIC is almost the same as the
UVꢀvis spectrum [absorption bands 310, 360, 410 nm (H2O)]
derived from nitrosylation of Fur. E. coli (containing four cysteines),24
As shown in Scheme 2a,b, chelate-cysteine-containing pep-
tide-bound RREs CnA-RRE (n = 1ꢀ4) were synthesized by
reacting peptide-bound DNICs CnA-DNIC with Fe(CO)2-
(NO)2 in aqueous solution at ambient temperature followed
by contact with O2. The transformation of peptide-bound
DNICs CnA-DNIC into peptide-bound RREs CnA-RRE via
the {Fe(NO)2}9{Fe(NO)2}10 reduced peptide-bound RRE
rCnA-RRE was monitored by UVꢀvis spectra in aqueous
solution; the shift of the electronic bands from 309 sh, 413 nm
[C1A-DNIC (H2O); Figure 3b] to 310, 369, 464, 613, 952 nm
(rC1A-RRE; Figures 3c and S4d in the SI) and then a further
shift to [312, 366, 448 sh nm (H2O); C1A-RRE; Figures 3d and
S4b in the SI] confirmed the conversion of C1A-DNIC to C1A-
RRE via the reduced-form rC1A-RRE (Table 1). In EPR spectra,
the disappearance of a rhombic EPR signal at g1 = 2.040, g2 =
2.032, and g3 = 2.014 [C1A-DNIC (H2O); Figures 4b and S6 in
the SI] accompanied by the simultaneous formation of the signal
at g^ = 2.006 and g = 1.970 [rC1A-DNIC (H2O); Figures 4c and
S9 in the SI] also confirmed formation of the {Fe(NO)2}9
{Fe(NO)2}10 reduced peptide-bound RRE rC1A-RRE when
C1A-DNIC was reacted with Fe(CO)2(NO)2 in aqueous solu-
tion at ambient temperature. All peptide-bound RREs KCAAK-
RRE and CnA-RRE were purified by gel-filtration chromatog-
raphy (Bio-Gel P-2 Gel, Bio Rad) and identified by ESI-MS
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Figure 4. EPR spectra (H2O) of (a) C2A-DNIC with g = 2.029 (A(N) = 2.45 G; A(H) = 1.90 G) at 298 K, (b) C1A-DNIC with g1 = 2.040, g2 = 2.032, and
g3 = 2.014 at 77 K, (c) rC1A-RRE with g^ = 2.006 and g = 1.970 at 77 K, and (d) [Fe(Cys)2(NO)2]ꢀ with g = 2.031 (A(N) = 2.2 G; A(H) = 1.18 G) at
298 K.
Scheme 1
Figure 5. CD spectra for CnA-DNIC (n = 1ꢀ4) at 298 K. All
measurements were conducted in the aqueous solution.
assigned to the combination of protein-bound {Fe(NO)2}9 (S =1/2)
and {Fe(NO)2}8 (S = 0) DNICs.24 Also, the combination of IR νNO
spectra of the peptide-bound RRE CnA-RRE (or KCAAK-RRE)
and peptide-bound DNIC CnA-DNIC, identical with the IR νNO
spectra of nitrosylation of Fur. E. coli (IR νNO stretching frequencies
1718, 1765, 1787, and 1815 cmꢀ1),24 suggests that nitrosylation of
Fur. E. coli may produce a mixture of protein-bound DNICs and
RREs. Therefore, in addition to EPR, X-ray absorption spectroscopy
(XAS), and nuclear resonance vibrational spectroscopy (NRVS)
reported to be adopted to characterize DNICs and RREs,22,25 it is
presumed that aqueous solution FTIR νNO spectra in combina-
tion with UVꢀvis spectra may serve as an efficient tool for
characterization and discrimination of the {Fe(NO)2}9 DNICs,
{Fe(NO)2}9-{Fe(NO)2}10 rRREs, and {Fe(NO)2}9{Fe(NO)2}9
RREs in biology.
Transfer of the {Fe(NO)2} Core of Cysteine-Containing
Peptide-Bound DNICs and RREs. Transfer of the {Fe(NO)2}9
core of peptide-bound DNICs was also investigated by the
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Scheme 2
Scheme 3
Scheme 4
reaction of peptide-bound DNICs C1A-DNIC and peptide CnA
(n = 2ꢀ4; or LMW cysteine) under N2 in aqueous solution at
ambient temperature (Scheme 3). In contrast to the inertness
of peptide-bound DNICs CnA-DNIC toward free cysteine
(Scheme 4a), the coexistence of C1A-DNIC and C2A-DNIC
was observed when C1A-DNIC was reacted with 1 equiv of C2A
in aqueous solution at ambient temperature (Figure S12 in the SI
and Scheme 3). As shown in Scheme 4, the relative stability of
CnA-DNIC (n = 1ꢀ4), KCAAK-RRE, and CnA-RRE (n = 1ꢀ4)
toward free cysteine was determined through the addition of free
cysteine to an aqueous solution of CnA-DNIC, KCAAK-RRE,
and CnA-RRE, respectively, at ambient temperature. The addi-
tion of 4 equiv of cysteine into an aqueous solution of mono-
dentate-cysteine-containing peptide-bound RREs KCAAK-RRE
resulted in the formation of DNIC [Fe(Cys)2(NO)2]ꢀ char-
acterized by the aqueous solution IR νNO (stretching frequencies
at 1727 and 1772 cmꢀ1; Figure 1a), UVꢀvis (electronic absorp-
tion bands at 308 and 408 nm; Figure S2e in the SI), and EPR
(a well-resolved 13-line hyperfine-splitting EPR g value of 2.031
with coupling constants AN(NO)= 2.2 G and AH(Cys)= 1.18 G)
spectra at 298 K (Figure 4d and Table 1).26 In contrast, the
reaction of CnA-RRE (n = 1ꢀ4) and cysteine in a 1:2 stoichi-
ometry yielded CnA-DNIC and [Fe(Cys)2(NO)2]ꢀ, identified
by aqueous solution IR, UVꢀvis, EPR, and ESI-MS (Scheme 4c).
These results illustrate one aspect of how the peptide-bound
DNICs/RREs function as an [Fe(NO)2]-donor species in the
presence of chelate-cysteine-containing peptides and free cy-
steine. Also, it is concluded that the relative stability of CnA-
DNIC, KCAAK-RRE, and CnA-RRE toward free cysteine fol-
lows the order of CnA-DNIC > CnA-RRE > KCAAK-RRE. That
is, the binding affinity of chelate-cysteine-containing peptide
CnA, free cysteine, and monodentate-cysteine-containing pep-
tide KCAAK toward the {Fe(NO)2}9 motif leading to the
formation of DNICs is in the order of chelate-cysteine-contain-
ing peptide > free cysteine . monodentate-cysteine-containing
peptide. This order, presumably, is related to the chelating effect
of the bound peptides.
Fe K-Edge XAS Studies of Peptide-Bound DNICs CnA-
DNIC (n = 1ꢀ4) and RRE KCAAK-RRE. To clarify the local
structures of a series of chelate-cysteine-containing peptide-
bound DNICs CnA-DNIC and monodentate-cysteine-contain-
ing peptide-bound RRE KCAAK-RRE, we conducted XAS
measurements. The experimental Fe K-edge spectra of pep-
tide-bound DNIC C4A-DNIC, RRE KCAAK-RRE, and model
complexes ([Fe(SEt)2(NO)2]ꢀ and [Fe(SEt)(NO)2]2) were
depicted in Figure 6. All peptide-bound DNICs CnA-DNIC
display the obvious peak within the range of 7113.4ꢀ7113.8 eV
(Table 1),17a which is generally assigned as a 1s f 3d transition.
On the basis of the Laporte selection rule,27 transitions are
forbidden if two states have the same parity. The relatively strong
preedge intensities indicate that the local geometry of the Fe
center is in a noncentrosymmetric environment (such as tetra-
hedral symmetry, Td). In Td symmetry, σ bonding between a
transition metal and a ligand can be described as the mixing
of metal orbitals of sp3 with sd3 hybridization. The mixing of a
p orbital (u parity) with a d orbial to relax the Laporte selection
rule rationalizes that the strong preedge peak within the range
of 7113.4ꢀ7113.8 eV was observed. This implies that the
local environment of the Fe site is bound by four-coordi-
nated atoms.
Extended X-ray absorption fine structure (EXAFS) data
analysis was performed to obtain the local structure of the Fe
site of peptide-bound DNICs CnA-DNIC and RRE KCAAK-
RRE, respectively (Figures 7 and S13 in the SI). The k3-weighted
EXAFS raw data (k3χ) used in the data analysis are displayed
in the inset. The fitting results are listed in Table S2 in the SI.
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Figure 6. Normalized XANES at the Fe K-edge of C4A-DNIC,
KCAAK-RRE, and model compounds ([Fe(SEt)2(NO)2]ꢀ and
[Fe(SEt)2(NO)2]2). The enlarged preedge region is depicted in
the inset.
The EXAFS fitting results of peptide-bound DNICs CnA-DNIC
indicate that the Fe is bound with two N and two S atoms at
average distances of 1.68ꢀ1.70 and 2.26ꢀ2.30 Å, respectively.
The third peak in the radial distribution function, including the
multiple scattering contribution of FeꢀNꢀO, is assigned as the
distance between Fe and O (2.80ꢀ2.82 Å). The EXAFS data and
fitting results are consistent with our previous single-crystal
structures of DNIC models.17a,28 In the peptide-bound RRE
KCAAK-RRE, the first and second peaks in the radial distribu-
tion function arise mainly from backscattering from two ligated
N atoms and two ligated S atoms, respectively. The third peak in
the radial distribution function is dominated by the single
scattering path between Fe and Fe as well as multiple scatter-
ing contributions from the FeꢀNꢀO path. The EXASF result
of peptide-bound RRE KCAAK-RRE is best described as a
[Fe(μ-SCys)2Fe] core with NO molecules coordinated to two
Fe sites. The average FeꢀN, FeꢀS, Fe---Fe, and Fe---O distances
are calculated as 1.70(2), 2.27(1), 2.70(1), and 2.76(3) Å, respec-
tively, consistent with those of [Fe(SEt)(NO)2]2 obtained from
the single-crystal X-ray structure.29
Figure 7. EXAFS data (k3χ shown in the inset) and the Fourier
transforms for (a) C4A-DNIC and (b) KCAAK-RRE. Open circles
and solid lines denote the experimental data and fitting results,
respectively.
a. Nitrosylation of the Oxidized/Reduced-Form [1Feꢀ0S]
Rubredoxin Bound by Monodentate-Cysteine-Containing
Proteins. As observed in the previous biomimetic study,21
nitrosylation of the oxidized/reduced-form [1Feꢀ0S] rubredox-
in bound by monodentate-cysteine-containing proteins may lead
to the proposed thermally unstable {Fe(NO)2}9 monodentate-
cysteine-containing protein-bound DNIC A intermediate via the
reductive elimination of disulfide and cysteine-containing pro-
tein (Scheme 5).18a This highly unstable {Fe(NO)2}9 DNIC A
intermediate is then driven to yield the {Fe(NO)2}9-{Fe(NO)2}9
monodentate-cysteine-containing protein-bound RREs via the
elimination of cysteine-containing protein (Scheme 5).
b. Nitrosylation of the Oxidized/Reduced-Form [1Feꢀ0S]
Rubredoxin Bound by Chelate-Cysteine-Containing Pro-
teins. Instead of formation of the protein-bound RREs derived
from nitrosative degradation of the monodentate-cysteine-con-
taining [1Feꢀ0S] rubredoxin, nitrosylation of the oxidized/
reduced-form [1Feꢀ0S] rubredoxin bound by chelate-cysteine-
containing proteins was presumed to lead to formation of the
thermally stable {Fe(NO)2}9 chelate-cysteine-containing pro-
tein-bound DNICs along with the reductive elimination of disulfide
and elimination of the proposed chelate-cysteine-containing protein,
’ DISCUSSION
Characterization of the nitrosylation products of [FeꢀS]
proteins in biological systems has been intensively studied.12ꢀ16
As shown in Schemes 1ꢀ4, the study on the binding affinity of
chelate/monodentate-cysteine-containing peptides toward the
[Fe(NO)2] motif and the transformation among chelate/mon-
odentate-cysteine-containing peptide-bound RREs, chelate-
cysteine-containing peptide-bound DNICs, and free-cysteine-
containing DNIC may lend credence to the proposal that
nitrosylation of [FeꢀS] proteins generates protein-bound RREs,
reduced protein-bound RREs, or protein-bound DNICs, modu-
lated by both the oxidation state of iron and the chelating effect of
the bound proteins of [FeꢀS] clusters, although in most cases
proteins are not structurally altered to a great extent upon interac-
tion with NO in vivo. Also, the local sequence of the proteins are
hardly similar to the peptide sequences used in this study. This
biomimetic study leading to initial insights concerning the degrada-
tion pathways and products of nitrosylating [FeꢀS] proteins may
be further classified as follows:
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Scheme 5
respectively (Scheme 6a,b). This study shows that the thermal stability
of peptide-bound DNICs/RREs is on the order of bidentate-cysteine-
containing peptide-bound DNICs > bidentate-cysteine-containing
peptide-bound RREs. Conclusively, the formation of protein-bound
RREs or protein-bound DNICs derived from nitrosylation of the
oxidized/reduced-form [1Feꢀ0S] rubredoxin is, obviously, regulated
by the chelating effect of the bound proteins of [FeꢀS] clusters.
c. Nitrosylation of the Oxidized/Reduced-Form Monoden-
tate-Cysteine-Containing [2Feꢀ2S] Ferredoxin. As shown
in Scheme 7a,28 nitrosylation of the monodentate-cysteine-con-
taining oxidized-form [2Feꢀ2S] ferredoxin yielded {Fe(NO)2}9-
{Fe(NO)2}9 protein-bound RREs accompanied by elimination of
sulfide via the thermally unstable {Fe(NO)2}9 monodentate-
cysteine-containing protein-bound DNICs. As observed in the
biomimetic study,21 NO radical binding to the electron-deficient
FeIIIꢀFeII center of the reduced-form monodentate-cysteine-con-
taining [2Feꢀ2S] ferredoxin may trigger reductive elimination
of sulfide accompanied by formation of the thermally unstable
{Fe(NO)2}9 and {Fe(NO)2}10 monodentate-cysteine-contain-
ing DNICs (Scheme 7b). The coordinative association of the
{Fe(NO)2}9 and {Fe(NO)2}10 DNICs leading to the dinuclear
{Fe(NO)2}9-{Fe(NO)2}10 reduced-form protein-bound RRE
intermediate followed by subsequent oxidation of the assembled
reduced-form protein-bound RRE intermediate rationalizes forma-
tion of the {Fe(NO)2}9-{Fe(NO)2}9 monodentate-cysteine-con-
taining RREs (Scheme 7b), supported by nitrosylation of Rieske-
type [2Feꢀ2S] ferredoxin ToMOC protein including two histidine
(His 45 and His 67) and two cysteine (Cys 47 and Cys 64) ligands,
yielding protein-bound RRE, reported by Lippard and co-workers.15
d. Nitrosylation of the Oxidized/Reduced-Form Chelate-
Cysteine-Containing [2Feꢀ2S] Ferredoxin. In contrast to
nitrosylation of the monodentate-cysteine-containing [2Feꢀ2S]
ferredoxin producing the {Fe(NO)2}9-{Fe(NO)2}9 protein-bound
RREs, nitrosylation of the oxidized-form chelate-cysteine-containing
[2Feꢀ2S] ferredoxin was proposed to yield the thermally stable
{Fe(NO)2}9 chelate-cysteine-containing protein-bound DNICs
(Scheme 8a). In a similar fashion, nitrosylation of the reduced-form
chelate-cysteine-containing [2Feꢀ2S] ferredoxin was presumed to
generate the thermally stable {Fe(NO)2}9 chelate-cysteine-containing
protein-bound DNICs via a consecutive process, oxidation of the
reduced-form protein-bound RREs and the subsequent splitting of
{Fe(NO)2}9-{Fe(NO)2}9 protein-bound RREs (Scheme 8b). In the
biological study,14 nitrosylation of a reduced-form SoxR protein-
containing [2Feꢀ2S] cluster leads to the formation of protein-
bound DNICs.
Scheme 6
DNICs, nitrosylation of chelate-cysteine-containing [4Feꢀ4S]2+
ferredoxin may exclusively yield protein-bound RREs via the
{Fe(NO)2}9-{Fe(NO)2}10 reduced-form protein-bound RRE
intermediate (Scheme 9a). Compared to nitrosylation of the
monodentate-cysteine-containing [1Feꢀ0S]/[2Feꢀ2S] yielding
protein-bound RREs, nitrosylation of monodentate-cysteine-
containing [4Feꢀ4S]2+ ferredoxin is presumed to result in
the formation of protein-bound RREs via the {Fe(NO)2}9-
{Fe(NO)2}10 reduced-form protein-bound RRE intermediate
(Scheme 9b), supported by nitrosylation of the [4Feꢀ4S]
protein (bound by four cysteines, Cys 23, Cys 53, Cys 56, and
Cys 62) in a WhiB-like family (WhiD), yielding protein-bound
RREs reported by Le Brun and co-workers.16
’ CONCLUSION AND COMMENTS
Syntheses of the cysteine-containing peptide-bound DNICs/
RREs/rRRE and study on the peptide-substitution reactions
among cysteine-containing peptide-bound DNICs, cysteine-
containing peptide-bound RREs, and free-cysteine-bound
DNICs have led to the following results.
1 The chelate-cysteine-containing peptide-bound DNICs,
characterized by aqueous solution IR, UVꢀvis, ESI-MS,
EPR, and CD spectra and XAS measurements, were
e. Nitrosylation of the Cysteine-Containing [4Feꢀ4S]2+
Ferredoxin. In contrast to nitrosylation of the chelate-cysteine-
containing [1Feꢀ0S]/[2Feꢀ2S] producing protein-bound
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Scheme 7
synthesized by the reaction of anionic water-soluble DNIC
1 and peptides CnA. In contrast, the chelate-cysteine-
containing peptide-bound RREs were prepared by the
reaction of {Fe(NO)2}10 [Fe(CO)2(NO)2] and peptide-
bound DNICs CnA-DNIC (n = 1ꢀ4) followed by O2
oxidation. As shown in Schemes 1 and 2, chelate-cysteine-
containing peptide-bound RREs and DNICs are chemically
reversible, in contrast to the irreversibility between mono-
dentate-cysteine-containing peptide-bound RREs and che-
late-cysteine-containing peptide-bound DNICs.
aqueous solution IR νNO spectra in combination with the
pattern of aqueous IR νNO spectra may serve as an efficient
tool to characterize and discriminate the existence of
protein-bound DNICs and RREs in a biological system, in
addition to EPR, XAS, and NRVS.23,26
3 The binding affinity of chelate-cysteine-containing peptides
and monodentate-cysteine-containing peptide toward the
[Fe(NO)2] motif may implicate that the stability of peptide-
bound/cysteine-bound RREs/DNICs is on the order of
bidentate-cysteine-containing peptide-bound DNICs >
free-cysteine-bound DNIC . monodentate-cysteine-con-
taining peptide-bound DNICs. Transformation of CnA-
RRE into CnA-DNIC under the presence of CnA explains
to some extent the stability of bidentate-cysteine-containing
peptide-bound DNICs over bidentate-cysteine-containing
peptide-bound RREs (∼monodentate-cysteine-containing
peptide-bound RREs). Namely, it is important not only that
chelate-cysteine-containing peptides stabilize the DNIC
form but also that chelate-cysteine-containing peptides
destabilize the RRE form.
Our results bridging the peptide-substitution reaction study
and investigation of the transformation between peptide-bound
RREs and DNICs may point the way to understanding nature’s
choice of forming chelate-cysteine-containing protein-bound
DNICs/RREs and monodentate-cysteine-containing protein-
bound RREs. That is, the results described in this study speak
2 It is noticed that the IR νNO spectra of monodentate-
cysteine-containing peptide-bound RRE KCAAK-RRE
and chelate-cysteine-containing peptide-bound RREs
CnA-RRE had the same pattern/position and the similar
separation of NO stretching frequencies (ΔνNO) (1764 s,
1788 s, 1820 w cmꢀ1 with ΔνNO = ∼24 cmꢀ1 (H2O) for
KCAAK-RRE and 1763 s, 1788 s, 1821 w cmꢀ1 with ΔνNO
=
∼25 cmꢀ1 for C1A-RRE in aqueous solution). In contrast
to separation (ΔνNO) of the NO stretching frequencies of
∼24 cmꢀ1 (H2O) for peptide-bound RREs containing
chelate/monodentate cysteines, the chelate-cysteine-con-
taining peptide-bound DNIC CnA-DNIC displays diagnos-
tic νNO stretching frequencies [e.g., 1722 s, 1767 s cmꢀ1
(H2O) for C1A-DNIC] with a separation (ΔνNO) of the
NO stretching frequencies of ∼45 cmꢀ1. In conclusion,
the relative position of the νNO stretching frequencies of
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Scheme 8
to the importance of cysteine residues being located in close
proximity along the protein backbone in order to stabilize
protein-bound DNICs over RREs. In particular, these results
may signify that free-cysteine-bound DNICs are only derived
from the transformation of protein-bound RREs in a biological
system (Scheme 4). Also, this study signifies that nitrosylation of
[FeꢀS] proteins producing protein-bound RREs, reduced-form
protein-bound RREs, or protein-bound DNICs in biological
systems may be regulated by both the oxidation state of Fe and
the chelating effect of the bound proteins of [FeꢀS] clusters.
3-tetramethyluronium hexafluorophosphate (Advanced Chemtech),
piperidine (Alfa Aesar), N,N-dimethylformamide (Fisher), N-methyl-
2-pyrolidinone (NMP; Mallinckrodt), triisopropylsilane (Alfa Aesar),
1,2-ethanedithiol (Acros), trifluoroacetic acid (TFA; Alfa Aesar), methyl
tert-butyl ether (TEDIA), 5,50-dithiobis(2-nitrobenzoic acid) (Alfa
Aesar), and 4,40-dipyridyl disulfide (Acros) were used as received.
Complexes [Na-18-crown-6-ether]2[Fe2(μ-S)2(NO)4] and Fe(CO)2-
(NO)2 were synthesized and characterized as described in published
literatures.30 UVꢀvis spectra were recorded on a Jasco V-570 spectro-
meter. Analyses of carbon, hydrogen, and nitrogen were obtained with a
CHN analyzer (Heraeus). ESI-MS spectra were obtained using a Varian
901 mass spectrometer.
’ EXPERIMENTAL SECTION
Peptide Synthesis and Purification. All peptides were prepared
on a 0.2 mmol scale by solid-phase Fmoc chemistry and standard
reaction cycles on an automated PS3 peptide synthesizer (Protein
Technologies Inc.). NMP was used as the major solvent in synthesis.
The coupling time for each cycle was 4 h. Each peptide has an acetylated
N-terminus. The use of a Rink amide resin with a MBHA linker
generated an amidated C-terminus following cleavage from the resin
with a 92.5% TFA/2.5% triisopropylsilane/2.5% H2O/2.5% ethane-
dithiol solution. All peptides were purified by reverse-phase HPLC
(Jasco) with a Thermo BioBasic semipreparative C18 column. H2O/
acetonitrile linear gradients with 0.1% (v/v) TFA as the counterion were
used for the purification at a flow rate of 3 mL/min. The identities of all
peptides were confirmed by using ESI-MS spectra: C1A observed 593,
expected 593.34; C2A observed 664, expected 664.51; C3A observed
Reagents and Chemicals. Manipulations, reactions, and transfers
were conducted under N2 according to Schlenk techniques or in a
glovebox (N2 gas). Solvents were distilled under N2 from appropriate
drying agents [diethyl ether from CaH2; acetonitrile from CaH2/P2O5;
hexane and tetrahydrofuran (THF) from sodium benzophenone] and
stored in dried, N2-filled flasks over 4 Å molecular sieves. N2 was purged
through these solvents before use. Solvent was transferred to the
reaction vessel via a stainless cannula under positive pressure of N2.
The reagents iron pentacarbonyl (Aldrich), sulfur powder (Showa),
sodium nitrite (Fluka), 18-crown-6-ether (TCI), sodium hydroxide
(Mallinckrodt), Fmoc-protected amino acids (Advanced Chemtech),
Rink amide MBHA resin (0.5 mmol/g; Novabiochem), 1-hydroxyben-
zotriazole (Advanced Chemtech), 2-(1H-benzotriazol-1-yl)-1,1,3,
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Scheme 9
735, expected 735.20; C4A observed 806, expected 806.33. Peptide
concentrations were determined by using a published assay with 4,40-
dipyridyl disulfide.31
Preparation of Peptide-Bound DNICs CnA-DNIC (n = 1ꢀ4).
To the 5 mL of phosphate buffer (20 mM, pH 8.5) of peptides [C1A
(0.01 mmol), C2A (0.01 mmol), C3A (0.01 mmol), C4A (0.01 mmol),
respectively] was added in a dropwise manner the 5 mL of phosphate
buffer (20 mM, pH 8.5) of complex 1 (0.008 g, 0.01 mmol). The
reaction solution was stirred under N2 at ambient temperature for 1 h.
The reaction was monitored by UVꢀvis spectra. The shift of the
absorption maximum from 400 nm (complex 1) to 412 nm implicated
the complete reaction of complex 1 and peptide. The resulting yellow-
brown solution was further purified by gel-filtration chromatography
(Bio-Gel P-2 Gel, Bio Rad). The formation of peptide-bound DNICs
was confirmed by the absorption spectra of the collected yellow fraction
with the presence of two absorption bands [C1A-DNIC (309 sh,
413 nm), C2A-DNIC (313 sh, 409 nm), C3A-DNIC (315 sh, 407 nm),
and C4A-DNIC (313 sh, 407 nm)] and by an EPR spectrum [g = 2.032
for C1A-DNIC; g = 2.029 for C2A-DNIC; g = 2.033 for C3A-DNIC;
g = 2.029 for C4A-DNIC (H2O)] at 298 K. The rhombic EPR g
Preparation of [Na-18-crown-6-ether][Fe(SPh-NO2CO-
OH)2(NO)2] (1). Complex [Na-18-crown-6-ether]2[Fe2(μ-S)2(NO)4]
(0.87 g, 1 mmol) and 5,50-dithiobis(2-nitrobenzoic acid) (0.793 g, 2 mmol)
were dissolved in CH3CN (10 mL) and stirred for 30 min under nitrogen at
ambient temperature. The reaction was monitored by FTIR. The IR νNO
shifting from (1667 s, 1647 s cmꢀ1) to [1759 s, 1732 s, 1712 cmꢀ1
(CH3CN)] was assigned to the formation of complex 1. Absorption
spectrum (H2O) [λmax, nm (ε, Mꢀ1 cmꢀ1)]: 400 (19 496). Anal. Calcd
for C26H32FeN4NaO16S2: C, 39.06; H, 4.03; N, 7.01. Found: C, 39.21; H,
4.33; N, 6.57. The reaction solution was dried under vacuum and then
redissolved in THF/diethyl ether (1:1 volume ratio). The mixture solution
was filtered through Celite to remove the insoluble solid, and hexane was
then added to precipitate the dark-red solid 1(1.28 g, 85%) characterized by
IR and UVꢀvis spectra and single-crystal X-ray diffraction.
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values are g1 = 2.040, g2 = 2.032, g3 = 2.014 for C1A-DNIC, g1 = 2.039,
g2 = 2.029, g3 = 2.014 for C2A-DNIC, g1 = 2.040, g2 = 2.030, g3 = 2.014
for C3A-DNIC, g1 = 2.040, g2 = 2.029, g3 = 2.014 for C4A-DNIC at 77
K. The peptide-bound DNICs were further characterized by ESI-MS:
C1A-DNIC observed 708, expected 708.3; C2A-DNIC observed 779,
expected 779.2; C3A-DNIC observed 850, expected 850.1; C4A-DNIC
observed 921, expected 921.3, respectively. The solution was dried
under vacuum, and the light-yellow solid was precipitated. The peptide-
bound DNICs CnA-DNIC were also identified by aqueous solution IR
{IRνNO: C1A-DNIC[1767s, 1722s cmꢀ1 (H2O)], C2A-DNIC[1768 s,
1722 s cmꢀ1 (H2O)], C3A-DNIC [1767 s, 1720 s cmꢀ1 (H2O)], C4A-
DNIC [1766 s, 1721 s cmꢀ1 (H2O)].
Preparation of Monodenate-Cysteine-Containing Pep-
tide-Bound RREs KCAAK-RRE and KCAAHK-RRE. The pure
liquid Fe(CO)2(NO)2 (1.2 μL, 0.01 mmol) was added dropwise into
the aqueous solution (5 mL) of peptide KCAAK (0.01 mmol) by a
microsyringe, and then the mixture solution was stirred under N2 at
ambient temperature for 1 h. The reaction was monitored by aqueous
solution IR and UVꢀvis spectra. The appearance of IR νNO stretching
frequencies 1764 s, 1788 s, and 1820 w cmꢀ1 (H2O) and the electronic
absorption bands of 312, 366, and 448 sh nm (H2O) were consistent
with the formation of peptide-bound RRE KCAAK-RRE. The red
mixture solution was further purified by gel-filtration chromatography
(Bio-Gel P-2 Gel, Bio Rad) and then dried under vacuum to precipitate
the yellow solid KCAAK-RRE. The peptide-bound RRE KCAAK-RRE
was also characterized by ESI-MS: KCAAK-RRE observed 1351,
expected 1350. In a similar fashion, the reaction of peptide KCAAHK
and Fe(CO)2(NO)2 (1:1 molar ratio) led to the formation of peptide-
bound RRE KCAAHK-RRE characterized by IR [1764 s, 1788 s, 1820 w cmꢀ1
(H2O)], UVꢀvis [310, 364, 448 nm (H2O)], and ESI-MS (observed
813, expected 1624).
Preparation of Chelate-Cysteine-Containing Peptide-
Bound RREs CnA-RRE (n = 1ꢀ4). To the aqueous solution
(5 mL) of peptide-bound DNICs CnA-DNIC [C1A-DNIC (0.0071 g,
0.01 mmol), C2A-DNIC (0.0078 g, 0.01 mmol), C3A-DNIC (0.0085 g,
0.01 mmol), C4A-DNIC (0.0092 g, 0.01 mmol), respectively] was
added in a dropwise manner the pure liquid Fe(CO)2(NO)2 (1.2 μL,
0.01 mmol) at ambient temperature. The reaction solution was stirred at
room temperature for 1/2 h and monitored by UVꢀvis and EPR spectra.
The shift of the electronic bands from (309 and 413 nm for C1A-DNIC,
313 and 409 nm for C2A-DNIC, 315 and 407 nm for C3A-DNIC, and
313 and 407 nm for C4A-DNIC) to (310, 369, 464, 613, and 952 nm for
rC1A-RRE, 306, 368, 467, 646, and 957 nm for rC2A-RRE, 312, 367,
464, 639, and 939 nm for rC3A-RRE, 310, 370, 462, 637, and 950 nm for
rC4A-RRE) in H2O and the EPR g values [g^ = 2.006 and g = 1.970 for
rC1A-RRE, g1 = 2.012, g2 = 1.999, and g3 = 1.988 for rC2A-RRE, g1 =
2.012, g2 = 1.999, and g3 = 1.989 for rC3A-RRE, and g1 = 2.012, g2 =
2.000, and g3 = 1.988 for rC4A-RRE at 77 K (H2O)] implicated
formation of the reduced peptide-bound RREs rCnA-RRE (n = 1ꢀ4).
The reaction solution was then stirred for 1 min after O2 gas was added
to the mixture solution at ambient temperature. The electronic absorp-
tions shifting to (312, 366, 448 sh nm) for C1A-RRE, (312, 364, 448
sh nm) for C2A-RRE, (314, 366, 448 sh nm) for C3A-RRE, (312, 364,
448 sh nm) for C4A-RRE and the disappearance of the EPR signal
suggested formation of the peptide-bound RREs CnA-RRE. All peptide-
bound RREs CnA-RRE were purified by gel-filtration chromatography
(Bio-Gel P-2 Gel, Bio Rad) and also characterized by ESI-MS: C1A-
RRE observed 823, expected 822; C2A-RRE observed 894, expected
893; C3A-RRE observed 965, expected 964; C4A-RRE observed 1036,
expected 1035. IR νNO (H2O): 1763 s, 1788 s, 1821 w cmꢀ1 (C1A-
RRE), 1764 s, 1788 s, 1822 w cmꢀ1 (C2A- RRE), 1763 s, 1788 s, 1822
w cmꢀ1 (C3A- RRE), and 1764 s, 1788 s, 1822 w cmꢀ1 (C4A-RRE).
The peptide-bound RREs CnA-RRE solution was dried under vacuum,
and the yellow solid was precipitated.
Conversion of Peptide-Bound RREs CnA-RRE (n = 1ꢀ4)/
KCAAK-RRE into Peptide-Bound DNICs CnA-DNIC (n = 1ꢀ4).
Complex KCAAK-RRE (0.0135 g, 0.01 mmol) was treated with 2 equiv
of peptide [C1A (0.118 g, 0.02 mmol), C2A (0.132 g, 0.02 mmol), C3A
(0.146 g, 0.02 mmol), C4A (0.162 g, 0.02 mmol), respectively] in 5 mL
of a phosphate buffer solution (20 mM, pH 8.5) at ambient temperature,
and then the reaction solution was stirred at room temperature for 10
min. IR νNO shifting from 1764 s, 1788 s, 1820 w cmꢀ1 (H2O; KCAAK-
RRE) to 1722 s, 1767 s, cmꢀ1 (H2O; C1A-DNIC) [(1722 s, 1768
s cmꢀ1) for C2A-DNIC, (1724 s, 1767 s cmꢀ1) for C3A-DNIC, (1723 s,
1766 s cmꢀ1) for C4A-DNIC], respectively, implicated the formation of
the peptide-bound DNICs CnA-DNIC. The mixture solution was
purified by gel-filtration chromatography (Bio-Gel P-2 Gel, Bio Rad)
and further characterized by ESI-MS. In a similar fashion, the reaction of
peptide-bound RREs C1A-RRE (or C2A-RRE) and peptide C1A (or
C2A) in the ratio of 1:1 led to the formation of peptide-bound DNICs
C1A-DNIC (or C2A-DNIC) characterized by IR and UVꢀvis.
Reaction of Peptide-Bound C1A-DNIC with Peptide CnA
(n = 2ꢀ4). The peptide-bound DNIC C1A-DNIC (0.0071 g, 0.01
mmol) and peptide C2A (0.01 mmol) were mixed in 5 mL of phosphate
buffer (20 mM, pH 8.5) and stirred under N2 at room temperature for
10 min. The reaction was monitored by ESI-MS. The coexistence of
peptide-bound DNICs C1A-DNIC and C2A-DNIC was characterized
by ESI-MS. In a similar fashion, the reaction of C1A-DNIC and C3A
also led to the formation of C1A-DNIC and C3A-DNIC identified by
ESI-MS.
Conversion of Peptide-Bound RREs CnA-RRE (n = 1ꢀ4)/
KCAAK-RRE into Cysteine-Bound DNIC [Fe(Cys)2(NO)2]ꢀ.
Peptide-bound RRE KCAAK-RRE (0.0135 g, 0.01 mmol) and
L-cysteine (0.0051 g, 0.04 mmol) were dissolved in 5 mL pf phosphate
buffer (20 mM, pH 8.5) and stirred under N2 at ambient temperature for
5 min. The reaction was monitored by aqueous solution IR, UVꢀvis, and
EPR spectra. The aqueous solution IR νNO shifting from [1764 s, 1788 s,
1823 w cmꢀ1 (H2O)] to [1727 s, 1772 s cmꢀ1 (H2O)] and the shift of
electronic bands to (310, 405 nm) were assigned to formation of the
cysteine-bound DNIC [Fe(Cys)2(NO)2]ꢀ with a well-resolved 13-line
EPR spectrum (g value 2.031). In a similar fashion, the reaction of
peptide-bound RREs CnA-RRE (n = 1ꢀ4) and L-cysteine (1:2 molar
ratio) led to formation of the cysteine-bound DNIC [Fe(Cys)2(NO)2]ꢀ
and peptide-bound DNICs CnA-DNIC (n = 1ꢀ4) characterized by IR,
UVꢀvis, EPR, and ESI-MS.
Aqueous Solution IR Spectroscopy. The samples were dis-
solved in degassed water and sealed between two CaF2 windows
containing a 6 μm Teflon-separated spacer. IR spectra were collected
using a Thermo Nicolet Nexus 470 spectrometer with a liquid-N2-
cooled HgꢀCdꢀTe detector. Spectra obtained from the accumulation
of 100 scans and Fourier-transformed data were treated with Happ-
Genzel apodization at a spectral resolution of 2 cmꢀ1
.
EPR Spectroscopy. EPR spectra were performed at the X band
using a Bruker EMX spectrometer equipped with a Bruker TE102 cavity.
A flat cell was used to record EPR spectra at 298 K. The microwave
frequency was measured with a Hewlett-Packard 5246 L electronic
counter.
CD Spectroscopy. CD experiments were performed with an AVIV
model 410 CD spectrometer. Far-UV CD spectra were obtained in
phosphate buffer (20 mM, pH 7.4), using a 1-mm-path length quartz
cuvette at 25 ꢀC. The concentrations of peptide and peptide-bound
DNICs were 100 μM in aqueous solution.
XAS Measurements and Data Analysis. All of the XAS
experiments were done on the wiggler Beamline BL-17C at the NSRRC,
Hsinchu, Taiwan. The incident X-ray photon energy was scanned using
a double crystal Si(111) monochromator [beam size: 4 mm (H) ꢁ 2 mm
(V)] with an incidence angle of 45ꢀ to the sample. The absorption
spectra were collected in the fluorescence mode [μ(E) = IF/I0] using a
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Inorganic Chemistry
ARTICLE
Lytle detector with the sample maintained at a temperature of 283 K by a
cold air jet stream. On the basis of comparisons of the first and last
spectra recorded for a given sample, there is no photoreduction or
radiation damage observed after repeated scans. The photon energy was
calibrated with respect to the absorption edge (7112.0 eV) of a reference
Fe foil, which was measured simultaneously with the sample.
(National Tsing Hua University, Hsinchu, Taiwan) for EPR
measurements.
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The data analysis procedure was based on the previous report
published by Li et al.34c The EXAFS χ(k) function (k = photoelectron
wavenumber) was obtained after preedge and postedge background
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program.32 χ(k) was then weighted by k3 and Fourier-transformed into
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n
2
2
f½ReðfjÞꢂ þ ½ImðfjÞꢂ g
∑
j¼1
Rfit
¼
ð1Þ
n
2
2
f½Reðχ~data jÞꢂ þ ½Imðχ~data jÞꢂ g
∑
j¼1
where~χ=k3χand nis the number of evaluations of fj, withfj =~χdata j ꢀ~χmodel j
(and, hence, Rfit), minimized in the nonlinear least-squares fitting algorithm.
Crystallography. Crystallographic data and structural refinement
parameters of complex 1 are summarized in Table S1 in the SI. The crystal
chosen for X-ray diffraction studies measured 0.18 ꢁ 0.07 ꢁ 0.02 mm3 for
complex 1. The crystal was mounted on a glass fiber and quickly coated in
epoxy resin. Unit cell parameters were obtained by least-squares refinement.
Diffraction measurements for complex 1 were carried out on a Bruker X8
APEX II CCD diffractometer with graphite-monochromatized Mo Kα
radiation (λ = 0.7107 Å) between 1.77ꢀ and 26.39ꢀ for complex 1. The
positional and anisotropic thermal parameters of all non-H and H atoms of
least-squares refinement were fixed at calculated positions and refined at
riding modes. A multiscan absorption correction was made. The SHELXTL
structure refinement program was employed.35
’ ASSOCIATED CONTENT
S
Supporting Information. X-ray crystallographic files in
b
CIF format and the structure determination of [Na-18-crown-6-
ether][Fe(SPhNO2COOH)2(NO)2], parameters used in fitting
the k3-weighted EXAFS data, and ESI-MS, IR, UVꢀvis, EPR,
CD, and EXAFS spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
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Corresponding Author
*E-mail: wfliaw@mx.nthu.edu.tw.
’ ACKNOWLEDGMENT
We gratefully acknowledge financial support from the National
Science Council of Taiwan. The authors thank Pei-Lin Chen for
single-crystal X-ray structural determinations and Juo-Chi Chen
10430
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Inorganic Chemistry
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