CuI and CuII complexes containing nitrite and tridentate aromatic amine ligand as models for the substrate-binding type-2 Cu site of nitrite reductase

Article abstract of DOI:10.1002/ejic.200400808

Cu^I and Cu^II complexes containing a tridentate aromatic amine compound [bis(6-methyl-2-pyridylmethyl)amine, Me₂bpa] in the absence and presence of nitrite have been prepared as models for the active site of dissimilatory copper-containing nitrite reductase (CuNIR). [Cu^II(Me₂bpa)(H₂O) (ClO₄)]ClO ₄ (1), [Cu^II(Me₂bpa)(NO₂)(ClO ₄)] (2), [Cu^I(Me₂bpa)(CH₃CN)]PF ₆ (3) and [Cu^I(Me₂bpa)(NO₂)] ₂·[(Ph₃P)₂NPF₆] (4) were prepared. The X-ray crystal structural analyses of 1, 2, and 4 reveal that the geometries of the Cu centers are distorted square pyramidal, distorted octahedral, and tetrahedral, respectively. The coordination modes of the nitrite ligands in 2 and 4 depend on the oxidation state of the copper ion: nitrite is coordinated to Cu^II and Cu^I through two oxygen atoms (O,O-coordination mode) and one nitrogen atom (N-coordination mode), respectively. A comparison of the absorption spectra of 1 and 2 in acetone solution indicates that the 387-nm absorption band (ε = 780 M^-1 cm^-1) of 2 is a charge-transfer transition. The broad absorption band at around 320 nm (ε ≈ 5000 M^-1 cm^-1) of 4 in dichloromethane is also due to a charge-transfer transition. Functional modeling of CuNIR has been accomplished by treating solutions of 4 with acid; the nitrite-binding Cu^I complex quantitatively gives the one-electron reduction product,NO. The nitrite reduction of 4 in dichloromethane obeys a second-order rate law, suggesting that the rate-determining step would be protonation of the nitrite ligand of 4. Electronic structure calculations of the Cu^I and Cu^II complexes containing Me₂bpa and nitrite by the density functional theory method demonstrate that the net charge of nitrite in Cu^I(Me₂bpa)(nitro) is larger than that in Cu^I(Me₂bpa)(nitrito). The mechanism of the nitrite reduction is discussed in comparison with the enzyme reaction mechanism of CuNIR.

Full text of DOI:10.1002/ejic.200400808

FULL PAPER
I
II
Cu and Cu Complexes Containing Nitrite and Tridentate Aromatic Amine
Ligand as Models for the Substrate-Binding Type-2 Cu Site of Nitrite
Reductase
[a]
[a]
[b]
[a]
Hiroshi Yokoyama, Kazuya Yamaguchi,* Manabu Sugimoto, and Shinnichiro Suzuki
Keywords: Bioinorganic chemistry / Copper / N ligands / Tridentate ligands
CuI and CuII complexes containing a tridentate aromatic
amine compound [bis(6-methyl-2-pyridylmethyl)amine,
Me bpa] in the absence and presence of nitrite have been
prepared as models for the active site of dissimilatory copper-
a charge-transfer transition. The broad absorption band at
–
1
–1
around 320 nm (ε Ϸ 5000
M
cm ) of 4 in dichloromethane
2
is also due to a charge-transfer transition. Functional model-
ing of CuNIR has been accomplished by treating solutions of
II
I
containing nitrite reductase (CuNIR). [Cu (Me
2
bpa)(H
2
O)
4 with acid; the nitrite-binding Cu complex quantitatively
II
I
(
ClO
bpa)(CH
NPF
4
)]ClO
4
(1), [Cu (Me
2
bpa)(NO
2
)(ClO
4
)] (2), [Cu (Me
)] ·[(Ph P)
2
-
-
gives the one-electron reduction product,NO. The nitrite re-
duction of 4 in dichloromethane obeys a second-order rate
law, suggesting that the rate-determining step would be pro-
tonation of the nitrite ligand of 4. Electronic structure calcula-
I
3
CN)]PF
6
(3) and [Cu (Me
2
bpa)(NO
2
2
3
2
6
] (4) were prepared. The X-ray crystal structural analy-
ses of 1, 2, and 4 reveal that the geometries of the Cu centers
are distorted square pyramidal, distorted octahedral, and tet-
rahedral, respectively. The coordination modes of the nitrite
ligands in 2 and 4 depend on the oxidation state of the cop-
I
II
tions of the Cu and Cu complexes containing Me
2
bpa and
nitrite by the density functional theory method demonstrate
I
that the net charge of nitrite in Cu (Me
2
bpa)(nitro) is larger
II
I
I
per ion: nitrite is coordinated to Cu and Cu through two
oxygen atoms (O,OЈ-coordination mode) and one nitrogen
atom (N-coordination mode), respectively. A comparison of
the absorption spectra of 1 and 2 in acetone solution indicates
than that in Cu (Me bpa)(nitrito). The mechanism of the ni-
2
trite reduction is discussed in comparison with the enzyme
reaction mechanism of CuNIR.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
–1
–1
that the 387-nm absorption band (ε = 780
M
cm ) of 2 is
Introduction
residues and one solvent water; the active site pocket con-
taining the Cu site lies at the bottom of a 16.5 Å cavity
formed at the interface between two adjacent monomers.
X-ray crystal structure analyses of nitrite-soaked oxidized
NIRs have demonstrated that the substrate is coordinated
The global inorganic nitrogen cycle sustained by bacteria
plays an important role in the animal and plant kingdoms.
Dinitrogen is introduced into the biosphere by the bio-
logical fixation of dinitrogen, and is removed from there
into the atmosphere again by denitrification. Denitrification
is the dissimilatory reduction of nitrate or nitrite to produce
dinitrogen by prokaryotic organisms. Nitrite reductase
II
to type-2 Cu in an asymmetric bidentate fashion through
[4–6]
two oxygen atoms instead of the water ligand,
enzymatic mechanism has been proposed.
and an
[7,8]
In the very
(NIR), a key enzyme of denitrification, catalyzes the re-
recent X-ray crystal structure of nitrite-soaked oxidized Al-
caligenes faecalis CuNIR at 1.4 Å resolution, NO2– coordi-
nates to the Cu primarily by one O atom, whereas the other
duction of nitrite to nitrogen monoxide. Cu-containing
NIR (CuNIR) is a 110-kDa homotrimer in which a mono-
mer contains one each of type-1 Cu (blue copper) and type-
[9]
O atom interacts more weakly with the metal center.
Cu (nonblue copper).[
1–3]
The interatomic distance be-
2
Moreover, the plane defined by the Cu and the two O atoms
and the plane defined by the N and the two O atoms form
a 75° angle and the NO2– binds to the Cu with an almost
tween the two Cu sites is 12.5 Å. The type-2 Cu site shows
a distorted tetrahedral geometry and is bound by three His
II
face-on interaction. Several nitrite adducts of Cu com-
[
[
a] Department of Chemistry, Graduate School of Science, Osaka
University,
plexes have been reported as model complexes for the active
[
10–14]
Toyonaka, Osaka 560-0043, Japan
Fax: +81-6-6850-5785
site of CuNIR.
Tolman et al. have reported the nitrite-
iPr
3 3
iPr
binding copper(i) complexes L Cu(NO ) (L
= 1,4,7-
2
E-mail: kazu@ch.wani.osaka-u.ac.jp
b] Department of Applied Chemistry and Biochemistry, Graduate triisopropyl-1,4,7-triazacyclononane) and [(LiPr Cu)
3
(μ-
2
School of Science and Technology, Kumamoto University,
Kurokami, Kumamoto 860-8555, Japan
Supporting information for this article is available on the
WWW under http://www.eurjic.org or from the author.
2+
NO2)] as structural and functional models for the nitrite-
binding type-2 Cu site and have discussed a mechanism for
NO generation involving these Cu complexes.
I
[15,16]
Eur. J. Inorg. Chem. 2005, 1435–1441
DOI: 10.1002/ejic.200400808
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1435

H. Yokoyama, K. Yamaguchi, M. Sugimoto, S. Suzuki
FULL PAPER
We report here the spectral and structural characteriza- N(1)N(2)N(3) plane of Me bpa by 19.6° and the O(1) ligat-
2
I
II
II
tion of Cu and Cu complexes containing nitrite and a ing atom binds axially to the Cu ion. The structural stud-
tridentate aromatic amine [bis(6-methyl-2-pyridylmethyl) ies of nitrite-binding Cu complexes have shown that nitrite
amine (Me bpa)] as a substrate-binding type-2 Cu site has a variety of coordination modes to a Cu center
model of CuNIR. Moreover, the nitrite reduction mecha- (Scheme 1). Complex 2 exhibits two distinct Cu –O
nism of the Cu complex is discussed based on the parame- bond lengths [Cu(1)–O(1) = 2.47(3) Å, Cu(1)–O(2) =
ters of activation of the transition state. The enzymatic re- 1.98(2) Å] and the coordination mode of NO (a in
duction mechanism of nitrite at the type-2 Cu is also pro- Scheme 1) is similar to those of the Cu –NO model com-
II
II
2
[
17]
II
nitrite
I
–
2
II
–
2
[
10–14]
II
posed in comparison with the present model complexes and plexes
and the nitrite-binding type-2 Cu site in ni-
the binding mode of nitrite observed in the native and mu- trite-soaked NIR at 1.4 Å resolution (Cu–O = 2.29–2.38,
[
9]
tant CuNIRs from Alcaligenes faecalis S-6 by X-ray crystal 2.04–2.08 Å). The Cu, N(4), and two O(1 and 2) atoms
[
9]
structure analysis.
lie in the same plane. The N–O bond lengths [N(4)–O(1) =
.24(4) Å, N(4)–O(2) = 1.28(4) Å] and the O(1)–N(4)–O(2)
angle [115.8(27)°] are similar to those in the free nitrite ion
1
[
18]
Results and Discussion
[O–N–O = 114.9(5)° and N–O = 1.240(3) Å].
II
Structural and Spectral Aspects of the Cu Model Complex
–
Containing NO2
II
The reaction of [Cu (Me bpa)(H O)(ClO )]ClO (1)
2
2
4
4
with one equivalent of NaNO in H O at room temperature
2
2
II
gives [Cu (Me bpa)(NO )(ClO )] (2), which was recrys-
2
2
4
Scheme 1.
tallized from aqueous methanol solution as green crystals.
Figure 1 shows the X-ray crystal structures of 1 and 2.
II
The geometry of Cu in 1 is distorted square pyramidal
The electronic absorption spectrum of 1 in acetone solu-
with three nitrogen atoms of the Me bpa ligand and one tion (Figure 2) shows one band at 675 nm (ε
=
2
–
–1
–1
oxygen atom from each of H O and ClO ; a 15.0° upside 190 m cm ), which is assigned to a ligand field transition.
2
4
–1
–1
deviation of the O(1) ligating atom of H O from the N(1) Complex 2 exhibits two bands at 378 nm (ε = 780 m cm )
N(2)N(3) plane of Me bpa is observed. The structure of 2 and 671 nm (ε = 200 m cm ), which are ascribed as a ni-
reveals the replacement of the H O ligand by nitrite and trite Ǟ Cu charge transfer (LMCT) and a d-d transition,
2
–1
–1
2
II
2
asymmetric O,OЈ-nitrite chelation to the distorted octahe- respectively. The small blue shift of the d-d band from
II
–
dral Cu ion along with the Me bpa and ClO ligands. 675 nm to 671 nm is due to the O-nitrite coordination of
2
4
The equatorial O(2) coordinating atom deviates from the nitrite. Moreover, the 77-K EPR spectra of 1 and 2 in ace-
Figure 1. ORTEP plots of 1 (left) and 2 (right) with 50% probability thermal ellipsoids. The hydrogen atoms have been omitted for
clarity. Selected distances [Å] and angles [°] for 1 are follows: Cu(1)–O(1) 2.017(7), Cu(1)–O(2) 2.441(7), Cu(1)–N(1) 2.001(6), Cu(1)–
N(2) 1.957(7), Cu(1)–N(3) 2.011(6); O(1)–Cu(1)–O(2) 111.6(3), O(1)–Cu(1)–N(1) 93.0(3), O(1)–Cu(1)–N(2) 153.0(3), O(1)–Cu(1)–N(3)
9
1
7.4(3), O(2)–Cu(1)–N(1) 90.5(3), O(2)–Cu(1)–N(2) 95.2(3), O(2)–Cu(1)–N(3) 91.7(3), N(1)–Cu(1)–N(2) 83.4(3), N(1)–Cu(1)–N(3)
67.8(3), N(2)–Cu(1)–N(3) 84.4(3). Selected distances [Å] and angles [°] for 2 are follows: Cu(1)–O(1) 2.47(3), Cu(1)–O(2) 1.98(2), Cu(1)–
O(3) 2.52(3), Cu(1)–N(1) 2.012(18), Cu(1)–N(2) 1.99(3), Cu(1)–N(3) 2.003(18), O(1)–N(4) 1.20(4), O(2)–N(4) 1.28(4); O(1)–Cu(1)–O(2)
5
8
4.9(10), O(1)–Cu(1)–O(3) 159.4(9), O(2)–Cu(1)–N(2) 155.6(10), N(1)–Cu(1)–N(2) 82.8(8), N(2)–Cu(1)–N(3) 82.1(9), N(1)–Cu(1)–O(3)
7.9(8), Cu(1)–O(1)–N(4) 83.7(19), Cu(1)–O(2)–N(4) 105.6(19), O(1)–N(4)–O(2) 115.8(27).
1436
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
Eur. J. Inorg. Chem. 2005, 1435–1441

I
II
Cu and Cu Complexes as Models for the Substrate-Binding Cu Site of Nitrite Reductase
FULL PAPER
tone solution reveal the typical axial signals (g Ͼ g ) of
|
|
Ќ
II
a tetragonal Cu chromophore having a d
x2–y2
ground state
(
(
Figure S1, Supporting Information). The parameters for 2
g = 2.25, g = 2.09, A = 16.8 mT) exhibit a slight de-
|
|
Ќ
||
crease in the g and A values with respect to the precursor
|
|
||
complex 1 (g = 2.27, g = 2.07, A = 17.2 mT). The differ-
|
|
Ќ
||
II
ence between the parameters of these Cu complexes sug-
gests the weak apical coordination [Cu(1)–O(3)] of nitrite
in 2.
I
Figure 3. ORTEP plots of the Cu complex in 4 with 50% prob-
6
ability thermal ellipsoids. The hydrogen atoms and [(Ph P) NPF ]
3
2
have been omitted for clarity. Selected distances [Å] and angles [°]
for 4 are follows: Cu(1)–N(1) 2.053(4), Cu(1)–N(2) 2.168(5), Cu(1)–
N(3) 2.033(5), Cu(1)–N(4) 2.087(5), O(1)–N(4) 1.076(8), O(2)–N(4)
1
.039(8); N(1)–Cu(1)–N(2) 82.4(2), N(1)–Cu(1)–N(3) 111.7(2),
Figure 2. Electronic absorption spectra of 1 (broken line) and 2
(solid line) in acetone at room temperature.
N(2)–Cu(1)–N(3) 81.0(2), N(1)–Cu(1)–N(4) 121.9(2), N(2)–Cu(1)–
N(4) 118.8(2), N(3)–Cu(1)–N(4) 124.2(2), Cu(1)–N(4)–O(1)
111.5(5) Cu(1)–N(4)–O(2) 114.8(5), O(1)–N(4)–O(2) 133.6(8).
The electronic absorption spectra of 3 and 4 in CH Cl
are shown in Figure 4. These Cu complexes have no ab-
2
2
I
I
sorption band in the visible region. Complex 4 exhibits an
Structural and Spectral Aspects of the Cu Model Complex
Containing NO2
–1
–1
–
intense broad band around 320 nm (ε Ϸ 5000 m cm ),
I
–
[10–14]
which is assigned to a Cu Ǟ NO2 MLCT transition.
iPr
3
The reduced complex of 1, the pale-yellow crystalline so- Tolman et al. have reported that L Cu(NO
lid [Cu (Me bpa)(CH CN)]PF (3), was prepared by treat- intense MLCT band at 308 nm (ε = 3800 m cm ) in
) exhibits an
2
I
–1
–1
2
3
6
ment of [Cu(CH CN)]PF with Me bpa in methanol. The CH
Cl
. The shift of the MLCT band could be due to the
2
3
3
6
2
2
iPr
nitrite-binding copper(i) complex, [Cu(Me bpa)(NO )] · different electronic structures of 4 and L Cu(NO
).
2
2 2
2
[
(Ph P) NPF ] (4) was obtained as a yellow powder from
3 2 6
an equimolar mixture of [Cu(CH CN)]PF , Me bpa, and
3
6
2
[
(Ph P) N]NO in acetone under nitrogen. The X-ray crys-
3 2 2
tal structure of 4 (Figure 3) reveals the N-nitrite coordina-
I
tion (d in Scheme 1) to the Cu center that adopts a dis-
torted tetrahedral geometry. Although such a binding mode
iPr
3
of nitrite in 4 is similar to that of L Cu(NO ), some im-
2
portant structural aspects are different.[
15,16]
The N–O bond
lengths [1.253(5) and 1.238(5) Å] and the O–N–O angle
iPr
3
[16]
[
116.6(4)°] of nitrite in L Cu(NO2) are almost the same
[18]
as those of free nitrite ion, but the nitrite N–O distances
O(1)–N(4) = 1.076(8) Å and O(2)–N(4) = 1.039(8) Å] and
[
the O(1)–N(4)–O(2) angle [133.6(8)°] of 4 are different from
the corresponding values of free nitrite. The Cu(1)–N(4)
bond length of 4 is 2.053(4) Å, which is longer than the
Figure 4. Electronic absorption spectra of 3 (broken line) and 4
(
solid line) in CH
2
Cl
2
at room temperature.
iPr
3
[16]
value of 1.903(4) Å in L Cu(NO2).
Unfortunately, the
large temperature factor of the O-atoms of the nitrite in 4
–
suggest the rotation/vibration of the NO2 ligand and lead
I
Nitrite Reductase Reactivity of the Cu Model Complex
I
to serious errors in the parameters of nitrite bound to Cu
Containing NO2–
[
19]
ion.
Therefore, it is difficult to discuss the structure of
nitrite-binding copper(i) complex in detail on the basis of
The cyclic voltammogram of 2 in H O shows a response
2
our present results.
with E1/2 = –95 mV vs. Ag/AgCl and a peak-to-peak sepa-
Eur. J. Inorg. Chem. 2005, 1435–1441
www.eurjic.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1437

H. Yokoyama, K. Yamaguchi, M. Sugimoto, S. Suzuki
FULL PAPER
ration, ΔE, of 74 mV in the presence of 500 mm NaNO2
(a in Figure 5). The ΔE value indicates a quasi-reversible
electron-transfer process. Addition of acid to the aqueous
solution of 2 brings about a strong catalytic current due to
–
the reduction of NO2 (b in Figure 5). Thus, 2 serves as an
–
electrocatalyst in the reduction of NO2 at pH 5.5.
Figure 6. Spectral changes observed during the anaerobic reaction
of 4 with trifluoroacetic acid in CH Cl at 213.0 K. The subsequent
2 2
traces, with progressively decreasing absorbance at 320 nm, refer to
spectra taken every second Inset: plots of the second-order rate law
for the reactions of 4 with CF
3
COOH at 213.0, 223.0, and 231.8 K.
[4] /[4] [acid] ).
The y-axis is (1/[4] [acid] )ln([acid]
0
0
0
x
0
x
Table 1. Second-order rate constants for nitrite reduction of 4.
T [K]
31.8
223.0
13.0
k
obsd. [m–1 s–1]
Figure 5. Voltammetric behavior of 2 (a) and after addition of
2
0.53± 0.04
0.34± 0.03
0.18± 0.02
HClO
4
(b) in 0.1 m TBAClO
4
aqueous solution at 25.0 °C (2:
–1
0
.5 mm; NaNO
2
: 500 mm; scan rate: 10 mVs ).
2
When four equivalents of trifluoroacetic acid was added
to a CH Cl solution of 4 at room temperature, the color
2
2
Table 2. Activation parameters for nitrite reduction of 4.
of the solution was changed from pale yellow to blue and
the stoichiometric evolution of NO gas (Ͼ95% vs. 4) was
observed by gas chromatography (Figure S2, Supporting
Information). No N O gas was detected, and [Cu(Me bpa)-
[kJmol ] ΔG϶ [kJmol–1] ΔH϶ [kJmol–1]
–
1
ΔS
϶
E
a
JK–1 mol ]
–1
[
23± 1 40± 2 25± 1
–51± 3
2
2
(
OCOCF ) ] was isolated from the reaction mixture. The
3 2
reaction of 4 with proton was monitored either by recording
the transient absorption spectra at one-second intervals or
by recording absorption vs. time at a fixed wavelength
(320 nm). At 213.0 K, the 320-nm band of 4 was observed
to be a sharp peak as shown in Figure 6. Introduction of
four equivalents of trifluoroacetic acid to the solution under
an Ar atmosphere induced a decrease in the absorbance at
Scheme 2.
3
7
4
20 nm and a slight increase in the absorbance at about
00 nm. As shown in the inset of Figure 6, the reaction of
with proton was found to be first-order both in [4] and in
I
II
Electronic-Structure Calculations of the Cu and Cu
Complexes Containing Me bpa and Nitrite by the Density
2
[
acid] (v = kobs[4][acid]) at 213.0, 223.0, and 231.8 K. The
Functional Theory (DFT) Method
rate constants (kobs) are listed in Table 1. The activation
parameters (Table 2) were estimated from Arrhenius–Eyr-
I
II
Electronic-structure calculations of the Cu and Cu
ing plots of the data (ln k = lnA – E /RT, ln(hk /k T) complexes containing Me bpa and nitrite were carried out
obs
a
obs
B
2
϶
϶
[20]
=
ΔS /R – ΔH /RT; Figure S3, Supporting Information).
with the DFT method using the Gaussian 98 program.
The mechanism of nitrite reduction of 4 with proton is The X-ray crystal data of 2 and 4 were employed for initial
II
illustrated in Scheme 2. The kinetic data of the reaction of geometries. The Cu (Me bpa) complex binding nitrite
2
II
–
4
with trifluoroacetic acid suggest that the rate-determining asymmetrically through two oxygen atoms (Cu –ONO ,
step is the first protonation of the nitrite ligand. Since a O,OЈ-nitrite coordination) is stabilized by 13.1 kcalmol–1
negative value of ΔS϶ generally corresponds to the ad- relative to that binding nitrite through one nitrogen atom
II
–
I
–
ditional loss of freedom of motion by the solvating mole- (Cu –NO , N-nitrite coordination), while Cu –NO is less
2
2
cule, the negative ΔS϶ value (–51 JK mol ) means that stable than Cu –ONO (1.0 kcalmol ). Therefore, nitrite is
–1
–1
I
–
–1
II
protonated 4 will be an active intermediate. The positive preferentially coordinated to Cu through two oxygen
϶
II
–
I
ΔH suggests that 4 is more stable than protonated 4. The atoms (Cu –ONO ), whereas the Cu complex could have
–
I
–
I
–
resonance structure of the nitrite ligand in 4 (O=N–O ↔ two nitrite-binding forms (Cu –ONO and Cu –NO ).
2
–
O–N=O) might contribute to the stability of 4.
These findings are consistent with the experimental data
www.eurjic.org Eur. J. Inorg. Chem. 2005, 1435–1441
1438
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

I
II
Cu and Cu Complexes as Models for the Substrate-Binding Cu Site of Nitrite Reductase
FULL PAPER
that the two LiPr -containing Cu complexes are bridged by would facilitate the N–O bond cleavage to produce NO gas
3
I
I
+
nitrite through both nitrogen and oxygen atoms in the di- through a Cu –NO intermediate (V Ǟ VI).
iPr
3
[16]
copper(ii) complex [(L Cu) (μ-NO )]PF .
The Cu–ni-
2
2
6
I
–
I
–
trite bond energies of Cu –NO and Cu –ONO were esti-
2
–
1
mated to be 103.2 and 104.2 kcalmol , respectively,
whereas those of Cu –NO and Cu –ONO were calcu-
lated to be 200.7 and 213.8 kcalmol , respectively. The
binding of nitrite to Cu in both the N-nitrite and the O,OЈ-
II
–
II
–
2
–
1
II
nitrite coordination is energetically favored over that of ni-
I
trite to Cu in the two forms. The net charges of the nitrite
I
–
I
–
II
–
II
ligands of Cu –NO , Cu –ONO , Cu –NO and Cu –
2
2
–
ONO were evaluated to be –0.649, –0.562, –0.314, and –
.403, respectively, suggesting that the N-coordinated nitrite
ligand of Cu –NO will be most easily attacked by proton
0
I
–
2
to produce NO and H O.
2
Proposed Mechanism of Nitrite Reductases
According to the crystallographic studies of the nitrite-
II
[10–16]
binding Cu complexes
and the nitrite-soaked oxid-
[
4–6]
ized CuNIR,
nitrite preferentially binds to the oxidized
type-2 Cu by asymmetric O,OЈ-coordination. We have pro-
posed a catalytic mechanism for CuNIR incorporating roles
for Asp98 hydrogen-bonded to the substrate and His255 hy-
drogen-bonded to Asp98 through H O, of which both the
2
Figure 7. Revized enzymatic reaction mechanism of CuNIR con-
[7]
residues form a hydrogen-bonding network around the taining type-1 Cu and type-2 Cu (cf. ref. ).
[
8]
type-2 Cu. In an intermediate species during nitrite re-
duction, one of the two oxygen atoms of nitrite coordinated
to type-2 Cu is probably hydrogen-bonded by both the pro-
tonated Asp98 and His255 residues. Very recently, Murphy
et al. have reported that the nitrite-bound CuNIR crystal
Conclusions
I
II
Cu and Cu complexes containing a tridentate aromatic
structure shows an angular orientation of nitrite bound to amine compound (Me bpa) and nitrite have been prepared
2
the type-2 Cu; the N atom is out of the plane defined by as models for the active site of dissimilatory copper-con-
the two O atoms and the Cu atom, which forms a 75° angle taining nitrite reductase (CuNIR). The X-ray crystal struc-
[
9]
with the plane defined by the N and the two O atoms.
tural analyses of 2 and 4 reveal that the geometries of the
Therefore, they proposed that electron transfer (ET) from Cu centers are distorted octahedral and tetrahedral, respec-
the type-1 Cu reduces the type-2 Cu to trigger a rearrange- tively, and the coordination modes of nitrite are O,OЈ-coor-
–
ment of NO2 (from O,OЈ-coordination to N-coordination) dination and N-coordination, respectively. The spectro-
I
+
to release water and form a Cu –NO intermediate, that is, scopic data of these complexes are consistent with their
the proximity of the N atom of nitrite to the Cu in the structural data. The reduction of nitrite by proton donation
substrate-soaked structure will facilitate this rearrangement to 4 gives NO. These findings imply that both structural
I
step. The present studies of the X-ray crystal structure, ni- and functional modeling of CuNIR are achieved with Cu
II
trite reduction kinetics, and electronic-structure calculation and Cu complexes containing the same aromatic amine
of 4 also suggest that nitrite is initially coordinated to the ligand, Me bpa. The kinetic results of the reaction of 4 with
2
oxidized type-2 Cu by the O,OЈ-coordination and then is acid suggest that the rate-determining step is protonation of
converted into N-coordination by the reduction of the type- the nitrite ligand of 4 and the resultant protonated species is
1
Cu. As shown in Figure 7, nitrite displaces a water ligand, the active intermediate. Moreover, electronic-structure cal-
–
I
II
which leaves a proton on Asp98 and is released as OH (I culations of the Cu and Cu complexes containing Me bpa
2
Ǟ II). When the type-2 Cu is reduced by an electron from and nitrite demonstrate that nitrite is preferentially coordi-
II
the type-1 Cu, the binding mode of nitrite is changed from nated to Cu through two oxygen atoms, but could bind to
I
O,OЈ-coordination to N-coordination and the proton of Cu by both O,OЈ- and N-coordination. Since the N-coordi-
His255, which has an imidazolium group, is donated to the nated nitrite ligand of 4 will be more easily attacked than
oxygen of N-coordinated nitrite (II Ǟ III Ǟ IV Ǟ V). N- the O,OЈ-coordinated one by proton, the nitrite reduction
I
Coordination of nitrite to the Cu ion and protonation to of 4 probably occurs through the N-coordination of nitrite
I
the nitrite ligand are supported by the kinetic analysis of 4 to Cu . According to the model complex studies for the
and the electronic-structure calculations of 2 and 4. Finally, active site of CuNIR, we have proposed a revised catalytic
[
7]
the electron transfer from the reduced type-2 Cu to NO H mechanism for CuNIR.
2
Eur. J. Inorg. Chem. 2005, 1435–1441
www.eurjic.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1439

H. Yokoyama, K. Yamaguchi, M. Sugimoto, S. Suzuki
FULL PAPER
probe (0.2-cm path-length) immersed in a small vessel containing
Experimental Section
a 5 mL CH
2 2
Cl solution of 4 (3.4 mg, 2.5 μmol) under an Ar atmo-
General Remarks: All reagents used were of the highest grade avail-
sphere. The reaction was started with the addition of 100 μL of a
able. Solvents were dried and distilled under N
2
. Bis(6-methyl-2-
]PF , and [(Ph P)
were synthesized according to the published procedures.
CH
2
Cl
2
solution containing CF
beforehand.
3
COOH (10 μmol), which had been
bpa),[ [Cu(CH
21]
CN)
pyridylmethyl)amine (Me
2
3
4
6
3
2
-
degassed with N
2
[
22]
N]NO
2
II
X-ray Crystallographic Study of [Cu (Me
(
2
bpa)(H
2
O)(ClO
4
)]ClO
4
All air-sensitive reactions were performed by using standard
Schlenk and vacuum-line techniques. Electronic absorption spectra
were recorded on a Shimadzu UV-2200 spectrophotometer. Low-
temperature absorption spectra were obtained with a Shimadzu
Multi-Spec 1500 spectrophotometer. X-band EPR spectra were re-
II
1) and [Cu (Me
carried out on a MAC Science MXC18 diffractometer with graph-
ite-monochromated Mo-K radiation (λ = 0.71069 Å). A blue (1)
2 2 4
bpa)(NO )(ClO )] (2): The X-ray experiment was
α
or green (2) crystal was mounted on a glass capillary. The reflection
intensities were monitored by three standard reflections at every
corded on
a JEOL JES-FE1X spectrometer at 77 K. Gas
150 measurements, and the decay of intensities was within 2%. The
chromatography was performed by using a Shimadzu GC14B ana-
lyzer with TCD detector (3-m molecular sieve 13X column,
unit-cell parameters used for the refinement were determined by
least-square calculations on the setting angles for 22 carefully cen-
tered reflections. An absorption correction was applied. Diffraction
data were corrected for both Lorentz and polarization effects. The
2
3.5 mLmin–1 flow rate, helium carrier gas, and at 50 °C). Cyclic
voltammetric analyses were carried out using a Bioanalytical Sys-
tems Model CV-50W voltammetric analyzer with a three-electrode
system consisting of a Ag/AgCl reference electrode, a gold wire
counter electrode, and a glassy carbon working electrode under an
Ag atmosphere at 25 °C.
[23]
structure was solved by direct methods SIR92 using the CRYS-
TAN-GM program system[ and refined anisotropically for non-
hydrogen atoms by full-matrix least-squares calculations. Each re-
finement was continued until all shifts were smaller than one-third
of the standard deviations of the parameters involved. Atomic scat-
tering factors and anomalous dispersion terms were taken from the
literature.[ All hydrogen atoms were located at their calculated
positions. The fundamental crystal data and experimental parame-
24]
II
[
Cu (Me
2
bpa)(H
2
O)(ClO
4
)]ClO
4
(1): A solution of Cu(ClO
4
)
2
·
6H
2
O (3.71 g, 10 mmol) in methanol (5 mL) was added to a solu-
tion of Me bpa (2.27 g, 10 mmol) in methanol (50 mL). The result-
ant solution was stirred for 1 h and stored for a few days at room
temperature to give blue crystals of
CuN (507.8): calcd. C 33.11, H 3.78, N 8.28; found
25]
2
1
(4.39 g, 86.5%). ters for structure determination are given in Table 3.
C
14
H19Cl
2
3
O
9
I
[
2 2 2 3 2 6
Cu (Me bpa)(NO )] [(Ph P) NPF ] (4): The X-ray experiment was
C 33.11, H 3.68, N 8.30.
carried out at –70 °C on a Rigaku Mercury CCD area detector
II
[
Cu (Me
2
bpa)(NO
2
)(ClO
4
)] (2): A solution of NaNO
2
(0.069 g, coupled with a Rigaku AFC-7R diffractometer with graphite mo-
1
1
mmol) in H
2
O (10 mL) was added to a solution of 1 (0.51 g,
α
nochromated Mo-K radiation (λ = 0.71069 Å). The pale-yellow
mmol) in H O (25 mL). The resultant solution was stirred for 1 h
2
crystal was mounted on a glass capillary. The data were corrected
for Lorentz and polarization effects, but not for secondary extinc-
tion. An empirical absorption correction was applied. The structure
was solved by direct methods using the Crystal Structure crystallo-
graphic software package[ and refined anisotropically for non-
hydrogen atoms by full-matrix least-squares calculations. Each re-
finement was continued until all shifts were smaller than one-third
of the standard deviations of the parameters involved. Atomic scat-
at room temperature to give 2 as a green precipitate. The green
complex 2 was recrystallized from methanol/H O (0.362 g, 83.0%).
17ClCuN (436.3): calcd. C 38.54, H 3.93, N 12.84; found
2
C
14
H
4 6
O
26]
C 38.37, H 3.95, N 12.83.
I
[
0
Cu (Me
2
bpa)(CH
3
CN)]PF
6
(3): [Cu(CH
3
CN)
atmosphere, to a
bpa (0.114 g, 0.5 mmol) in methanol (5 mL). After
stirring at 60 °C for 10 min, the resultant solution was stored at
20 °C. The pale-yellow crystalline solid thus obtained was filtered
off, washed with dry diethyl ether, and dried under N (0.196 g,
2.2%). C16 20CuF P (476.9): calcd. C 40.29, H 4.24, N 11.75;
4
]PF
6
(0.167 g,
.5 mmol) was added as a solid, under an N
2
solution of Me
2
tering factors and anomalous dispersion terms were taken from the
[25]
literature.
All hydrogen atoms were located at their calculated
–
positions. The maximum and minimum peaks on the final differ-
2
–3
ence Fourier map corresponded to 0.73 and –0.52 eÅ , respec-
8
H
6 4
N
tively. The fundamental crystal data and experimental parameters
for structure determination are given in Table 3.
found C 40.09, H 4.23, N 11.89.
I
[
(
Cu (Me
2
bpa)(NO
2
)]
2
·[(Ph
3
P)
2
NPF
6
]
(4):
[Cu(CH
atmosphere,
bpa (0.114 g, 0.5 mmol) in acetone (3.5 mL).
(0.292 g, 0.5 mmol) was then added to the resultant
solution, under an N atmosphere. After stirring for 1 h 4 precipi-
tated as a yellow powder (0.301 g, 88.7%). For X-ray analysis, yel-
low crystals of 4 were obtained from CH Cl /acetone. C64
(1357.3): calcd. C 56.63, H 4.76, N 9.29; found C 56.64,
3 4 6
CN) ]PF
CCDC-251363 (for 1), -251364, (for 2) and -251365 (for 4) contain
the supplementary crystallographic data for this paper. These data
can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
0.167 g, 0.5 mmol) was added as a solid, under an N
to a solution of Me
(Ph P) N]NO
2
2
[
3
2
2
2
Electronic-Structure Calculations: Electronic-structure calculations
I
II
of the Cu and Cu complexes containing Me
2
bpa and nitrite were
2
2
2
H64Cu -
[
27]
carried out with the DFT method in the Gaussian 98 program.
6 9 4 3
F N O P
For all molecules, the geometries were fully optimized without any
H 4.90, N 9.41.
constraints. The three-parameter Becke–Lee–Yang–Parr (B3LYP)
Measurement of NO Generated from 3: A solution of 4 (10 mg,
functional was adopted as the exchange-correlation functional.[28]
7
.5 μmol) in CH
with a rubber septum. A degassed solution of CF
in CH Cl (0.05 mL) was then introduced with a syringe at room
2
Cl
2
(0.45 mL) was prepared in a small vial capped
The core electrons of Cu were replaced with the effective core po-
tential (ECP) of Hay and Wadt.[29] The basis set for Cu was the
3
COOH (6 μL)
split-valence set (LANL2DZ[21]) for the ECP. The Huzinaga–Dun-
2
2
temperature. The solution changed immediately from yellow to
blue. Analysis of the head-space gas by a GC analyzer indicated
that NO had been generated (7.3± 0.2 μmol).
ning (9s5p)/[4s2p] and (4s)/[2s] basis sets were used for the first row
elements and hydrogen, respectively.[30]
Supporting Information (see also footnote on the first page of this
article): Further figures illustrating the ESR spectra of 1 and 2, the
GC chart, and the Arrhenius–Eyring plot for the reaction of 4 with
proton are provided.
2 2
Kinetics: The kinetic studies of nitrite reduction of 4 in CH Cl
were carried out by monitoring the intensity decrease of the 320-
nm band. The absorbance was detected by a fiber-optics quartz
1440
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
Eur. J. Inorg. Chem. 2005, 1435–1441

I
II
Cu and Cu Complexes as Models for the Substrate-Binding Cu Site of Nitrite Reductase
FULL PAPER
Table 3. Crystal data for 1, 2, and 4
1
2
4
Formula
Formula mass
Color
Crystal size [nm]
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [°]
β [°]
C
507.80
blue
0.75×0.30×0.30
triclinic
P1
8.558(5)
8.650(7)
7.820(5)
113.18(5)
110.95(4)
71.86(5)
487.1(5)
1
14
H
19Cl
2
CuN
3
O
9
C
436.30
green
0.45×0.10×0.03
triclinic
P1
8.972(8)
12.395(9)
8.516(8)
93.13(7)
109.33(7)
77.97(6)
874.010010(1)
2
14
H
17ClCuN
4
O
6
C
64 2 6 9 4 3
H64Cu F N O P
1357.27
pale yellow
1.00×0.50×0.10
monoclinic
C2/c
30.72(1)
12.950(5)
15.596(6)
-
96.29(1)
–
¯
γ [°] 3
V [Å ]
6166.7(3)
4
1.462
Z
D
–3
calcd. [gcm ]
1.731
1.657
Radiation
μ [cm ]
Mo-K
14.51
259.00
θ–2θ
52.94
2220
2205
0.069
0.087
α
(λ = 0.71073 Å)
Mo-K
α
(λ = 0.71073 Å)
Mo-K
8.42
2800.00
ω–2θ
53.46
6690
4312
0.064
0.097
α
(λ = 0.7107 Å)
–1
14.435
446.00
θ–2θ
52.86
1566
1454
0.069
0.091
F
000
Scan method
2θmax [°]
No. reflections obsd.
No. reflections used
[
a]
R
R
[
b]
w
|)2/ΣwF
2 1/2
]
; w = 1/[σ (F
2
) + 0.03F
2
] for 1 and 2, w = 1/[σ (F
2
) + 0.005F
2] for 4.
[a] R = Σ||F
o
| – |F
c
||/Σ|F
o
|. [b] R
w
= [Σw(|F
o
| – |F
c
o
o
o
o
o
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2
[
We thank Prof. Susumu Kitagawa and Dr. Mitsuru Kondo, Kyoto
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ray crystallographic experiments. This work was support by
Grants-in-aid for Scientific research (B) (no.11440198 to S. S.) and
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Eur. J. Inorg. Chem. 2005, 1435–1441
www.eurjic.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1441