Single-turnover intermolecular reaction between a FeIII- superoxide-CuI cytochrome c oxidase model and exogeneous Tyr244 mimics

Article abstract of DOI:10.1039/b607277a

An Fe^III-superoxide-Cu^I cytochrome c oxidase model reacts intermolecularly with hindered phenols leading to phenoxyl radicals, as was observed in the enzyme and evidence for the formation of an Fe ^IV-oxo is presented. The

Full text of DOI:10.1039/b607277a

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III
Single-turnover intermolecular reaction between a Fe –superoxide–Cu
I
cytochrome c oxidase model and exogeneous Tyr244 mimics{
James P. Collman,* Richard A. Decr e´ au and Christopher J. Sunderland
Received (in Austin, TX, USA) 23rd May 2006, Accepted 24th June 2006
First published as an Advance Article on the web 9th August 2006
DOI: 10.1039/b607277a
III
I
An Fe –superoxide–Cu cytochrome c oxidase model reacts
intermolecularly with hindered phenols leading to phenoxyl
radicals, as was observed in the enzyme and evidence for the
We previously reported 1 to be a true biomimetic CcO model:
3 B
(a) not only it does reproduce the structural heme a –Cu motif
3
a
3b
Fig. 1), (b) but also it carries out reduction of O to H O, and
(
2 2
3c,d
III
I
IV
formation of an Fe –oxo is presented.
2
(c) it binds O in an Fe –superoxide–Cu fashion exactly as the
2
I
3e
2
enzyme, with Cu not binding to O , and not being oxidized.
3c
Cytochrome c oxidase (CcO) is a respiratory enzyme that catalyzes
The reaction of EPR-silent 1 with 3–12 equiv. of the phenols
the reduction of O
2
to H O without releasing highly-toxic, partially
2
2–H in dichloromethane under careful exclusion of O leads to two
2
1
reduced oxygen species (PROS). To carry out the 4e reduction, a
2
?
equiv. of phenoxyl radical 2 which displays an intense EPR signal
2
fully reduced enzyme has 6e available stored in five redox active
4a
at g = 2.0082 (Fig. 2). The yield of 2 was determined by
?
1
and a tyrosine Tyr244. The
centers: heme a, Cu
A
, heme a , Cu
3
B
comparing the integration of the EPR signal to a standard solution
catalytic cycle begins when dioxygen binds to the reduced CcO
of this phenoxyl radical generated by the reaction of the phenol
4b,c
II
Fea3 –Cu active site to form a heme–superoxide complex with no
I
III
with K Fe CN and quantified by iodometric titration. { Both
phenols 2a/b–H were chosen based on the stability of their
3
6
I
oxidation of Cu (Fea3
III 2?
I
2 2
–Cu ). Regardless of the sequence,
O
I
B
II
?
phenoxyl radicals 2 resulting in a slow decay of the EPR
4a–c
signals.
two more electrons are provided, one from Cu , leading to Cu
IV
,
B
and another one from heme a
3
, leading to iron LO, respectively.
However there is still controversy about the role of the tyrosine
The kinetics of this reaction studied by EPR, best fit in a pseudo
first-order rate law (Fig. 3A–C); the pseudo-first-order constant
(kobs) is proportional to the concentration of 2 (Fig. 3C). The
residue, illustrated by two different mechanisms proposed in the
2
literature. In the fully reduced mechanism, the 4th electron and a
proton come from heme a–CuA and proton transduction occurs
across the membrane, leaving the tyrosine unreacted. In contrast,
in the mixed-valence mechanism, the tyrosine provides both the
proton and the 4th electron, leading to the resonance Raman and
second-order rate constant k obtained from the slope was found
2
21
21
to be 0.012 M
min
for the reaction with 2a–H. This
kinetic behavior demonstrates that the reaction between 1
?
and 2 is a bimolecular process. The rate law is: d[2 ]/dt = k [2][1]
2
EPR signatures of the so-called P
M
intermediate: a ferryloxo, a
with kobs = k [2].
2
2
copper(II) and a tyrosyl radical. The present study is designed to
demonstrate spectroscopically the validity of this scenario by
showing that soluble hindered phenols such as 2–H/D can act as
an efficient source of a proton and an electron when reacting
intermolecularly with a dioxygen complex of CcO model 1 (Fig. 1).
H
A small kinetic isotope effect of k /kD = 2 was observed from
the reaction between 1 and 2–D.{ This result implies proton
III
Fig. 1 (A) Proposed intermolecular reaction between a Fe –superoxide–
I
Cu CcO complex model 1 and phenols (2–H/D). Details of this reaction
are shown in Scheme 1. (B) Structural comparison of 1 with the active site
of CcO.
Fig. 2 Spectroscopic evidence for a biomimetic reaction between an
Stanford University, Chemistry Department, Stauffer II, Stanford, CA-
94306, USA. E-mail: jpc@stanford.edu
{ Electronic supplementary information (ESI) available: Experimental
procedures, MS and UV spectra. See DOI: 10.1039/b607277a
III
Fe –superoxide–Cu CcO model (1) and phenols 2–H (3–12 equiv.)
I
?
leading to the phenoxyl radicals 2 : X-band EPR spectrum of phenoxyl
?
radical 2a (g = 2.0082).{
3
894 | Chem. Commun., 2006, 3894–3896
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Fig. 3 Intermolecular reaction between 1 and an excess of 2–H/D in
?
CH Cl
2
2
at room temperature. A. Concentration of 2 at different times.
Each dot is the mean of two sets of duplicates. B. Pseudo-first-order plots
?
?
?
Fig. 4 Ferryloxo models 7a and 7b.
based on the change of concentration of 2 : [2 ]
?
f
final concentration of 2 ,
?
?
[
2 ]
t
concentration of 2 at time t. C. Plots of kobs against [2 ].
prepared with a slight deficit of iodosylbenzene to ensure that 2
would primarily react with 7a. The ferryloxo nature of 7a was
established using methods previously reported for other ferryloxo
transfer occurs in the rate-determining step from the phenol 2–H
to the O -bound heme 1, consistent with formation of a
2
hydroperoxo species 5. Compound 5 has never been observed
8a–c,f
porphyrins lacking superstructures:
(a) spectroscopic investi-
spectroscopically in the enzyme, but was previously postulated
5
based on DFT calculations (Scheme 1). The rate of the reaction
1
gations carried out at low temperature showed dramatic H-NMR
paramagnetic upfield shifts, and 4–10 nm shifts for the Q bands in
the absorption spectrum, (b) the reaction of 7a/b with triphenyl-
phosphine at low temperature led to triphenylphosphine oxide,
whereas reaction with ammonium hydroxide led to a species
measured with 2b–H is three times slower than for 2a–H. This
result indicates that the weaker O–H bond strength of the
p-methoxy phenol 2a–H (bond dissociation energy (BDE) ca. 3
2
1
kcal mol smaller) is more reactive than p-alkylamide phenol
III
2b–H which is more acidic (by ca. 1 pK unit). The latter is closer
a
(presumably an Fe -only version of 6) that no longer oxidizes
to the values found for the pK of the cross-linked histidine–
a
triphenylphosphine.
4d–f
tyrosine models (6.6–8.4).
The yield of the phenoxyl radical implies that 2 electrons are
abstracted from the phenol pool. It is expected that the Cu would
II
be oxidized to Cu under these conditions accounting for the
The rate of this reaction is also influenced by the polarity of the
solvent: the reaction is 4 times slower in a non-polar dichloro-
methane–toluene mixture (1 : 2 vol.) than in a polar dichlor-
omethane–acetonitrile mixture (1 : 2 vol.) that is more relevant to
additional electron. The reaction product 6 is presumably
II
magnetically coupled, as no Cu signal was observed in the EPR
physiological conditions (dielectric constants (e25uC) for: water:
6
spectrum of the mixture at the end of the reaction.
78.5; acetonitrile: 36.6; dichloromethane: 9.1; toluene: 2.3).
Primary evidence for the formation of an oxoferryl species 4
comes from the formation of triphenylphosphine oxide (tppo)
following treatment of the mixture containing the putative species
I
The expected intramolecular reaction of a hydroperoxo–Cu
species 5 leads to rupture of the O–O bond and subsequent
formation of 4. This intramolecular reaction is expected to be
faster than another intermolecular reaction between 5 and 2–H,
especially when the concentration of 2–H is low. The reduction of
the ferryloxo species 4 by one molecule of phenol 2–H is
reminiscent of the reactivity of the peroxidase non radical
?
4 with triphenylphosphine (tpp, after ca. 1 equiv. of 2 was
formed). The mixture was analyzed by TLC and GC against
authentic standards.{ High yields of tppo were obtained at 240 uC
or even at room temperature, although greater stability of the
8a–d,f,g
ferryloxo species is expected at low temperature.
unambiguous evidence of an oxygen-atom transfer from 4 to
This result is
7
compound-II species with phenols. Such a reaction would lead
8f
to a ferric-hydroxo species 6 and is favored thermodynamically.
?
Evidence of such a reaction is given by the formation of 2 from
triphenylphosphine.
A series of careful control reactions
conducted at room temperature between 1 and triphenylphosphine
showed that no tppo is formed. Overall the yield in tppo is
satisfactory, given the possible competition between this inter-
molecular oxidation reaction and another intermolecular reduction
of 4 by 2–H as well as the low stability expected for 4 at room
temperature.
the reaction of 2–H with in situ generated ferryloxo porphyrin
species such as model 7a and ferryloxo tetrakismesitylporphyrin
8a–c
TMP) 7b
(
(Fig. 4). The ferryloxo 7a was synthesized at ca.
2
60 uC (acetonitrile–acetone–dry ice bath) by adaptation of a
8d
method described for simple porphyrins, oxygen-atom transfer
from a soluble iodosylbenzene (1-iodosyl-2-(2-methyl-propane-2-
Further evidence for the formation of an oxoferryl species 4
comes from mass spectrometry.{ A nanospray infusion technique
using a low ionization potential at room temperature was
employed to analyze a cold mixture resulting from the reaction
between 1 and 2–H leading to 4 (240 to 278 uC, by injection from
a pre-cooled syringe). The spectrum was carefully examined taking
8e
sulfonyl)-benzene) to a deoxy-Fe-only precursor form of 1. It was
into account the charges of the species resulting from the loss of
II
the PF counterion or the hydroxyl Cu species. The major peak
6
had an isotope distribution of peaks centered at m/z = 1661.43.
This pattern is consistent with the theoretical spectrum of a singly
IV
charged potassium chloride adduct of a Fe LO/Cu form of 4, that
may result from the loss of HO from Cu followed by Cl insertion
III
I
Scheme 1 Intermolecular reaction between Fe –superoxide–Cu (1) and
phenols (2–H/D). Proposed mechanism and intermediates formed.
during ionization.
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Chem. Commun., 2006, 3894–3896 | 3895

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Nanospray Infusion Experiments. Jungjoo Yoon and Pr Edward
I. Solomon for preliminary resonance Raman studies.
Notes and references
1
2
S. Ferguson-Miller and G. T. Babcock, Chem. Rev., 1996, 96, 2889.
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Acad. Sci. USA, 1998, 95, 8020; (b) F. MacMillan, A. Kant, J. Behr,
T. Prisner and H. Michel, Biochemistry, 1999, 39, 9179; (c)
D. A. Proshlyakov, M. A. Pressler, C. DeMaso, J. F. Leykam,
D. L. DeWitt and G. T. Babcock, Science, 2000, 290, 1588.
3
(a) J. P. Collman, C. J. Sunderland and R. Boulatov, Inorg. Chem., 2002,
41, 2282; (b) R. Boulatov, J. P. Collman, I. M. Shiryaeva and
C. J. Sunderland, J. Am. Chem. Soc., 2002, 124, 11923; (c)
J. P. Collman, C. J. Sunderland, K. Berg, M. A. Vance and
E. I. Solomon, J. Am. Chem. Soc., 2003, 125, 6648; (d) J. P. Collman,
K. E. Berg, C. J. Sunderland, A. Aukauloo, M. A. Vance and
E. I. Solomon, Inorg. Chem., 2002, 41, 6583; (e) Compound 1 is best
described as a heme–superoxide complex in which the distal Cu does not
participate in covalent bonding to the coordinated oxygen, and it is
reasonably stable at room temperature. As such it is a uniquely stable
analog of the initial heme-bound dioxygen intermediate of CcO. This
III
I
Fig. 5 UV-Vis absorption of Fe –superoxide–Cu (1) before reaction
with phenol 2a (dotted line) and after (dark line). Inset: magnification of
the Q bands.
N
is unlike other CcO and other electron rich Cu(L ) complexes, where
3f–j
Cu–dioxygen complexes are typical. The Cu does not directly react
with oxygen: the Zn–CuMP complex appears to be air stable and low
temperature (250 uC, 5% MeCN–THF) NMR spectra are unchanged
2 2
upon exchange of a N head gas with 1 atm O ; (f) J.-G. Liu, Y. Naruta,
F. Tani, T. Chishiro and Y. Tachi, Chem. Commun., 2004, 120; (g)
J.-G. Liu, Y. Naruta and F. Tani, Angew. Chem., Int. Ed., 2005, 44, 1836;
The intermolecular reaction between 1 and 2–H was also
monitored by UV-vis spectroscopy (Fig. 5) and analyzed in light of
3c
(
h) E. Kim, J. Shearer, S. Lu, P. Mo e¨ nne-Loccoz, M. E. Helton,
S. Kaderli, A. D. Zuberb u¨ hler and K. D. Karlin, J. Am. Chem. Soc.,
004, 126, 12716; (i) E. Kim, K. Kamaraj, B. Galliker, N. D. Rubie,
the absorption spectra of 1 and of an authentic phenoxyl radical
?
2
generated in situ.{ Of the four bands characteristic of phenoxyl
2
?
radical 2 (beside the absorption of the parent molecule 2–H at
P. Moenne-Loccoz, S. Kaderli, A. D. Zuberbuhler and K. D. Karlin,
Inorg. Chem., 2005, 44, 1238; (j) E. Kim, M. E. Helton, S. Lu, P. Moenne-
Loccoz, C. D. Incarvito, A. L. Rheingold, S. Kaderli, A. D. Zuberbuhler
and K. D. Karlin, Inorg. Chem., 2005, 44, 7014.
(a) M. Lucarini, V. Mugnaini, G. F. Pedulli and M. Guerra, J. Am.
Chem. Soc., 2003, 125, 8318; (b) J. T. Lai, Tetrahedron Lett., 2001, 42,
557; (c) E. M u¨ ller and K. Ley, Chem. Ber., 1955, 88, 601; (d)
F. G. Bordwell and X.-M. Zhang, J. Phys. Org. Chem., 1995, 8, 529;
(e) J. P. Collman, Z. Wang, M. Zhong and L. Zeng, J. Chem. Soc.,
Perkin Trans. 1, 2000, 1217; (f) J. A. Cappuccio, I. Ayala, G. I. Eliott,
I. Szundi, J. Lewis, J. P. Konopelski, B. A. Barry and O. Einarsdottir,
J. Am. Chem. Soc., 2002, 124, 1750.
5 M. R. A. Blomberg, P. E. M. Siegbahn, G. T. Babcock and
M. Wikstr o¨ m, J. Am. Chem. Soc., 2000, 122, 12848.
Handbook Chemistry and Physics, ed. R. C. Weast, CRC Press,
Cleaveland, Ohio, 1977, E-44.
7 J. E. Critchlow and H. B. Dunford, J. Biol. Med., 1972, 247, 3703.
8 (a) D.-H. Chin, A. L. Balch and G. N. La Mar, J. Am. Chem. Soc., 1980,
3
00 nm), only the most intense absorption band that gradually
3
21
21
appears at 339 nm (e = 3.8 6 10 M cm ) could be used to
correlate the UV-vis data with the kinetic data from EPR. The
absorptions at 388 nm and 405 nm were barely visible at the end of
the reaction as shoulders on the Soret band. The broad band
4
?
21
centered at 540 nm in 2 and typical of phenoxyl radicals was too
21
weak (e = 120 M cm ) to be distinguished from the porphyrin
Q bands. Moreover, significant shifts in the Q bands were
observed during the reaction.
III
I
This study demonstrates that 1, an iron –superoxo–copper
2
biomimetic model of the oxy intermediate observed in CcO, reacts
6
with an exogeneous soluble Tyr244 mimic 2 to generate a phenoxyl
2
radical 2 as occurs in the enzyme. However unlike the tyrosyl
?
radical in the enzyme, the phenoxyl radical formed (2 ) is well
?
1
02, 1446; (b) A. L. Balch, Y.-W. Chan, R.-J. Cheng, G. N. La Mar,
L. Latos-Grazynski and M. W. Renner, J. Am. Chem. Soc., 1984, 106,
779; (c) A. L. Balch, G. N. La Mar, L. Latos-Grazynski, M. W. Renner
resolved and does not couple nor interfere with the neighboring
II
Cu species. Strong evidence is provided regarding the formation
7
of an hydroperoxo-type intermediate 5 and of a heme ferryloxo
product 4. Thorough mechanistic studies of this intermolecular
reaction and complete characterization of the ferryloxo species 4
and 7a (resonance Raman) are in progress and should pave the
way to future studies of a similar intramolecular reaction in more
and V. Thanabai, J. Am. Chem. Soc., 1985, 107, 3003; (d) J. T. Groves,
R. C. Haushalter, M. Nakamura, T. E. Nemo and B. J. Evans, J. Am.
Chem. Soc., 1981, 103, 2884; (e) J. P. Collman, L. Zeng, H. J. H. Wang,
A. Lei and J. I. Brauman, Eur. J. Org. Chem., 2006, 2707; (f) D.-H. Chin,
G. N. La Mar and A. L. Balch, J. Am. Chem. Soc., 1980, 102, 5945; (g)
R. A. Ghiladi, R. M. Kretzer, I. Guzei, A. L. Rheingold, Y.-M. Neuhold,
K. R. Hatwell, A. D. Zuberbuller and K. D. Karlin, Inorg. Chem., 2001,
9
advanced CcO models having a covalently attached phenol.
4
0, 5754.
(a) J. P. Collman, R. A. Decr e´ au and S. Costanzo, Org. Lett., 2004, 6,
033; (b) J. P. Collman, R. A. Decr e´ au and C. Zhang, J. Org. Chem.,
2004, 69, 3546.
RAD is thankful for a Lavoisier Fellowship. Fruitful discussion
with Neal K Devaraj and Dr Xavier Ottenwaelder. Dr Allis Chien
Head of the Stanford University Mass Spectrometry (SUMS) for
9
1
3
896 | Chem. Commun., 2006, 3894–3896
This journal is ß The Royal Society of Chemistry 2006