Spectroscopic evidence for a heme-superoxide/Cu(I) intermediate in a functional model of cytochrome c oxidase

Article abstract of DOI:10.1021/ja034382v

A superstructured tetraphenylporphyrin with a covalently attached proximal imidazole axial base and three distal imidazole pickets has been developed as a model for the active site of terminal oxidases such as cytochrome c oxidase. The oxygen adduct of the Fe-only heme (at low temperature) has a diamagnetic NMR and is EPR silent, which taken together with a resonance Raman oxygen isotope sensitive band (νFe-O) at 575/554 cm^-1 (¹⁶O₂/¹⁸O₂) indicates formation of a six-coordinate heme-superoxide complex. Unexpectedly, the Fe/Cu complex, where the copper is in a trisimidazole environment ～5 A above the heme plane, displays similar characteristics: a diamagnetic NMR, EPR silence, and νFe-O at 570/544 cm^-1. This indicates the dioxygen adduct of this Fe/Cu system is also a superoxide. This contrasts with previously characterized partially reduced dioxygen intermediates of binuclear heme/copper complexes that form Fe/Cu μ-peroxo complexes. Copyright

Full text of DOI:10.1021/ja034382v

Published on Web 05/10/2003
Spectroscopic Evidence for a Heme-Superoxide/Cu(I) Intermediate in a
Functional Model of Cytochrome c Oxidase
James P. Collman,* Christopher J. Sunderland, Katja E. Berg, Michael A. Vance, and
Edward I. Solomon
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080
Received January 28, 2003; E-mail: jpc@stanford.edu
Chart 1. Structural Comparison of the Active Site of CcO with the
Model Complex Fe/Cu[NMePr]+ a
Cytochrome c oxidase (CcO) is an example of a heme/copper
oxidase that catalyzes the 4e reduction of molecular oxygen to
water, a pivotal reaction in aerobic respiration. The catalytic cycle
II
is initiated when dioxygen binds to the reduced active site (Fea3
/
/
I
•-
CuB ) of CcO to form a heme-superoxide complex (Fea3IIIO2
I
CuB ).1 No bioinorganic complex has yet replicated this motif of a
heme-superoxide held in proximity to a copper(I) center. Com-
monly, the reaction of dioxygen with heterobinuclear heme/Cu
model complexes is an Fe-O2-Cu (µ-peroxo) product.2-8 Although
once proposed to be part of the catalytic cycle of CcO, spectroscopic
evidence no longer supports µ-peroxo intermediates in CcO.9,10
Thus, existing biomimetic systems may be inappropriate models
for elucidating mechanistic details of dioxygen reduction by CcO.
Herein we report spectroscopic evidence for the first heterobinuclear
biomimetic heme/Cu oxidase model forming a heme-superoxide/
CuI moiety. These preliminary studies indicate that contrary to other
CcO models, reduced heme/Cu centers placed at a distance and in
a ligation sphere similar to that in CcO need not form peroxide-
bridged complexes. Indeed, a distal metal ion can lead to stabiliza-
tion of a heme dioxygen complex relative to the Fe-only porphyrin.
This suggests CuB may also have nonredox roles in O2 binding
and reduction.
1
a Numbers refer to H resonances in Figure 1.
Fe[NMePr] and Fe/Cu[NMePr]+ were synthesized as previously
reported.11 A combination of 1H and 19F NMR, EPR, and IR
spectroscopies indicates that the representation in Chart 1 is a
reasonable depiction of the structure in solution, with the Fe
coordinated by the proximal imidazole, the distal tris-imidazole
N-donors directed toward the molecular cavity, and the copper of
Fe/Cu[NMePr]+ in a tris-imidazole environment in both the CuI
and CuII oxidation states.11
Spectrophotometric titration of the Fe/Cu complex with O2
indicates a 1:1 [Fe/CuNMePr]+:O2 binding stoichiometry. Forma-
tion of the Fe/Cu‚O2 adduct is rapid (within the cuvette mixing
time) and is marked by a shift in the Soret band at 424 nm to 422
nm which also diminishes in intensity (ꢀ 1.8 × 105 to 1.0 × 105
M-1 cm-1), while the Q-band at 538 nm shifts to 542 nm with a
small increase in intensity.
Exposure of Fe/Cu[NMePr]+ (in 5% d3-MeCN:d8-THF) to
dioxygen rapidly (<15 s) leads to a single major diamagnetic
species as evidenced by the 1H NMR spectroscopy (Figure 1). The
sample is EPR silent. All resonances were assigned by H-H COSY
and NOE experiments and support the proposition that the oxygen-
ated adduct possesses the expected mirror-plane symmetry on the
NMR time scale. The minor impurity observed in the NMR spectra
of Fe/Cu[NMePr]+‚O2 is assigned as the CO adduct, FeCO/Cu-
[NMePr]+.11 Since the affinity of Fe/Cu[NMePr]+ for CO is very
high (essentially irreversible), trace CO adduct formation can be
attributed to contamination of the Fe/Cu system during synthesis.
Indeed, this minor impurity is observed in the NMR spectrum of
the Fe/Cu system prior to oxygenation. Oxygenation was also
Figure 1. NMR spectra of Fe/Cu[NMePr]+ in 5% d3-MeCN/d8-THF before
(upper) and after (lower) exposure to 1 atm O2 at 22 °C. Full assignments
are in Supporting Information and ref 11.
followed by 19F NMR spectroscopy, as the CF3 marker resonance
in Fe/Cu[NMePr]+ (δCF3 -63.4 ppm) shifts upon O2 addition to
-65.8 ppm which upon slow decomposition (hours), becomes a
broad signal at -65.1 ppm.
Typically, diamagnetism of oxygenated heme/Cu complexes is
attributed to formation of CuII antiferromagnetically coupled to the
2-
FeIII center via a bridging O2 unit.2,6 Our experience with the
Co-porphyrins of this ligand,12 led us to believe that an alternate
O2 adduct, a picket-bound CuI above the (magnetically coupled)
FeIIIO2 superoxide may be a viable formulation.
-
Resonance Raman spectroscopy (1 mM in CH2Cl2, λex 407 nm,
20 mW) failed to detect an oxygen isotope sensitive band in the
(Por)M-O-O-M′(L) νO-O region (∼800 cm-1).13 However, an
oxygen isotope sensitive band was observed at 570/544 cm-1 (16O2/
18O2) (Figure 2). Oxymyoglobin,14 oxy-picket-fence porphyrin15
(Im[TpivPP]FeO2) and oxy-CcO,16 all well-characterized examples
of heme-superoxide complexes, manifest Fe-16O2 stretches at 569,
572, and 571 cm-1 respectively.17 More importantly, upon oxy-
genation of the copper-free complex Fe[NMePr], an oxygen isotope
sensitive band appears at 575/554 cm-1, indicating formation of
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10.1021/ja034382v CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
NMR characterization, whereas FeO2/Cu[NMePr]+ has a lifetime
of several hours.
Previous studies have demonstrated that these heme/Cu catalysts
are functional (electrocatalytically reducing O2 to H2O under
physiologically relevant conditions, with minimal leakage of
partially reduced oxygen species).18 Those studies did not find a
direct role for Cu in O-O bond activation. We now have
spectroscopic evidence that the Fe-O2 and CuI centers of FeO2/
Cu[NMePr] are not covalently interacting, in contrast to the usual
finding that binuclear heme/Cu complexes form FeIII/CuII µ-peroxo
complexes.2-8 Few dioxygen complexes of heme/Cu CcO models
are stable at room temperature, especially when coordinated with
an axial nitrogen base. These results indicate that stabilization of
partially reduced dioxygen intermediates of a heme/Cu complex
does not require µ-peroxo formation. These observations of
nonredox contributions from copper to the thermodynamic and
decompositional stabilization of a heme/Cu dioxygen adduct may
be insights into some of the reactivity-tuning roles of CuBI early in
the catalytic cycle of CcO. We are currently investigating the
stereoelectronic characteristics of Fe/Cu[NMePr]+ that leads it to
so closely mimic the reactivity of the Fe/Cu site of CcO with respect
to its dioxygen binding and reduction chemistry.
Figure 2. Resonance Raman (22 °C) of Fe/Cu[NMePr]+ after oygenation,
with 16O2 or 18O2 and the difference spectra.
the expected heme-superoxide. The νFe-O value of oxy-Fe/Cu-
[NMePr]+ is therefore consistent with O2 as a superoxide ligand
in this metalloporphyrin.
Further evidence congruent with the FeO2/Cu[NMePr]+ (super-
oxide) formulation evolves from a consideration of the correspond-
ing Co porphyrin analogues, where superoxides are known to be
more stable and resonance enhancement of both M-O and O-O
occurs.12 In CoO2/Cu[NMePr]+, νO-O and νCo-O are observable at
1148/1073 cm-1 (16O2/18O2) and 528/504 cm-1 (16O2/18O2), respec-
tively.12 The νO-O frequency is characteristic of a cobalt-superoxide,
as is the νCo-O value. The Co-O stretch is ∼46 cm-1 lower than
in FeO2/Cu[NMePr]+, typical for Co vs Fe (Por)M-superoxide νM-O
stretches (cf νFe-O/νCo-O, 568/516 cm-1, for (Im)M[TpivPP]O2).
Thus, we conclude that the dioxygen adduct formed with this
heme/Cu system is an imidazole ligated (six-coordinate) heme-
superoxide, FeO2/Cu[NMePr]+, an analogue for oxy-CcO.
FeO2/Cu[NMePr]+ is stable for several hours at room temper-
ature. The oxygenated complex does not revert to deoxy Fe/Cu-
[NMePr]+ under vacuum (three freeze/pump/thaw cycles), although
exposure to a head-gas of CO rapidly produces FeCO/Cu[NMePr]+.
This indicates FeO2/Cu[NMePr]+ is a thermodynamically stable
but kinetically labile O2 adduct.
Acknowledgment. This material is based upon work supported
by the NSF under Grant No. CHE-013206. Support from NIH Grant
No. 017880 also contributed to this work.
Supporting Information Available: Further experimental data
including NMR and Raman data (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
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Without distal copper, Fe[NMePr] behaves as expected for an
imidazole-ligated heme. In coordinating solvents the complex is a
six-coordinate and low-spin complex (λmax 428 nm, δpyrr: 8.5-8.6
ppm). By contrast to the Fe/Cu complex, Fe[NMePr] does not form
an observable (by NMR) dioxygen complex at room temperature
(1 atm O2), although binding can be driven to completion at low
temperature (-60 °C). In coordinating solvents Fe[NMePr] binds
oxygen reversibly: The low-temperature oxy- species reverts to
deoxy-Fe[NMePr] by warming to room temperature or upon
removal (at -60 °C) of the O2 head-gas.
Clearly there is qualitative difference in the dioxygen affinity
of these complexes: FeO2/Cu[NMePr]+ is stable at room temper-
ature under vacuum, Fe[NMePr]O2 dissociates without an O2 head-
gas and requires low temperature for complete binding. Thus, it is
apparent that CuI in the distal site strongly enhances dioxygen
binding to iron, paralleling behavior recently described for the Co-
porphyrins of this ligand system.12 In the Co analogues, H-bonding
from the terminal oxygen of the bound superoxide to the picket
amide NH’s was found to be stronger in Co/Cu[NMePr]+ than in
Co[NMePr], and a similar contribution may also be in effect for
Fe/Cu[NMePr]+. The Fe/Cu superoxide is also more stable to
degradation than is the Fe-only superoxide. For example, at room
temperature in CD2Cl2, Fe[NMePr]O2 decomposes too rapidly for
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