Formation and spectroscopic characterization of the dioxygen adduct of a heme–Cu complex possessing a cross-linked tyrosine–histidine mimic: modeling the active site of cytochrome c oxidase

Abstract

A binucleating porphyrin with covalently appended copper chelates having a cross-linked imidazole–phenol group as the novel active site model of cytochrome c oxidase has been prepared, and the dioxygen adduct of its iron(II)–copper(I) complex was spectroscopically characterized.

---

Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, catalyzes the 4e/4H⁺ reduction of dioxygen to water without generating toxic reactive intermediates, conserving the released energy for the synthesis of ATP.¹ The active site of O₂ reduction is comprised of heme a₃/Cu_B binuclear moiety in which one of the copper-bound histidines is covalently cross-linked to a tyrosine residue between the C6 of Tyr244 and the ε-nitrogen of His 240 (in the bovine enzyme sequence).² This unprecedented Tyr-His cross-link is proposed either to function as an electron and a proton donor to the dioxygen bound to heme a₃ or to fix Cu_B in a certain configuration and distance from heme a₃ during the catalytic O₂ reduction.³ A number of heme-based dinuclear Fe–Cu complexes have been reported as model compounds in the hope of unraveling the mechanism of O₂ reduction in the active site of CcO.⁴ However, the reported heme-based models are devoid of the Tyr-His cross-linkage. There are a few recent reports⁵ about the syntheses and physicochemical investigations of cross-linked phenol–imidazoles. Very recently, Karlin and co-workers⁶ reported copper complexes with imidazole–phenol cross-links as an initial synthetic model for the Cu_B site in CcO. Herein we report the first example of constructing a heme-containing model with covalently appended copper chelates having a cross-linked imidazole–phenol group as a novel CcO model compound.

The synthetic routes to the desired compounds are shown in Scheme 1.‡ The aldehyde 3 is prepared firstly from the coupling of methoxymethyl (MOM)-protected 2-hydroxyphenylboronic acid (1) with 4(5)-(tert-butyldiphenylsilanyloxymethyl)-1H-imidazole (2),⁷ followed by removing the silyl protecting group and then oxidized by activated MnO₂. It is noteworthy that none of the desired product is obtained when 1H-imidazole-4-carbaldehyde is employed for the coupling. The MOM-protected phenylboronic acid (1) can be obtained in 65% overall yield by sequential reactions involving metalation (n-BuLi/ether/−70 °C) of MOM-protected 2-bromo-4-methylphenol, and treatment with B(OMe)₃, followed by acidic work-up. Treatment of 3 with 2-aminomethylimidazole in methanol generates the corresponding Schiff base intermediate, which is consequently reduced by NaBH₄in situ to give 4 in a yield of 65%. The tripodal ligand 5 is isolated by reacting the amine 4 with methyl 6-chloromethylnicotinate in the presence of K₂CO₃ in CH₃CN, and then hydrolyzing in a KOH solution. The prepared tripodal ligand is an important building block in assembling CcO active site models. The condensation reaction between 5 and the porphyrin 6 (2-[10,15,20-tris-(2,4,6-trimethyl-phenyl)-porphyrin-5-yl]-phenylamine) is performed in the presence of Et₃N/2-chloromethylpyridinium iodide in CH₂Cl₂ to give the covalent conjugate L^OMOM (7) in 63% yield. Finally, the MOM group is removed with bromotrimethylsilane in CH₂Cl₂ at − 30 °C to regenerate the phenolic hydroxyl group and the hydroxyl free ligand L^OH (8)‡ is obtained in a moderate yield (60%).

Scheme 1 Reagents and conditions: (i) cat. [Cu(OH)TMEDA]₂Cl₂, CH₂Cl₂, O₂, rt, 75%; (ii) n-BuN⁺F⁻, THF, rt, 93%; (iii) MnO₂, CHCl₃, reflux, 85%; (iv) a, 2-aminomethylimidazole, Et₃N, MeOH, b, NaBH₄, 65%; (v) methyl 6-chloromethylnicotinate, K₂CO₃, CH₃CN, rt, 61%; (vi) KOH, THF, rt, 86%; (vii) 2-chloromethylpyridinium iodide, 5, Et₃N, CH₂Cl₂, rt, 63%; (viii) Me₃SiBr, CH₂Cl₂, − 30 °C, 60%.

Stepwise metalation of the porphyrins, 7 and 8, begins with addition of excess FeBr₂ in THF at reflux, followed by extraction with an aqueous Na₂EDTA solution, yielding the corresponding mononuclear Fe^II porphyrins, L^OMOMFe^II and L^OHFe^II, respectively. Addition of copper salt [Cu(CH₃CN)₄]⁺ CF₃SO₃⁻ gives the desired Fe^II/Cu^I complexes with similar UV–vis spectra to those of their mononuclear Fe^II complexes, [L^OMOMFe^IICu^I]⁺ (9), ESI–MS m/z = 1330.5 (M⁺); [L^OHFe^IICu^I]⁺ (10), ESI–MS m/z = 1286.5 (M⁺).

Both 9 and 10 react with O₂ at − 30 °C in CH₃CN to give the dioxygen adducts [L^OMOMFe^III–O₂–Cu^II]⁺ (11) and [L^OHFe^III–O₂–Cu^II]⁺ (12), respectively. The formation of the corresponding peroxo species was evidenced by the following observations: (1) upon exposure of the reduced form 9 or 10 to O₂, its UV–vis spectra show distinctive changes with clear isosbestic points. The Soret band shifts from 429 nm to 421 nm, and the Q-band at 533 nm disappears (Fig. 1), which indicates the formation of a dioxygen adduct as described for those of our previously isolated peroxo-bridged Fe–O₂–Cu species.⁸ (2) ESI mass spectra of the dioxygen adducts show a distribution of peaks centered at 1362.5 (M⁺) for 11, and 1318.5 (M⁺) for 12. The observed isotope distribution of peaks agrees very well with the simulated pattern based on the ratio of Fe^II–Cu^I : O₂ = 1 : 1. The expected increase in mass of 4 is observed when 11 (m/z, M⁺, 1366.5) or 12 (m/z, M⁺, 1322.5) forms from ¹⁸O₂. (3) The resonance Raman spectra of 11 shows an isotope dependent peak at 801 cm⁻¹ which shifts to 755 cm⁻¹ with ¹⁸O-labeled dioxygen, and 12 displays a similar isotope sensitive band at 799 (¹⁶O₂) and 752 cm⁻¹ (¹⁸O₂), respectively (Fig. 2). The observed isotopic shifts are in good agreement with the value calculated from the harmonic oscillator approximation of the O–O stretching vibration [Δ_calcd(¹⁶O₂/¹⁸O₂) = 46 cm⁻¹]. These observed ν(O–O) values are similar to those of previous reported dioxygen adducts in the peroxy state.^4,8 (4) Both 11 and 12 are EPR silent in a frozen solution (CH₃CN, 77K), which indicates the presence of the strong antiferromagnetic coupling between the two metals.

Fig. 1 UV–visible spectral changes of 10, [L^OHFe^IICu^I]⁺, to 12 upon exposure to dioxygen in CH₃CN at −30 °C.

Fig. 2 Resonance Raman spectra of 12 formed from ¹⁶O₂ (A) and ¹⁸O₂ (B). The difference spectra A minus B is shown as trace C (3% toluene in CH₃CN, −30 °C, 413 nm excitation).

The formed peroxo species are stable at − 30 °C in CH₃CN, and on warming of the solution to room temperature (after removal of excess O₂in vacuo), the dioxygen adducts [L^OMOMFe^III–O₂–Cu^II]⁺ (11) and [L^OHFe^III–O₂–Cu^II]⁺ (12) exhibit interesting differences. For 11, the major decomposed product is the μ-oxo complex formulated as [L^OMOMFe^III–O–Cu^II]⁺ [m/z, 1346.6 (M⁺)] with UV–vis features [λ_max = 440 nm (Soret)] similar to the reported μ-oxo analogues.^8,9 By contrast, no μ-oxo final species is observed for 12. The final decomposed product demonstrates features like that of the hydroxo ferric porphyrin derivatives.¹⁰ The EPR spectrum (MeCN, 77 K) of the product shows signals at g = 5.56 and 1.99 corresponding to a high spin iron(III) porphyrin, and signals at g_∥ = 2.23 and g_⊥ = 2.06, which are assigned to a S = 1/2 Cu(II) ion in a tetragonal field.¹¹ We tentatively formulate the product as [L^OHFe^III–OH, Cu^II]²⁺. The decomposition mechanism and further product characterization are in progress.

In summary, a novel heme-based binucleating ligand incorporated with N-(2′-hydroxyphenyl)imidazole moiety as a CcO's Cu_B site mimic has been designed and successfully prepared. The oxygenation reaction with its iron(II)–copper(I) complex has been preliminarily investigated by various spectroscopic methods.

This work was financially supported by the Grant-in-Aid for COE Research (#08CE2005) and for Scientific Research on Priority Areas (#09235225 and 11228207) from MEXT and for Scientific Research (A) (#14204073) from JSPS. P&P project, Green Chemistry, of Kyushu University partly supported this research. J.-G. Liu gratefully acknowledges JSPS for providing postdoctoral fellowship.

Notes and references

1. S. Ferguson-Miller and G. T. Babcock, Chem. Rev., 1996, 96, 2889; G. T. Babcock, Proc. Natl. Acad. Sci. USA, 1999, 96, 12971.
2. S. Yoshikawa, K. Shinzawa-Itoh, R. Nakashima, R. Yaono, E. Yamashita, N. Inoue, M. Yao, M. J. Fei, C. P. Libeu, T. Mizushima, H. Yamaguchi, T. Tomizaki and T. Tsukihara, Science, 1998, 280, 1723; C. Ostermeier, A. Harrenga, U. Ermler and H. Michel, Proc. Natl. Acad. Sci. USA, 1997, 94, 10547.
3. D. A. Proshlyakov, M. A. Pressler, C. DeMaso, J. F. Leykam, D. L. DeWitt and G. T. Babcock, Science, 2000, 290, 1588; E. Pinakoulaki, U. Pfitzner, B. Ludwig and C. Varotsis, J. Biol. Chem., 2002, 277, 13563.
4. J. P. Collman, R. Boulatov and C. J. Sunderland, in The Porphyrin Handbook, eds. K. M. Kadish, K. M. Smith and R. Guilard, Academic Press, San Diego, 2003, Vol. 11, pp. 1–49 and references cited therein..
5. J. P. Collman, Z. Wang, M. Zhong and L. Zeng, J. Chem. Soc., Perkin Trans. 1, 2000, 1217; K. M. McCauley, J. M. Vrtis, J. Dupont and W. A. van der Donk, J. Am. Chem. Soc., 2000, 122, 2403; J. A. Cappuccio, I. Ayala, G. I. Elliott, I. Szundi, J. Lewis, J. P. Konopelski, B. A. Barry and O. Einarsdottir, J. Am. Chem. Soc., 2002, 124, 1750; M. Aki, T. Ogura, Y. Naruta, T. H. Le, T. Sato and T. Kitagawa, J. Phys. Chem. A, 2002, 106, 3436.
6. K. Kamaraj, E. Kim, B. Galliker, L. N. Zakharov, A. L. Rheingold, A. D. Zuberbuhler and K. D. Karlin, J. Am. Chem. Soc., 2003, 125, 6028.
7. J. P. Collman and M. Zhong, Org. Lett., 2000, 2, 1233 . The coupling reaction affords two isomers, N-1 and N-3 aryl products, which can be readily separated by silica-gel column chromatography in 75% (N-1) and 7% (N-3) yields, respectively..
8. T. Chishiro, Y. Shimazaki, F. Tani, Y. Tachi, Y. Naruta, S. Karasawa, S. Hayami and Y. Maeda, Angew. Chem., Int. Ed., 2003, 42, 2788; Y. Naruta, T. Sasaki, F. Tani, Y. Tachi, N. Kawato and N. Nakamura, J. Inorg. Biochem., 2001, 83, 239.
9. E. Kim, M. E. Helton, I. M. Wasser, K. D. Karlin, S. Lu, H.-W. Huang, P. Moenne-Loccoz, C. D. Incarvito, A. L. Rheingold, M. Honecker, S. Kaderli and A. D. Zuberbuhler, Proc. Natl. Acad. Sci. USA, 2003, 100, 3623; M.-A. Kopf and K. D. Karlin, Inorg. Chem., 1999, 38, 4922.
10. Data for the decomposed product [L^OHFe^III–OH, Cu^II]²⁺: UV–vis (CH₃CN) λ_max = 418, 560 nm. ESI–MS m/z = 651.9 (M²⁺)..
11. F. Tani, Y. Matsumoto, Y. Tachi, T. Sasaki and Y. Naruta, Chem. Commun., 1998, 1731; B. Andrioletti, D. Richard and B. Boitrel, New J. Chem., 1999, 23, 1143 . As comparison, the EPR spectrum of the dinuclear complex [L^OHFe^III–Cu^II]OTf₃ shows signals at g = 5.45 and 2.01, g_∥ = 2.24 and g_⊥ = 2.09, respectively..

---

Footnotes

† Electronic supplementary information (ESI) available: experiment procedures for preparing metal porphyrins and oxygenation reaction. See http://www.rsc.org/suppdata/cc/b3/b311538k/

‡ Synthetic details will be reported elsewhere. All new compounds were fully characterized by spectroscopic methods. Stated yields refer to isolated compounds and the purity was guaranteed by chromatography. Data for L^OH (8), ¹H NMR (400 MHz, CDCl₃) δ 8.84 (d, J = 8.0, 1 H), 8.75 (d, J = 4.8, 2 H), 8.67 (d, J = 4.4, 2 H), 8.63 (d, J = 3.6, 4 H), 8.03 (d, J = 6.0, 1 H), 7.85 (d, J = 7.2, 1 H), 7.83 (s, 1 H), 7.76 (s, 1 H), 7.54 (t, 1 H), 7.24 ∼ 7.26 (m, 6 H), 7.20 (s, 2 H), 7.14 (s, 1 H), 6.82 (d, J = 8.0, 1 H), 6.77 (d, J = 6.0, 1 H), 6.70 (d, J = 8.0, 1 H), 6.59 (d, J = 8.0 Hz, 1 H,), 6.28 (s, 1 H), 6.03 (s, 1 H), 3.29 (s, 2 H), 3.25 (s, 2 H), 3.16 (s, 2 H), 2.86 (s, 3 H), 2.60 (s, 3 H), 2.58 (s, 6 H), 2.13 (s, 3 H), 1.85 (s, 3 H), 1.82 (s, 6 H), 1.79 (s, 3 H), 1.75 (s, 6 H), − 2.54 (s, 2 H). IR (KBr) 3411, 3318, 3026, 2916, 2855, 1697, 1683, 1674, 1652, 1599, 1578, 1558, 1520, 1472, 1457, 1446, 1399, 1377, 1344, 1284, 1257, 1217, 1188, 1131, 1070, 968, 804 cm⁻¹. HR-MS (FAB, NBA) Found: 1170.5869. Calcd for C₇₆H₇₂N₁₁O₂: [M + H]⁺, 1170.5870.

---