A water-soluble supramolecular complex that mimics the heme/copper hetero-binuclear site of cytochrome c oxidase

The O2 adduct of an aqueous synthetic heme/copper model system built on a porphyrin/cyclodextrin supramolecular complex has been characterized.


Introduction
Cytochrome c oxidase (CcO) is the terminal enzyme in the mitochondrial respiratory chain. CcO consumes most of the molecular oxygen (O 2 ) processed by living organisms by reducing it to water (H 2 O). 1 The four-electron/four-proton reduction process (O 2 + 4e À + 4H + / 2H 2 O) takes place at the heme a 3 /Cu B hetero-binuclear active centre of CcO (Fig. 1a). [1][2][3][4][5] For the catalytic O 2 reduction reaction, the reaction mechanism schematically depicted in Fig. 1b has been proposed. [1][2][3] In the catalytic cycle, the fully reduced heme a 3 /Cu B site (Fe II /Cu I , compound R) reacts with O 2 to form an oxymyoglobin-like superoxo complex of heme a 3 (Fe III -O 2 À /Cu I , compound A). 3,6 Compound A is rapidly ($0.5 ms) converted to an oxoferryl intermediate (Fe IV ]O/Cu II -OH, compound P) via O-O bond cleavage assisted by H atom injection from a vicinal tyrosine residue. [3][4][5][6] Mechanistic investigations have suggested that one or more water molecules near the bound O 2 can facilitate the conversion of compound A to compound P. 7,8 To understand the reaction mechanism, synthetic heme/ copper models have been constructed using tetraarylporphinatoiron(II) (PFe II ) combined with Cu I complexes (Cu I L n , where L is a nitrogen donor ligand; n (coordination number) ¼ 3 or 4). 4,5 However, upon oxygenation of the PFe II / Cu I L n model systems in anhydrous organic solvents, m-peroxotype bridged structures, i.e., PFe III -O 2 -Cu II L n complexes, tend to form instead of compound A-like superoxo species. [9][10][11][12] In native CcO, the m-peroxo-type bridged structure has not been experimentally identied, although it has been proposed as a transitional precursor of compound P. 3,12,13 The structural differences between the native and model systems (superoxo vs. m-peroxo) 14 might be attributed to the inuence of water. 7,8,13 A model study by Naruta and co-workers demonstrated that the m-peroxo complex (PFe III -O 2 -Cu II L 3 ) formed at À70 C was converted to the superoxo complex (PFe III -O 2 À /Cu I L 3 ) at À30 C by the action of water molecules. 15 In native CcO, highly ordered water molecules have been detected in the vicinity of heme a 3 /Cu B . 7,16 A quantum chemical calculation suggested that a water molecule in the vicinity of Cu B decreases the energy barrier of the transformation of compound A to compound P. 8 In this context, a water-soluble PFe II /Cu I L n model compound would be useful to investigate the role of water on the reactivity of the Fe/Cu hetero-binuclear complex with O 2 . However, very few heme/copper mimics functioning under aqueous conditions have been prepared so far, except for the system constructed in the engineered heme pocket of myoglobin. 17,18 In this study, we describe an aqueous synthetic PFe/CuL 3 hetero-binuclear model system built on a porphyrin/ cyclodextrin supramolecular complex (Scheme 1). This system takes advantage of the very stable formation of a selfassembling 1 : 2 complex of 5,10,15,20-tetrakis(4sulfonatophenyl)porphinatoiron (FeTPPS) with per-O-methylated b-cyclodextrins (CDs). 19 We have previously studied the porphyrin/cyclodextrin complexes as simple biomimetic models of heme proteins that function under aqueous conditions, [20][21][22][23] where the molecular cage of per-O-methylated b-CDs provided a microscopic hydrophobic environment for FeTPPS similar to the heme pocket of heme proteins. 24 Here, we have synthesised a per-O-methylated b-CD dimer linked by a Cu IIterpyridine complex (Cu II TerpyCD 2 , Scheme 1) to replicate the distal tridentate Cu B site of CcO. The structural characterisation of the supramolecular FeTPPS/CuTerpyCD 2 complex and its reactivity towards O 2 are described.

Results and discussion
Synthesis of a water-soluble Fe III /Cu II hetero-binuclear complex The synthetic route of a supramolecular Fe III TPPS/Cu II TerpyCD 2 complex is shown in Scheme 1 and experimental details are described in (ESI ‡). Briey, the terpyridyl ligand was inserted as a linker of the CD dimer (TerpyCD 2 ) by the reaction of 5,5 00bis(mercaptomethyl)-2,2 0 :6 0 ,2 00 -terpyridine with 2,3-monoepoxyper-O-methylated b-CD (Epo-OMe-b-CD). 20 The addition of CuSO 4 $5H 2 O to TerpyCD 2 in an aqueous solution generated two absorption bands at 336 and 350 nm (Fig. 2a), which corresponded to the ligand to metal charge transfer bands of the terpyridyl-Cu II 1 : 1 complex. 25 In the UV-vis titration, a biphasic spectral change was observed ( Fig. 2a inset), indicating that the 1 : 2 complex of Cu 2+ with TerpyCD 2 (l max ¼ 333 nm) was rst formed and then it was converted to the thermodynamically stable 1 : 1 complex upon further addition of Cu 2+ . The spectral Scheme 1 Preparation of the supramolecular Fe III TPPS/Cu II TerpyCD 2 complex. changes were completed at one equivalent of Cu 2+ . The complexation between TerpyCD 2 and Cu 2+ was also monitored by electrospray mass spectroscopy. In the 1 : 1 mixture of CuSO 4 and TerpyCD 2 in H 2 O, the 1 : 1 complex (Cu II TerpyCD 2 ) was observed at m/z 1577 and 1059 (Fig. 2b), which corresponds to [Cu II TerpyCD 2 ] 2+ and [(H 2 O)Cu II TerpyCD 2 + H] 3+ , respectively. The 1 : 2 complex was also detected as a small ion peak when the 1 : 2 mixture of CuSO 4 and TerpyCD 2 in H 2 O was analysed by electrospray mass spectroscopy (data not shown).
The Cu II TerpyCD 2 complex was then titrated with Fe III TPPS (Fig. 3a). The Soret band of Fe III TPPS shied from 408 nm to 418 nm, indicating that a m-oxo-dimer of Fe III TPPS dissociated to the monomeric monohydroxo complex (Fe III (OH À )TPPS) 19 through interaction with Cu II TerpyCD 2 . The spectral changes were completed upon addition of one equivalent of Cu II TerpyCD 2 to Fe III TPPS, indicating a quantitative 1 : 1 complexation. The obtained complex was then analysed by electrospray mass spectroscopy. The two main ion peaks were detected at m/z 1385 and 2078 as tri-and di-anionic species, respectively (Fig. 3b). Considering total charges of the complexes, the peaks at m/z 1385 and 2078 were assigned to the m-oxo and m-hydroxo Fe III TPPS/Cu II TerpyCD 2 complexes, i.e., [PFe III -O-Cu II CD 2 ] 3À and [PFe III -(OH)-Cu II CD 2 ] 2À , respectively. The assignments were conrmed by isotope pattern simulations ( Fig. 3b inset). Evidence of the m-oxo (Fe III -O-Cu II ) structure was also provided by its characteristic absorption bands at 453 and 567 nm, which appeared when the pH of the solution was increased (Fig. S3 ‡). The red-shied Soret band at alkaline conditions indicates formation of the PFe III -O-Cu II moxo complex. [26][27][28] The pH titration revealed the acid-base equilibrium of [PFe III -O-Cu II CD 2 ] 3À and [PFe III -(OH)-Cu II CD 2 ] 2À with pK a ¼ 8.8. This pK a value is consistent with that previously predicted by Karlin and Blackburn (pK a ¼ 8 AE 2.5). 28 The electron paramagnetic resonance (EPR) spectra showed signicantly weak signals at g ¼ 6.09 and 2.08 in the Fe III TPPS/ Cu II TerpyCD 2 complex (Fig. S4 ‡) because of the antiferromagnetic coupling between the two metal ions as a result of their close proximity. The optimized molecular structure (Fig. 4)    enough to reduce both Fe III and Cu II to Fe II and Cu I . 29,30 Aer the reduction, the solution was passed through a short gel-ltration column (Sephadex G-25) under aerobic conditions to remove excess S 2 O 4 2À and its oxidised products. The UV-vis spectrum of the resulting solution showed absorption maxima at 419 nm and 542 nm (Fig. 5, blue line); the Q-band was very different from that of the oxidised state (Fe III TPPS/Cu II TerpyCD 2 , l max (Qband) ¼ 570 nm, green line) and similar to that of the O 2 complex of the previously reported Fe II TPPS/CD dimer system. 20 Introduction of CO gas into the solution caused further spectral changes with absorption maxima at 418 nm and 535 nm (Fig. 5, red line). The sharp Soret band is characteristic of the CO-Fe II TPPS complex, 20 indicating that a ligand exchange from O 2 to CO occurs in this system. The O 2 complex was further characterized by EPR and resonance Raman (rR) spectroscopic analyses. The EPR spectrum of the O 2 adduct of Fe II TPPS/Cu I TerpyCD 2 measured at 77 K was completely silent (Fig. S4 ‡), which was consistent with the spectra of other O 2 complexes of the PFe II /Cu I L n heterobinuclear systems. [31][32][33] The rR analysis at 77 K (frozen solution of the O 2 adduct) using 405 nm excitation revealed a characteristic band at 578 cm À1 , which shied to 551 cm À1 under an 18 O 2 atmosphere (Fig. 5 inset). The isotope shi (Dn ¼ 27 cm À1 ) corresponds to the expected value for the n Fe-O stretching mode. 15 The wavenumber is quite similar to those of the PFe III -O 2 À /Cu I L n superoxo complexes in the previously reported native 34 and synthetic model systems as listed in Table 1. 14,15,35 Furthermore, the O-O bond stretching mode (n O-O ) was not enhanced in this system. This is a relevant observation as the n O-O band is oen observed in the range of 750-900 cm À1 in the PFe III -O 2 -Cu II L n m-peroxo complexes, but not in the case of the Fe III -O 2 À /Cu I L n superoxo complexes (Table 1). 14 (Fig. 6), which is the same coordination mode as in compound A of native CcO. 3,14,38 The superoxo PFe III -O 2 À /Cu I CD 2 complex was gradually converted to another state when the solution was allowed to stand at pH 7 and 25 C under aerobic conditions (Fig. 7). The absorption spectra showed several isosbestic points and the nal spectrum (shown as a green line in Fig. 7) was coincident to that of the oxidised Fe III TPPS/Cu II TerpyCD 2 complex (Fig. 5). EPR spectral changes also support oxidation of the superoxo PFe III -O 2 À /Cu I CD 2 species to the Fe III TPPS/Cu II TerpyCD 2 complex (Fig. S4 ‡). The rst-order rate constants (k obs ) for the conversion were determined from the absorbance change at various pH conditions. Interestingly, the superoxo complex was more rapidly converted at lower pH (Fig. 7 inset). The linear pH/ log k obs dependency at pH 7-10 (slope ¼ À0.11) suggests that the conversion is partially accelerated by a proton-coupled process. 39 Collman et al. have reported that the rate of the O 2 reduction catalysed by their PFe/CuL n model complex is pHdependent and increases at lower pH. 40 We have previously reported that the autoxidation rate of the O 2 complex in the PFe II / CD dimer system without any distal functions is independent of pH in the neutral pH region (7-10), whereas it is accelerated at   pH below 6 and above 10. 24 Therefore, the pH-rate dependency at the neutral pH region suggests that the water molecules gathered at the distal Cu site promote the conversion of the PFe III -O 2 À /Cu I CD 2 complex to the oxidised PFe III -(OH)-Cu II CD 2 complex.
The quantum chemical study on native CcO 8 proposes that a water molecule coordinating to the distal copper ion facilitates the conversion of compound A to compound P through the formation of the hydroperoxo Fe III -OOH intermediate that has not been experimentally detected. Thus, the involvement of a water molecule in the present PFe III -O 2 À /Cu I CD 2 complex is likely to occur. In addition, molecular modelling suggests that a water molecule bound to the distal copper ion can induce protonation of the superoxo complex (Fig. 8a), where the methoxy groups of the CD dimer are suitable to provide two hydrogen bonding sites to the water. The pH-dependent decomposition of the superoxo complex, as shown in Fig. 7, might be explained by the acid-base equilibrium of the water molecule (Fig. 8b), where the proton-donation to the superoxo complex is likely to induce the O-O bond cleavage as proposed in CcO 8 and/or the proton-assisted autoxidation reaction similar to myoglobin. 41,42 The O 2 binding in the present complex was practically irreversible; the O 2 complex of Fe II TPPS/Cu I TerpyCD 2 was never converted to its Fe II /Cu I deoxy complex, even when the O 2 complex once formed was dissolved in a deoxygenated buffer (Fig. S5 ‡). In contrast, the deoxy complex was observed in the Fe II TPPS/TerpyCD 2 complex without copper under the same experimental conditions. 43 This result indicates that the O 2 bound to PFe II is tightly held by the distal Cu I L 3 complex, as previously demonstrated by the Fe/Cu superoxo complex. 14 The tight O 2 binding was also conrmed by observing ligand exchange with CO. The ligand exchange occurred slowly over $30 min when the Fe/Cu superoxo complex was dissolved in a CO saturated buffer (Fig. S5 ‡), whereas it occurred instantaneously in the absence of distal Cu complex or in the absence of O 2 (Fig. S5 ‡). The ligand exchange of O 2 with CO also rapidly occurs in the previous Fe II TPPS/CD dimer systems. 20

Electrochemical analysis for the O 2 reduction
To evaluate the CcO-like function of this system, we monitored the electrocatalytic O 2 reduction reaction. [45][46][47] The cyclic voltammogram (CV) of the Fe III TPPS/Cu II TerpyCD 2 complex immobilized on a glassy carbon electrode showed a reversible redox couple at E 1/2 ¼ À0.21 V (vs. Ag/AgCl) in a deoxygenated buffer solution (under Ar, Fig. 9a, black line). The result is similar to those of the previously reported PFe/CuL n heterobinuclear systems; the Fe III /Fe II and Cu II /Cu I redox waves appear at the same potentials. 31,46 In an air-saturated buffer, the CV of the Fe III TPPS/Cu II TerpyCD 2 complex showed a large catalytic current below À0.25 V because of O 2 reduction (Fig. 9a, blue line). A comparison of the CVs of the Fe III TPPS/ Cu II TerpyCD 2 complex with those of the reference samples, i.e., Fe III TPPS and Fe III TPPS/TerpyCD 2 (Fig. 9b), clearly indicates the effect of the Fe/Cu hetero-binuclear structure in the O 2 reduction; the Fe III TPPS/Cu II TerpyCD 2 complex showed a very large catalytic current starting from a lower onset potential (DE onset ¼ À40 mV). The O 2 reduction process was then studied by linear sweep voltammetry (LSV) using a rotating disk electrode (RDE, Fig. 9c). The LSVs of the Fe III TPPS/Cu II TerpyCD 2 and Fe III TPPS/ TerpyCD 2 complexes showed diffusion limited catalytic O 2reduction currents below À1.0 V vs. Ag/AgCl. In the case of FeTPPS without the CD dimer, the current was never saturated in LSV due to a slow reaction rate of the iron porphyrin with O 2  on the disk electrode (Fig. S6 ‡). The saturated currents observed in the Fe III TPPS/Cu II TerpyCD 2 and Fe III TPPS/TerpyCD 2 complexes at various rotation rates were analysed using the Koutecky-Levich equation to determine the average number of electrons (n) used in the O 2 reduction (Fig. 9d). 48 A signicant increase in the n value was observed for the Fe/Cu heterobinuclear complex (n ¼ 3.03 AE 0.01) compared to the control sample without copper (n ¼ 1.63 AE 0.03). 49 Therefore, we conclude that the terpyridyl Cu complex associated with FeTPPS in our model system facilitates the catalytic O 2 reduction as an electron source, as proposed in the mechanism of native CcO 3 and as proven using the synthetic model systems. 5,45,48

Conclusions
In conclusion, we have synthesized a water-soluble biomimetic model complex for the heme a 3 /Cu B hetero-binuclear active centre of CcO by utilizing a supramolecular complexation, and characterised its reactivity with O 2 . To the best of our knowledge, this is the rst example of a totally synthetic CcO model that works in a completely aqueous solution. In common with compound A of native CcO, we have identied the PFe III -O 2 À / Cu I CD 2 superoxo complex as the O 2 adduct in our model system in aqueous solution, whereas the PFe III -O 2 -Cu II L n m-peroxo complexes tend to form in the other synthetic model systems in anhydrous organic solvents. The pH-dependent conversion of the PFe III -O 2 À /Cu I CD 2 superoxo complex to its oxidised m-hydroxo PFe III -(OH)-Cu II CD 2 complex suggested the involvement of water molecules in the formation of the superoxo complex in aqueous solution. We believe that our aqueous model system will help to clarify the long-standing arguments with regard to the native and synthetic model systems in CcO chemistry.

Conflicts of interest
The authors declare no conict of interest. Fig. 9 (a, b) CV of the FeTPPS/CuTerpyCD 2 complex and its reference samples absorbed on the glassy carbon electrode with Nafion (5 wt% dispersion, 10 mL) in pH 7 phosphate buffer at a scan rate of 0.1 V s À1 using Ag/AgCl and Pt wire as the reference counter electrodes, respectively. (c) LSV data for the FeTPPS/CuTerpyCD 2 complex (10 nmol) coated with Nafion (5 wt% dispersion, 10 mL) on a glassy carbon electrode in air saturated pH 7.0 phosphate buffer at a scan rate of 10 mV s À1 at multiple rotations using Ag/AgCl and a Pt wire as the reference and counter electrodes, respectively. (d) Koutecky-Levich plots for the FeTPPS/CuTerpyCD 2 and FeTPPS/TerpyCD 2 complexes at the potentials of À1.0, À1.1 and À1.2 V to determine the average number of electrons (n) used for the O 2 reduction reaction.