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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Am Chem Soc. Author manuscript; available in PMC 2010 April 15.
Published in final edited form as:
PMCID: PMC2743311

Catalytic Reduction of O2 by Cytochrome c Using a Synthetic Model of Cytochrome c Oxidase


Cytochrome c oxidase (CcO) catalyzes the four-electron reduction of oxygen to water, the one-electron reductant Cytochrome c (Cytc) being the source of electrons. Recently we reported a functional model of CcO that electrochemically catalyzes the four-electron reduction of O2 to H2O (Collman et. al. Science, 2007, 315, 1565). The current paper shows that the same functional CcO model will catalyze the four-electron reduction of O2 using the actual biological reductant Cytc in a homogeneous solution. Both single and steady-state turnover kinetic studies indicate that O2 binding is rate determining and O-O bond cleavage and electron transfer from reduced Cytc to the oxidized model complex are relatively fast.

Cytochrome c oxidase (CcO) performs a four electron reduction of oxygen to water, in the last step of respiration.1 The active site in CcO is comprised of a heme/Cu site having a post translationally modified tyrosine residue covalently bound to one of the histidine ligands of the distal Cu (Fig. 1A).2 Cytochrome c (Cytc), a small electron transfer protein, is the source of electrons for CcO. Cytc has a co-ordinatively saturated low-spin heme active site (Fig. 1B).1 Mimicking the structure and function of CcO using synthetic model complexes has attracted significant attention over the last two decades.3,4,5 Recently a model of CcO that incorporates a heme with a covalently attached proximal imidazole, a tris-imidazole distal binding pocket for Cu, and a covalently modified tyrosine (Y244) analogue has been reported (FeCuPhOH, Fig. 1C).6 This functional model catalyzes the selective four electron reduction of oxygen at physiological pH, using an electrode as the source of electrons and generates negligible (<4%) partially reduced oxygen species (PROS) during catalytic turnover.7 Herein we report the selective catalytic four electron reduction of oxygen by the biological one-electron reductant Cytc (from horse heart) using this functional CcO model in a homogeneous mixed solvent system.

Figure 1
Active site structures of A) CcO B) Cytc and C) synthetic model of CcO (Fe Cu and phenol analogue).

This homogeneous reaction was monitored by following the oxidation of reduced Cytc by O2 in the presence of 2% FeCuPhOH catalyst. Fig. 2 shows the kinetic traces. These data show a decrease in the percentage of reduced Cytc at a pseudo 1st order rate of 1.3×10−3 s−1, which is much faster than the slow auto-oxidation of reduced Cytc (~1×10−5 s−1).8 Monitoring the O2 concentration of the solution before and after the reaction shows that 3.9±0.1 equivalents of reduced Cytc are oxidized per equivalent of O2 consumed, indicating that this oxidation process is stoichiometric within error. The rate of catalysis (kobs/[Catalyst]) is pH dependent decreasing from 3.9 ± 0.2 × 103 M−1s−1 between pH 6–7 to 1.8 ± 0.1 × 103 M−1s−1 at pH 8 (Fig. S1) indicating that protonation may be rate limiting at high pH.

Figure 2
Kinetic traces of oxidation of the 550 nm band of reduced Cytc in the presence of 2% FeCuPhOH by aerated (dark blue) and O2 saturated (light blue) 50:50 aqueous buffer acetonitrile solvent at pH 7 and 250C. Inset: absorption spectra of reduced (black) ...

Due to limitations in the solubility of Cytc under these experimental conditions only 25 turnovers (i.e. 1 equivalent catalyst:100 equivalents Cytc) could be obtained.9 The turnover number for this catalyst has been determined by studying its electrocatalytic O2 reduction. The catalyst modified with an alkyne linker was “clicked” on to a C16SH thiol in a self assembled monolayer (SAM, details in SI). The SAM limits the rate of electron transfer from the electrode to the catalyst to 4 s−1.7 Electrolysis of this covalently bound catalyst at physiological potentials gives a turnover number of 9±1 × 103.

Reduction of O2 using reduced Cytc with the catalyst was studied at various conditions with the aim of elucidating the mechanism and identifying the rate determining step (rds) during catalytic turnover. The proposed mechanism for the four electron reduction of O2 by Cytc is presented in Scheme 1. In the first step, O2 binds to the reduced catalyst forming an FeIII-superoxo species (step A, Scheme 1). Since the catalyst contains four electrons (two from FeII, one from CuI and one from phenol), the next step leads to the formation of “PM” intermediate (step B, Scheme 1) which is comprised of an oxidized CuII, an FeIV=O ferryl and a phenoxide radical.6b This oxidized intermediate is then reduced by four equivalents of Cytc regenerating the fully reduced active catalyst (steps C and D). The rds could involve (a) O2 binding, (b) O-O bond cleavage, or (c) electron transfer from reduced Cytc to the oxidized catalyst.

Scheme 1
Possible mechanism of O2 reduction by Cytc in the presence of the FeCuPhOH CcO model complex.

The turnover rate increases by more than two fold (Fig. 2) upon using an O2 saturated solution instead of an air saturated solution. This indicates that O2 binding may be the rds. The catalytic reaction shows a modest inverse deuterium isotope effect (kH/kD) of 0.82. Also there is no change in the rate of O2 reduction by varying the concentration of reduced Cytc with a constant catalyst concentration (Fig. S1). This indicates that electron transfer from reduced Cytc is rapid and is not involved in the rds during catalytic turnover. Single turnover kinetic experiments were performed with the FeCuPhOH and the “Fe-only” complex (without Cu and phenol) to obtain the rates of O2 binding and electron transfer.

The Fe-only complex acts as a control to measure the rate of O2 binding to the catalyst (step A, Scheme 1), as this complex lacks the necessary reducing equivalents for the O-O bond cleavage (step B, Scheme 1). The reaction of O2 with the reduced FeII complex was monitored following the characteristic FeII absorption at 434 nm, which shows an O2 binding rate of 0.5 s−1 (Fig. 3) forming a ferric superoxide species, followed by its slow hydrolysis with a rate of 0.05 s−1 (Fig. S3). Parallel monitoring of the FeII state of the FeCuPhOH catalyst at 434 nm shows a monophasic O2 binding with a rate constant of 0.1 s−1 (Fig. 3). EPR and UV-Vis spectra of the reaction product at 40s indicate it is identical to the chemically produced FeIIICuII species (Fig. S3, S4). Since the FeCuPhOH catalyst reduces O2 stoichiometrically (vide supra) and the amount of hydrolysis side reaction is negligible (PROS <4%)7, the monophasic kinetics of the O2 reaction (i.e. no intermediates observed) implies that the O-O bond cleavage (step B, Scheme 1) and decay of the high valent intermediate (step C, Scheme 1) must be much greater than 0.1 s−1. The rate of O2 binding to the fully reduced FeCuPhOH catalyst is slower than the Fe-only complex possibly because of greater steric hindrance in the former due to the phenol substituent. Note that the O2 binding rates (step A, Scheme 1) of these complexes are much slower that those reported for CcO and other O2 binding heme proteins and model complexes.10,11,12 We have recently shown that this slow O2 binding is due to the presence of an axial water ligand, which H-bonds to additional H2O molecules in the distal pocket, making the ferrous catalyst low-spin in nature (Fig. S5), in contrast to the high-spin five coordinate ferrous active site of the enzyme.1,13 The small inverse kinetic isotope effect observed during catalytic turnover is consistent with displacement of a bound water in the rds.14

Figure 3
Kinetic traces showing the decrease of 434 nm absorption intensity of the Fe-only (red) complex and FeCuPhOH (blue) catalyst in the presence of O2. Inset: absorption spectra of reduced Fe-only (red), and FeCuPhOH (blue) complexes. The black arrow indicates ...

The second order rate constants (k2nd) for electron transfer between reduced Cytc and oxidized FeIIICuIIPhOH catalyst or the FeIII-only complex (step D, Scheme 1), under anaerobic conditions, were independently estimated to be ~4±0.1 × 104 M−1s−1 (Fig. S6). This translates to a pseudo first-order rate of ~1.2 s−1 (k2ndx[Cytc2+]) under catalytic turnover. The rate of O2 binding (0.1 s−1) is at least 10 times slower than the rate of electron transfer from reduced Cytc to the FeIIICuIIPhOH catalyst. The rates of reduction of the ferryl and tyrosyl radical species (step C, Scheme 1) are arguably greater than the rate of reduction of FeIIICuII (step D, Scheme 1) as they have higher driving forces for electron transfer. Comparison of these rates indicates that O2 binding is the rds during catalytic turnover at physiological pH; whereas both the O-O bond cleavage and electron transfer steps are relatively fast.

In summary, we have demonstrated that our functional model of CcO can catalyze the selective four electron reduction of O2 using the biological reductant Cytc. The rate limiting step in the catalytic cycle is O2 binding to the catalyst. The rate of O-O bond cleavage is >> 0.1 s−1. The oxidized products are rapidly reduced back to the active form by reduced Cytc. We believe this is the first report where kinetically inert O2 has been reduced to H2O by Cytc using a synthetic functional model as a catalyst.

Supplementary Material


Supporting Information Available:

The experimental details, pH dependence, Cytc concentration dependence, turnover number, UV-Vis, EPR, resonance Raman spectra and electron transfer rate plots are available free of charge via the Internet at


This research was funded by NIH GM-17880-38 (J.P.C.).


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