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

Abstract

Cytochrome c oxidase (CcO) catalyzes the four-electron reduction of oxygen to water, the oneelectron 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 O 2 to H 2 O (Collman et. al. Science, 2007, 315, 1565. The current paper shows that the same functional CcO model will catalyze the four-electron reduction of O 2 using the actual biological reductant Cytc in a homogeneous solution. Both single and steady-state turnover kinetic studies indicate that O 2 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. This homogeneous reaction was monitored by following the oxidation of reduced Cytc by O 2 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 O 2 concentration of the solution before and after the reaction shows that 3.9±0.1 equivalents of reduced Cytc are oxidized per equivalent of O 2 consumed, indicating that this oxidation process is stoichiometric within error. The rate of catalysis (k obs /[Catalyst]) is pH dependent decreasing from 3.9 ± 0.2 × 10 3 M −1 s −1 between pH 6-7 to 1.8 ± 0.1 × 10 3 M −1 s −1 at pH 8 ( Fig. S1) indicating that protonation may be rate limiting at high pH.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 jpc@stanford.edu....

Transition Metal Complexes and the Activation of Dioxygen

“…Mechanistically, the 4e À reduction of O 2 proceeds in essentially the same manner regardless of the electron source (chemical versus electrolytic; Figure 22). Catalysis with 48 performed at a 2 % catalyst loading in a 1:1 water:acetonitrile mixture (pH ¼ 7) at 25 C was shown to be stoichiometric with respect to the reductant [143]. Interestingly, O 2 binding was shown to be rate limiting as opposed to electron transfer in this case, in contrast to what is proposed in the native enzyme [142].…”

Electrocatalytic O2-Reduction by Synthetic Cytochrome c Oxidase Mimics: Identification of a “Bridging Peroxo” Intermediate Involved in Facile 4e–/4H+ O2-Reduction

“…C c O has attracted paramount interest of synthetic inorganic and bioinorganic chemists due to its great fundamental and practical importance in understanding dioxygen activation/reduction, and proton translocation, etc. 5–10 The X-ray structures of C c O reveal the active site responsible for O 2 binding and reduction, to be a binuclear site comprising of heme- a 3 , with a proximal histidine (His) along with a distal side histidine bound copper (Cu B ). 3,11,12 One of these three ligating His residues is covalently cross-linked to a nearby tyrosine (Tyr) residue which is said to act as a source of electron and proton in the reduction of O 2 and proton translocation.…”

Factors That Control Catalytic Two- versus Four-Electron Reduction of Dioxygen by Copper Complexes

“…7 Meanwhile, multicopper oxidases (MCO’s) 1a,1c,8 and heme-copper oxidases (HCO’s) 9 facilitate 4 e − /4H + reduction of dioxygen to water; the latter reactivity is analogously a fuel cell reaction. 10–16 …”