Protonation of the oxo-bridged heme/copper assemblies: Modeling the oxidized state of the cytochrome c oxidase active site

Abstract

In this study on model compounds for the resting oxidized state of the iron‑copper binuclear center in cytochrome c oxidase (CcO), we describe the synthesis of a new μ-oxo-heme/Cu complex, [(TPP)Fe^III-O-Cu^II(tmpa)][B(C₆F₅)₄] (2) {TPP: tetraphenyl porphyrinate(2-); TMPA: tris(2-pyridylmethylamine)}, as well as two protonation events for three μ-oxo-heme/Cu complexes with varying peripheral substituents on the heme site. The addition of increasing amounts of strong acid to these μ-oxo-heme/Cu systems successively led to the generation of the corresponding μ-hydroxo, μ-aquo, and the dissociated complexes. The heme/Cu assemblies bridged through a water ligand are reported here for the first time and the ¹H NMR and ¹⁹F NMR spectral properties are consistent with antiferromagnetically coupled high-spin iron(III) and copper(II) centers. By titration using a series of protonated amines, the pK_a values for the corresponding μ-hydroxo-heme/Cu species (i.e., the first protonation event) have been reported and compared with the pK_a ranges previously estimated for related systems. These synthetic systems may represent structural models for the oxidized Fe^III-X-Cu^II resting state, or turnover intermediates and can be employed to clarify the nature of proton/electron transfer events in CcO. SYNOPSIS: The resting oxidized state of the cytochrome c oxidase active site contains an Fe_a3-OH_x-Cu_B moiety. Here, we investigated two successive protonation events, for a series of μ-oxo-heme/Cu assemblies and reported the pK_a values for the first protonation event. The μ-aquo-heme/Cu complexes described here are the first examples of such systems.

Keywords: Acid dissociation constant; Antiferromagnetic coupling; Bridging ligand; Cytochrome c oxidase; Paramagnetic NMR; Protonation.