Cu(II)-coordination in the bimetallic sites of laccase and cytochrome oxidase

Abstract

Abstract Molecular oxygen plays a key role in many biological oxidation reactions. Dioxygen is reduced to peroxide by most oxidases but a few enzymes can reduce it all the way to two water molecules. Among these latter enzymes are laccase and cytochrome oxidase. They contain four metal centers per functional unit. Although laccase contains only copper ions while cytochrome oxidase has two copper ions together with two heme irons, they show marked similarities with respect to the prosthetic metal groups. Thus, only two of the metal centers, type 1 and 2 Cu(II) in laccase and Cu A (II) and cytochrome a in cytochrome oxidase are detected by EPR. These metal sites serve as primary electron-accepting sites which are reduced by one-electron donating substrates [1, 2]. In both enzymes the other two metal ions form a bimetallic unit, type 3 copper in laccase and cytochrome α 3 -Cu B in cytochrome oxidase. In the resting enzymes these metals are believed to be in the Cu(II) and Fe(III) states, respectively, which are ERP-silent due to strong exchange coupling between the contiguous metal ions. These sites form the dioxygen-reducing centers which are reduced by intramolecular electron-transfer from the two EPR-detectable metal sites. Since the bimetallic units are EPR-nondetectable under most experimental conditions, their metal coordination and role in the enzyme mechanisms are little known but of great interest. However, new EPR signals, originating from one of the type 3 Cu(II) ions in laccase and from Cu B (II) in cytochrome oxidase, have recently been generated [3, 4]. Further exploration of these metal sites has therefore been possible. Both proteins show very similar EPR signals which are characterized by a rhombic g -tensor and a hyperfine coupling which is intermediate between that of type 1 and 2 Cu(II). Rhombic Cu(II) EPR signals of a similar kind are also observed in other proteins with copper in bimetallic sites, such as superoxide dismutase and half-met hemocyanin. Together with a pronounced amino acid sequence homology between a blue oxidase (ceruloplasmin) and cytochrome oxidase with the copper-binding site in superoxide dismutase, the similarity in Cu(II) EPR signals suggests that the metals may be coordinated in a similar way in these different proteins. The type 3 Cu(II) in laccase and the Cu B (II) in cytochrome oxidase have been studied by the ENDOR technique [5]. The laccase data show the presence of at least three nitrogeneous ligands. At least one of these is an imidazole and this is further supported by pulsed EPR studies [6]. The hyperfine coupling to the three nitrogens differs significantly indicating a low coordination symmetry for this Cu(II) in agreement with the rhombic g -tensor. An exchangeable proton suggests a nearby H 2 O or OH − , perhaps as a fourth ligand. Comparative 14 N ENDOR studies of the Cu B (II) site in cytochrome oxidase show that the resonance from three distinct nitrogenous ligands with hyperfine couplings are essentially the same as those of the type 3 Cu(II) site in laccase. These results are therefore a further indication of a possible similar metal binding in both enzymes.