Scheme 1.2: Representative examples of oxygenation of organic substrates catalysed by dioxygen-activating copper metalloproteins, categorised by copper centre type.
Copper containing proteins can be classified into seven different types based on the spectroscopic properties of their active sites.1,8 Those which use dioxygen as an electron acceptor and are primarily involved in oxidation and oxygenation of organic substrates are types 2, 3 and 4 (T2, T3 and T4, respectively, Scheme 1.2). All utilise the copper(I)/copper(II) redox pair, whereby the copper(I) state facilitates initial dioxygen binding by electron transfer. Owing to its d10 configuration copper(I) is relatively silent spectroscopically, and so the type classification described above is based on the spectroscopic features of the oxidised d9 copper(II) states.1,12 Copper(III) has traditionally not been considered of biological relevance because of its redox inaccessibility when ligated solely by amino acid side chains.13,14 However, several mechanistic scenarios have more recently emerged which
propose involvement of copper(III)-oxygen species as reactive intermediates.15,16 The three types of copper centres mentioned above possess a histidine rich coordination environment, containing at least two of these aromatic nitrogen donors per copper ion in all but a single case (Section 1.2.3).1,17,18 The number and arrangement of copper ions in each type of centre differs, as does the oxygen binding mode, both of which are discussed in more detail below.
1.2.1 Type 2 Sites
Figure 1.1: Active site of PHM showing the end-on binding mode of dioxygen (left), and the second distal T2 copper centre (right, PDB Code: 1SDW).19
Type 2 centres are also referred to as 'normal' as a result of their distinguishing electron paramagnetic resonance (EPR) spectroscopic signals, which are similar to those of 'normal' tetragonal copper(II) complexes.1,12,17 Representative metalloenzymes possessing T2 centres include galactose oxidase,1,8 amine oxidase,6,12 dopamine β-monooxygenase (DBM)1,8,17 and peptidylglycine α-hydroxylating monooxygenase (PHM).12,17 The oxidase enzymes are truly mononuclear, requiring an additional redox-active organic cofactor to shuttle the extra electron needed for oxidation of their respective substrates and reduction of dioxygen to peroxide.17,20 On the other hand DBM and PHM, which both catalyse aliphatic hydroxylation reactions,21 contain two T2 centres separated by at least 7 Å.20 This separation is evidenced by their lack of magnetic interaction, and they are thus also known as non-coupled dinuclear centres.17,20 One of these copper ions is coordinated by three histidine side chains, whereas the second is ligated by two histidine donors in combination with a methionine residue (Figure 1.1). While both copper ions are involved in storage and transfer of the electrons required to achieve reactivity, dioxygen binding and substrate transformation occurs exclusively at the T2 site ligated by methionine.21 Dioxygen initially undergoes single electron reduction upon binding to the copper(I) ion at this site to give an end-on copper(II)-superoxo (CuII-O2•, ES, Scheme 1.4) adduct which is thought to then react directly with the substrate.16,18,19
1.2.2 Type 3 Sites
Figure 1.2: Active site of Hc showing the side-on binding mode of dioxygen (PDB Code: 1JS8).22
Type 3 centres contain two closely spaced copper ions which show strong antiferromagnetic coupling in the copper(II) state. This interaction leads to EPR silent behaviour,12,16 and hence these sites are also known as coupled binuclear centres. The three members of this class, hemocyanin (Hc), tyrosinase (Tyr) and catechol oxidase (CO), all bind dioxygen between their two copper ions reversibly, with the same bridging mode.23 This results from their almost identical active site structures, with both copper ions ligated by three histidine residues each (Figure 1.2). Dioxygen binding causes a significant contraction of the Cu···Cu distance (from ca. 4.5 Å to 3.3 Å)1,14,17 and establishes the strong superexchange pathway that couples the two copper(II) ions.16,18,24 Although Hc acts only as an oxygen carrier, both CO and Tyr can oxidise catechols to o-quinones, and Tyr additionally hydroxylates monophenols.1,25 These differences in function are in part thought to arise from restricted substrate access to the active site, imposed by flanking amino acid residues in Hc and CO.17,18,23 Dioxygen receives one electron from each of the two copper(I) ions in the active site upon binding, resulting in a side-on di-copper(II)-peroxo (µ-η2:η2-O2, SP, figure Scheme 1.4) adduct. However, the actual species responsible for oxygen transfer in Tyr is still under debate (see Section 1.3.3 for more detail).
1.2.3 Type 4 Sites
Figure 1.3: Active site of pMMO showing the unusual N-terminal donor (PDB Code: 3RGB).26
Type 4 copper centres consist of a mononuclear T2 site and a coupled binuclear T3 site.1,8 These are clustered together in a triangular arrangement, at which the four-electron reduction of dioxygen occurs to give two molecules of water.18 Representative examples of proteins containing T4 sites include ascorbate oxidase and the large and diverse family of laccase metalloenzymes.1,8,17,18 The T4 site itself is not of exceptional relevance to the current work, however, a related configuration of copper ions has been identified in particulate methane monooxygenase (pMMO).27 This metalloenzyme possesses a T2-like mononuclear site, and an unusual dinuclear copper centre more than 20 Å away. The dinuclear centre has a short Cu···Cu distance of 2.6 Å, with one copper ion coordinated by two histidine side chains. The second active site copper ion is chelated by a single histidine residue, which coordinates through the side chain and additionally exhibits unusual N-terminal ligation (Figure 1.3).27 It has recently been shown that oxidation of methane occurs at this dinuclear copper centre,3 yet the mode of dioxygen binding and resulting active oxygenating species are still unknown (Section 1.3.2). That the remarkable reactivity of pMMO stems from an atypical dinuclear copper site helps to emphasise the importance of copper-mediated dioxygen activation, and has provided fresh motivation for bioinorganic research efforts, including in the field of model chemistry.11,15