Structure activity relations in copper-dependent metalloenzymes [electronic resource] : insight into dioxygen activation by mono- and trinuclear active sites
Keywords: Chemistry.

Activation of molecular oxygen is often accomplished by Cu-dependent metallo-enzymes in Nature. Two important classes are the Multicopper Oxidases (MCOs) and the Polysaccharide Monooxygenases (PMOs), also known as auxiliary activity enzymes 9-11 (AA9-11). MCOs utilize four Cu's arranged in a T1, a T2, and binuclear T3 site. The T2 and T3 sites collectively form a trinuclear Cu cluster (TNC), that is the location for the four-electron reduction of O2 to water, while the T1 functions as an electron shuttle from the substrate. In the PMOs, a mononuclear Cu site activates either cellulose or chitin by insertion of oxygen from O2, which subsequently leads to their degradation. The reduction of O2 in the MCOs occurs via two, two-electron transfers from the 4xCu(I) active site. The first two-electron transfer is rate-limiting and it has been debated which TNC pair performs this reactivity. By mutating the His residue to Gln of the T3 [alpha] and T3 [beta] Cu's, we found that only the T3 [alpha] variant allowed for fast O2 reactivity. Furthermore, the two-electron reduced Peroxy Intermediate (PI) formed in the T3 [alpha] variant was spectroscopically identical to that formed in the Wild-type enzyme. Importantly, this proves experimentally that the first two electrons in O2 reduction are provided by the T2 and the T3 [beta] Cu's which are near an anionic carboxylate residue that is required for O2 reduction. In a follow-up study, we expanded the mutations of the T3 [beta] Cu to include a His to Glu variant. While fast two-electron reduction of O2 was turned off in this variant, a slow one-electron oxidation of the T3 [beta] Cu was observed. By comparison to the similar slow one-electron oxidation of the His to Gln T3 [beta] variant by EPR, we found that this occurred via a common intermediate where neither of the mutated residues was bound to the Cu(II). This was followed by re-coordination of the mutated residue to generate the respective resting forms of the variants. This behavior was consistent with observations by Cu K-edge XANES showing that the mutated residues did not coordinate to the reduced T3 [beta] Cu. The lack of fast two-electron reactivity was further evaluated by DFT calculations of the reduced Wild Type (WT) TNC compared to the reduced TNC of the variants. Here it was found that while the 3-coordinate WT T3 [beta] Cu(I) has a high energy dxy filled orbital that provides excellent overlap with one of the two [pi] *-LUMOs of O2 that is bridged to the T2 Cu, the variants have a highest energy dz2 orbital that does not allow for efficient overlap with the O2 acceptor orbitals. This shows the importance of having the correct geometric- and electronic configuration for activating O2 by two-electron reduction. The nature of the resting form in MCOs is important for reactivating the enzymes for catalysis. Two resting forms with different spectroscopic features have been described in the literature: the resting oxidized (RO) form and the alternative resting (AR) form. We were able to generate both forms in a high-potential Bilirubin oxidase (BOD) and show how they could be interconverted. Importantly, only RO could be activated for catalysis by reduction via the T1 Cu, whereas AR was only activated by low-potential electron donors, i.e. dithionite or an electrode poised at < 400mV. From XANES we showed that this activation-difference relates to RO having a fully oxidized TNC, while AR has a singly oxidized TNC with a lower electron affinity than the T1 Cu, which explains its lack of reactivation. It is an important task to understand how the three oxidized TNC Cu's in RO are able to accept electrons via a high potential T1 Cu, while this is not the case in AR. We therefore systematically studied the outersphere reduction and oxidation behaviors of RO and AR in a high potential (~700mV) laccase. We found that the T3 pair accepts two electrons in an essentially concerted process, indicating a similar or slightly higher driving force for the second electron versus the first. In contrast, while the singly oxidized Cu in the TNC of AR would not reduce via the T1 Cu, outersphere oxidation of the fully reduced TNC led to the same singly oxidized TNC Cu as in AR, which has a redox potential of ~400mV. These observations were evaluated by DFT calculations, where a destabilized half-reduced T3 pair was optimized in the reduction of RO, and a different, ~17kcal/mol more stable, half-reduced T3 pair was optimized in the one-electron oxidation of the reduced TNC. Importantly, a linear transit calculation revealed a significant energy barrier on going from the destabilized to the more stable half-reduced T3 structure, which allows the second electron into the T3 pair to be delivered via the high-potential T1 Cu, in agreement with the experimental results. This elucidates the properties of the TNC and its surroundings that control the efficient three-electron reduction of the resting TNC from a high-potential T1 Cu, and how potential energy traps, i.e. AR are avoided. Several crystal structures of the newly identified PMOs have been reported, but these have not been able to show details of the active site Cu that allows for activation of O2 in solution. The coordination environment of the mononuclear active site in an AA9 PMO was therefore evaluated by Cu K-edge XANES and EXAFS. It was shown that while the oxidized Cu has a four-coordinate tetragonal geometry, a water-derived ligand is lost upon reduction, resulting in a T-shaped 3-coordinate Cu(I). The reactivity of the Cu(I) with dioxygen was probed by EPR and stopped-flow spectroscopy, and a fast regeneration of the Cu(II) resting enzyme form was observed. This was proposed, based on Marcus Theory, to occur via innersphere superoxide formation, which was supported by a computational comparison to well-defined Cu-superoxide model complexes. Furthermore, the optimized structure of the Cu(I) active site showed how the enzyme is able to perform the difficult one-electron reduction of O2 by driving the reaction with the favorable interaction between the formed Cu(II) and superoxide. This provides important insight into how mononuclear Cu sites in general can activate O2 for catalysis.