The two oxidized forms of the trinuclear Cu cluster in the multicopper oxidases and mechanism for the decay of the native intermediate

Abstract

Multicopper oxidases (MCOs) catalyze the 4e ؊ reduction of O2 to H 2O. The reaction of the fully reduced enzyme with O2 generates the native intermediate (NI), which undergoes a slow decay to the resting enzyme in the absence of substrate. NI is a fully oxidized form, but its spectral features are very different from those of the resting form (also fully oxidized), because the type 2 and the coupled-binuclear type 3 Cu centers in the O 2-reducing trinuclear Cu cluster site are isolated in the resting enzyme, whereas these are all bridged by a 3-oxo ligand in NI. Notably, the one azide-bound NI (NIAz) exhibits spectral features very similar to those of NI, in which the 3-oxo ligand in NI has been replaced by a 3-bridged azide. Comparison of the spectral features of NI and NI Az, combined with density functional theory (DFT) calculations, allows refinement of the NI structure. The decay of NI to the resting enzyme proceeds via successive proton-assisted steps, whereas the ratelimiting step involves structural rearrangement of the 3-oxo-bridge from inside to outside the cluster. This phenomenon is consistent with the slow rate of NI decay that uncouples the resting enzyme from the catalytic cycle, leaving NI as the catalytically relevant fully oxidized form of the MCO active site. The all-bridged structure of NI would facilitate electron transfer to all three Cu centers of the trinuclear cluster for rapid proton-coupled reduction of NI to the fully reduced form for catalytic turnover.laccase ͉ superexchange ͉ electron paramagnetic resonance ͉ magnetic circular dicroism ͉ density functional theory M ulticopper oxidases (MCOs) catalyze the 4e Ϫ reduction of O 2 to H 2 O by using four Cu centers. The electrons are taken up at the blue type 1 (T1) Cu site and transferred Ϸ13 Å to the trinuclear Cu cluster, composed of a normal type 2 (T2) and a coupled-binuclear type 3 (T3) site, where the O 2 reduction occurs (1, 2). The T2 Cu site is held in the protein by two His and has a water-derived OH Ϫ ligand external to the cluster, whereas the OH Ϫ -bridged T3 Cu site is held by three His on each Cu. The reaction of O 2 with the fully reduced enzyme generates the native intermediate (NI) (3-7). A combination of Cu K-edge x-ray spectroscopy (XAS) and magnetic circular dichroism (MCD) has unambiguously demonstrated that NI is a fully oxidized form with fully reduced O 2 (3). Recent model studies combined with calculations have further demonstrated that the three Cu(II) centers in the trinuclear site are all bridged by a 3 -oxo ligand (8, 9). In the absence of reducing substrate, NI slowly decays to the resting enzyme, in which the one remaining O atom of the O 2 is terminally bound as OH Ϫ to the T2 site, as indicated by 18 O isotope ratio MS (IRMS) (10, 11) and 17 O EPR (12) experiments. Thus, this process requires the 3 -oxo-bridged NI to undergo a large rearrangement. Importantly, the slow rate of NI decay conflicts with the much higher turnover number, indicating that the resting enzyme is not involved in the catalytic cyc...

O2 Reduction to H2O by the multicopper oxidases

“…From figure 28B this rotation removes one proton from the H 2 O at T3Cu B to reform the T3μ 2 OH bridge and as the T2-T3Cu A bridging H 2 O rotates out of the cluster it donates one proton to the terminal OH at the T2 Cu, weakening this bond and allowing its substitution by the rotated OH from O 2 reduction. 47 This process is calculated to have a barrier of ~ 8.5 kcal/mol which is reasonable given that the experimental barrier for the conversion of NI to resting is 8.8-13.9 kcal/mol. 48 Importantly, this rate of conversion (k = 0.05 s −1 ) 27 is orders of magnitude slower than the turnover rate of the enzyme (k= 350 s −1 ).…”

On the Possibility of Uphill Intramolecular Electron Transfer in Multicopper Oxidases: Electrochemical and Quantum Chemical Study of Bilirubin Oxidase

“…Whereas processes 1 and 2 are in general reversible (Fig. 4B, insert [20,83], is a proton-independent, essentially irreversible, and very fast process [8,10]. Bioelectrocatalytic current densities from BOx-modified electrodes, when limited by the kinetics of the enzyme, can be calculated using the following equation:…”

Structural insight into the oxidation of sinapic acid by CotA laccase

“…Yoon and colleagues have proposed a possible mechanism for the reduction of TNC based on spectral data and calculations. It was believed the all-bridged structure would facilitate electron transfer to all three copper atoms in TNC and the proton-coupled reduction of TNC (Yoon et al, 2007). Here, in the molecule A of CotA/SA complex, Cu3a, Cu3b and Cu2 are observed all bridged to the oxygen, which will favor the reduction of TNC.…”

“…Here, structural analysis on the oxygen binding in the molecule A of CotA/SA complex suggests, during the molecule oxygen reduction and splitting, the two oxygen atoms are coordinated asymmetrically with copper ions in TNC, in which one oxygen atom is coordinated with 3 copper ions, and 2 copper ions for the other oxygen atom. Probably, the structural asymmetry on Cu-Cu and Cu-O and the difference in electron transfer drop a hint about sequentially reduction of three Cu centers in TNC (Yoon et al, 2007). Further investigation should be performed to confirm if this differential reduction is related to the photon assisted O-O bond cleavage (Ferraroni et al, 2007).…”