Tuning the Type 1 Reduction Potential of Multicopper Oxidases: Uncoupling the Effects of Electrostatics and H-Bonding to Histidine Ligands

摘要

In multicopper oxidases (MCOs), the type 1 (T1) Cu acceptselectronsfrom the substrate and transfers these to the trinuclear Cu cluster(TNC) where O-2 is reduced to H2O. The T1 potentialin MCOs varies from 340 to 780 mV, a range not explained by the existingliterature. This study focused on the similar to 350 mV difference inpotential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range ofspectroscopies performed on the oxidized and reduced T1 sites in theseMCOs shows that they have equivalent geometric and electronic structures.However, the two His ligands of the T1 Cu in Fet3p are H-bonded tocarboxylate residues, while in TvL they are H-bonded to nonchargedgroups. Electron spin echo envelope modulation spectroscopy showsthat there are significant differences in the second-sphere H-bondinginteractions in the two T1 centers. Redox titrations on type 2-depletedderivatives of Fet3p and its D409A and E185A variants reveal thatthe two carboxylates (D409 and E185) lower the T1 potential by 110and 255-285 mV, respectively. Density functional theory calculationsuncouple the effects of the charge of the carboxylates and their differencein H-bonding interactions with the His ligands on the T1 potential,indicating 90-150 mV for anionic charge and similar to 100 mVfor a strong H-bond. Finally, this study provides an explanation forthe generally low potentials of metallooxidases relative to the widerange of potentials of the organic oxidases in terms of differentoxidized states of their TNCs involved in catalytic turnover.