Spectroscopic Studies of Perturbed T1 Cu Sites in the Multicopper Oxidases Saccharomyces cerevisiae Fet3p and Rhus vernicifera Laccase:  Allosteric Coupling between the T1 and Trinuclear Cu Sites

Abstract

The multicopper oxidases catalyze the 4e- reduction of O2 to H2O coupled to the 1e- oxidation of 4 equiv of substrate. This activity requires four Cu atoms, including T1, T2, and coupled binuclear T3 sites. The T2 and T3 sites form a trinuclear cluster (TNC) where O2 is reduced. The T1 is coupled to the TNC through a T1-Cys-His-T3 electron transfer (ET) pathway. In this study the two T3 Cu coordinating His residues which lie in this pathway in Fet3 have been mutated, H483Q, H483C, H485Q, and H485C, to study how perturbation at the TNC impacts the T1 Cu site. Spectroscopic methods, in particular resonance Raman (rR), show that the change from His to Gln to Cys increases the covalency of the T1 Cu-S Cys bond and decreases its redox potential. This study of T1-TNC interactions is then extended to Rhus vernicifera laccase where a number of well-defined species including the catalytically relevant native intermediate (NI) can be trapped for spectroscopic study. The T1 Cu-S covalency and potential do not change in these species relative to resting oxidized enzyme, but interestingly the differences in the structure of the TNC in these species do lead to changes in the T1 Cu rR spectrum. This helps to confirm that vibrations in the cysteine side chain of the T1 Cu site and the protein backbone couple to the Cu-S vibration. These changes in the side chain and backbone provide a possible mechanism for regulating intramolecular T1 to TNC ET in NI and partially reduced enzyme forms for efficient turnover.

Theoretical studies of the active-site structure, spectroscopic and thermodynamic properties, and reaction mechanism of multicopper oxidases

“…Still, it is clear that there is significant communication between the two redox sites in MCOs. These results are in accordance with the experimental observation that the redox potentials of Cu-T1 changes by up to 75 mV when two His ligands of the TNC were mutated [82].…”

“…Augustine et al [82] have investigated the influence of the mutation of the two His Cu-T3 ligands that lie along the Cu-T1 → Cu-T23 ET pathway to Gln and Cys residues. By using resonance Raman spectroscopy, they showed that these mutations increase the covalency of the Cu-S bond at the Cu-T1…”

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

“…Laccases are generally found in plants and fungi [6], but they have also been reported in a few bacteria including Azospirillum lipoferum [7], Bacillus sphaericus [1], Marinomonas mediterranea [8] and Streptomyces griseus [9]. Although fungal laccases have a higher reduction potential of type I copper than bacterial laccases and this makes them more suitable for commercial applications [10] their use present the following drawbacks: slow growth and difficulty in controlling the glycosylation degree [11]. In addition, bacterial laccases are more amenable to genetic manipulation than fungal laccases [12].…”

Novel multienzyme oxidative biocatalyst for lignin bioprocessing

“…[2][3][4] In Nature the selective oxidation of lignin is carried out by white-rot basidiomycetes fungi that produce a pool of extracellular ligninolytic enzymes such as laccases and peroxidases. 5,6 In particular laccases can easily oxidise phenolic groups 7 and in presence of radical mediator, such as 1-hydroxybenzotriazole, their reactivity can be extended towards other functional groups as phenyl-aryl ethers. 8 Mn-peroxidase and lignin peroxidases are able to oxidize lignin at the phenolic and non-phenolic aryl-ether positions respectively.…”