Mechanistic investigations of a recombinant laccase from Thermus thermophilus HB27

Abstract

Laccases belong to the family of multicopper oxidases (MCOs) that couple oxidn. of a wide spectrum of substrates to the four-electron redn. of dioxygen (O_2) to water. Laccases typically contain four copper ion sites arranged in two centers: one type 1 blue copper center and one trinuclear cluster (TNC) consisting of one type 2 copper and a binuclear type 3 copper pair. Dioxygen binding, activation, and redn. are believed to proceed at the TNC with electrons delivered from the type 1 site. The precise mechanism of coupling O_2 redn. to substrate oxidn. is a subject of considerable fundamental and technol. significance. Of particular interest is the mechanism by which laccases oxidize refractory substrates such as phenols and lignin. To gain deeper insight into laccase catalysis, we have cloned the gene for a recombinant multicopper oxidase from the hyperthermophilic bacterium Thermus thermophilus HB27 (Tth-MCO), and expressed the enzyme as a 6Histagged protein in Escherichia coli. Several mutants of the recombinant enzyme have been prepd. by sitedirected mutagenesis and investigated using various structural and spectroscopic techniques. Our recent results on the reaction pathways and intermediates involved in the enzymic reaction will be discussed.