Fluorescence and active site engineering studies of copper-containing oxidoreductases

Abstract

Copper-containing proteins are involved in a wide range of biological processes mainly via oxidation and reduction reactions. The oxidation state of these proteins can be monitored via Förster resonance energy transfer (FRET) between a covalently attached fluorescent dye and the protein’s redox active centre. Consequently, changes in absorbance upon reduction or oxidation of the protein can be related to changes in the fluorescence intensity and lifetime. This FRET-based approach has been applied to study the catalytic activity of the copper-containing blue nitrite reductase (bNiR) from Alcaligenes xylosoxidans at the single molecule level by means of scanning confocal microscopy combined with fluorescence lifetime imaging (FLIM). bNiR catalyzes the reduction of nitrite to nitric oxide during denitrification. The active centre of bNiR consists of a type 1 (T1) Cu site, which acts as the initial port of entry for electrons, and a type 2 (T2) Cu site, where nitrite reduction occurs. Detailed analysis of single molecules of immobilized, fluorescently labeled bNiR has allowed two populations of molecules to be identified that turn over with different catalytic rates. Previous studies of the catalytic mechanism of copper-containing NiRs distinguished two possible reaction pathways. The single molecule results imply these occur as a consequence of heterogeneity in the enzyme population. Fluorescent labeling of laccases, which catalyze the oxidation of a range of substrates coupled to the four electron reduction of O, with fluorescent dyes was 2 investigated for their potential use in the development of a FRET-based biosensor. A novel expression system for the Trametes versicolor laccase Lcc1 in Schizophyllum commune was developed. The recombinant protein is similar to another native laccase (Laccase A) from T. versicolor and both exhibit significantly higher catalytic efficiency with phenolic compounds than the bacterial small laccase (SLAC) from Streptomyces coelicolor. Regardless, preliminary data indicate involvement of a tyrosyl radical in the catalytic activity of fungal laccases, similar to what is observed in SLAC. bNiR and SLAC are trimers, with each monomer consisting of two cupredoxin- like domains. The structure of the catalytic site and the location of a T1 Cu site are different in these two enzymes. A crystal structure provides detailed insight into why an attempt to introduce the SLAC active site into bNiR was unsuccessful. Attempts to introduce T1 Cu sites into the cupredoxin-like domains of bNiR and SLAC, which normally lack this site, resulted in the introduction of a tetragonal thiolate-containing T2 Cu site.