Shedding light on biological redox processes - Single molecule studies of Cu-containing oxidoreductases

The investigation of electron-transfer (ET) processes, as well as redox reactions is important to understand a whole series of biochemical processes. Single-molecule techniques are a precious tool whose diffusion and technical evolution made them available for the study of biologically relevant reactions. In this thesis an approach to the study of electron-transfer and redox reaction by means of single-molecule techniques is presented. Two proteins were investigated: azurin, from Pseudomonas aeruginosa, which contains a type-1 copper center, and the blue nitrite reductase (bNiR), from Alcaligenes xylosoxidans, which contains a type-1 and a type-2 copper center. By means of a resonant energy transfer (FRET) __ based approach it was possible to use a fluorescent label attached to the surface of the proteins to obtain information about their redox state. By using fluorescence correlation spectroscopy in solution it was possible to study ET processes between azurin and the label and between two covalently linked azurin monomers. Nitrite reductase was immobilized in an agarose matrix and single enzyme molecules were investigated by using scanning confocal microscopy: new details about the internal ET between the two copper centers of bNiR were revealed and new light was shed on the catalytic cycle of the enzyme