Seeing enzymes in action
Single-molecule studies of electron-transfer between copper centers of small laccase (SLAC) from S. coelicolor
Single molecule enzymology provides an unprecedented level of detail about aspects of enzyme mechanisms which have been difficult to probe in bulk. One such aspect is intramolecular electron transfer (ET) which is a recurring theme in the research on oxidoreductases. Recently, we introduced a technique to study ET in enzymes at single molecule level by means of confocal fluorescence microscopy (PNAS. 2008, 105, 3250).
I will present recent results on an enzyme, small laccase (SLAC), from S. coelicolor which converts O2 to H2O with concomitant oxidation of organic substrate(s). SLAC is unique, among commonly studied multicopper oxidases (MCO), in its structure (being a homo-trimer of two-domain monomers) and function where it employs a so-called type 1 (T1) Cu site, a trinuclear Cu centre (TNC) and a tyrosine residue (Y108) to catalyze this process which has a high activation barrier. I have measured, for the first time, intramolecular ET rates between the T1 and TNC of SLAC during turnover, one molecule at a time. The distribution across many molecules shows an average ET rate ~450 s-1 independent of substrate concentration, consistent with the proposed enzyme mechanism and with the results of transient kinetics experiments. The activation energy for ET amounts to 350 meV and varies from molecule to molecule with a spread of ±25 meV. Experiments are underway to measure other microscopic rate constants in the enzymatic cycle which have never been measured in bulk. The method is suitable to study ET in a wide range of redox active enzymes in-vitro as well as in-vivo.