INSTITUTO DE QUIMICA, FISICA DE LOS MATERIALES, MEDIOAMBIENTE Y ENERGIA
Unidad Ejecutora - UE
Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes
DURÃO, P.; CHEN, Z.; FERNANDES, A. T.; HILDEBRANDT, P.; MURGIDA, D. H.; TODOROVIC, S.; PEREIRA, M. M.; MELO, E. P.; MARTINS, L. O.
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY
Año: 2008 vol. 13 p. 183 - 183
The copper content of recombinant CotA laccasefrom Bacillus subtilis produced by Escherichia colicells is shown to be strongly dependent on the presence ofcopper and oxygen in the culture media. In copper-supplementedmedia, a switch from aerobic to microaerobicconditions leads to the synthesis of a recombinant holoenzyme,while the maintenance of aerobic conditionsresults in the synthesis of a copper-depleted population ofproteins. Strikingly, cells grown under microaerobicconditions accumulate up to 80-fold more copper thanaerobically grown cells. In vitro copper incorporation intoapoenzymes was monitored by optical and electron paramagneticresonance (EPR) spectroscopy. This analysisreveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and thetype 3 copper centers. The copper reconstitution of holo-CotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.