The state of copper in Neurospora laccase

1. Neurospora crassa laccase has been prepared from the growth medium and studied by optical absorption, circular dichroism and electron paramagnetic resonance (EPR) spectroscopy. The molecular weight, the copper content and the amino acid composition have also been determined. 2. The molecular weight as determined by gel filtration in 6 M guanidine hydrochloride and by sodium dodecyl sulfate gel electrophoresis is found to be 64 000. The enzyme contains 3.8 copper ions per 64 000. 3. The visible and the near ultraviolet difference absorption spectrum shows two maxima, at 330 and 595 nm, and a shoulder at about 720 nm. The circular dichroism spectrum between 300 and 760 nm contains five bands in the oxidized enzyme. After reduction of the enzyme with ascorbate there remains only a band at 305 nm. 4. EPR measurements show that 52% of the total copper in the protein is paramagnetic. Two EPR signals of equal intensity with different hyperfine splitting constants, of 9 and 18.5 mT, are present, which are assigned to Type 1 Cu2+ and Type 2 Cu2+, respectively, as found in other blue copper-containing oxidases.