The role of copper in the catalytic action of laccase and ceruloplasmin

Abstract

1. 1. The rate of reoxidation of ascorbic acid-reduced copper in laccase (EC 1.10.3.2) and ceruloplasmin has been measured spectrophotometrically in a stopped-flow apparatus. The kinetics of reduction of Cu 2+ by various substrates has been studied by electron-spin resonance absorption. The rate of formation and decay of a free radical formed with p-phenylenediamine as substrate has also been measured, and the radical has been identified as the positive ion. 2. 2. The rate of O 2 consumption, calculated from the kinetic parameters on the basis of a mechanism involving reduction of Cu 2+ by the substrate followed by reoxidation by O 2, agrees well with the experimentally determined rate, thus providing strong evidence for the correctness of this mechanism. 3. 3. In a few cases, the experimental curves for Cu 2+ and free radical have been compared with calculated curves, obtained by numerical integrations of kinetic equations with a digital computer. The results indicate that the radical decay occurs by a reaction not involving the enzymes. 4. 4. Electron-spin resonance measurements of a bacterial copper protein, azurin, indicate that small hyperfine splittings, as observed with laccase and ceruloplasmin, may occur without an exchange interaction between Cu 2+ and Cu 1+. It is suggested that the Cu 1+ of laccase and ceruloplasmin may bind the substrates by interacting with their π-electrons.