SOLUBILIZATION AND PROPERTIES OF COPPER REDUCING ENZYME-SYSTEMS FROM THE YEAST-CELL SURFACE IN DEBARYOMYCES-HANSENII

Copper reducing materials could be solubilized with Zymolyase-100 T from the surface of yeast cells (cell wall) in the presence of 1.0 M sorbitol as a stabilizer of spheroplasts formed. Copper reduction by solubilized cell wall materials (SCWM) was biphasic: the first was independent on NAD(P)H, and the second was dependent. The NAD(P)H-independent copper reduction may be nonenzymatic and the SH groups of cell wall components were the main electron donors. On the other hand, the properties of NAD(P)H-dependent copper reduction of SCWM revealed that solubilized cell wall materials contained a NAD(P)H-copper reducing enzyme, which was designated as a copper reductase. Copper reduction was affected by ligands to copper such as amino acids and ethylenediaminetetraacetic acid (EDTA). Copper reducing velocity could be measured by simultaneous addition of EDTA and bathocuproinedisulfonic acid (BCDS) to the reaction mixture. EDTA completely stopped the copper reduction by SCWM. The optimum pH of copper reductase was about 5.0. Copper-dependent NAD(P)H oxidase activity was also found in copper reducing materials. These results suggest that both NADH and NADPH serve as an electron donor of Cu(II) reduction in the copper reductase and that the copper uptake in the yeast may be enzymatically controlled by this copper reductase and SH groups.