Evidence Against Reduction of Cu2+ to Cu+ during Dioxygen Activation in a Copper Amine Oxidase from Yeast
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Abstract
The role of metal in copper amine oxidases was studied by removal and replacement of the copper in the enzyme from Hansenula polymorpha. The active-site cupric ion was reduced with the substrate, methylamine, and removed by anaerobic dialysis against cyanide and EDTA. Metal-free protein was found to have little or no activity in oxidizing methylamine and was unreactive toward phenylhydrazine. Addition of cobaltous ion to the metal-free protein restored 19% of the native specific activity and 75% of the native reactivity toward phenylhydrazine. A detailed kinetic study of the cobalt-substituted amine oxidase revealed that the major cause of the reduced specific activity was a 68-fold increase in the apparent Km for oxygen. Saturation of the cobalt enzyme with oxygen gave a kcat equivalent to the native kcat. This study of a cobalt-substituted amine oxidase supports the mechanism proposed by Su and Klinman [Su; Klinman Biochemistry 1998, 37, 12513−12525] in which electrons are passed directly from the reduced TPQ cofactor into pre-bound oxygen without the need for a prior reduction of the metal. Instead of transferring electrons, copper is proposed to provide electrostatic stabilization during a rate-limiting reduction of molecular oxygen to superoxide anion.
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