Coordination environment and fluoride binding of type 2 copper in the blue copper protein ascorbate oxidase

Abstract

The coordination environment of the type 2 (nonblue) copper in native ascorbate oxidase (L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) and of a derivative of the enzyme having the type 1 (blue) copper reversibly bleached has been examined by electron paramagnetic resonance (EPR) spectroscopy. In the g\perp region of the spectrum of bleached ascorbate oxidase, a seven-line superhyperfine pattern is seen that is attributed to the presence of three nitrogen-donor ligands to a type 2 copper having tetragonal geometry. The superhyperfine splitting patterns in the g|| region of the EPR spectra of native and bleached ascorbate oxidase show that as many as two fluorides may bind to type 2 copper. Because fluoride inhibits the enzyme competitively with respect to ascorbic acid, it is proposed that the type 2 copper is part of the ascorbate binding site.

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