TRIVALENT COPPER, SUPEROXIDE, AND GALACTOSE OXIDASE

In a detailed mechanistic investigation of the reaction catalyzed by the monocopper enzyme, galactose oxidase, the following observations (among others) have been made: (1) in kinetic experiments, O2 uptake vs. time plots are frequently not linear but show induction periods or bursts depending on conditions, and enzyme inactivation by the product H2O2 occurs unless catalase is present; (2) the rate of O2 uptake is inhibited by superoxide dismutase at pico-to nanomolar concentrations, and during this inhibition the intensity of the EPR signal given by the galactose oxidase Cu (II) increases; (3) ferrocyanide also inhibits the enzymic reaction and causes an increase in the intensity of the EPR signal given by the enzymic Cu (II); (4) the rate of the enzyme-catalyzed reaction of O2 is increased by adding superoxide, or oxidants such as ferricyanide, chloroiridate, or manganic ions; (5) excess ferricyanide causes a complete disappearance of the EPR signal given by the enzymic copper but has no effect on the molecular weight of the enzyme; (6) in the presence of ferricyanide-ferrocyanide redox buffers the rate of O2 uptake, and the intensity of the EPR signal given by the enzymic Cu (II), define a one-electron redox potential of 0.41 V above pH 7.5 but which increases by 0.06 V per pH unit at pHs below 7 (pK3 of the group involved is 7.25); (7) large changes in the optical spectrum of the enzyme are observed on adding oxidants, but the same spectrum (absorption maxima at 440 and 800 nm with molar extinction coefficients of ca. 4900 and 2800, respectively, and a large increase in absorption at 320 nm [Δ⋍≃ 2800]) is obtained irrespective of the oxidant used; (8) titration of galactose oxidase with hexachloroiridate indicates that the species giving the above spectrum is one electron further oxidized than the major species present in the resting enzyme; (9) with oxidants present the rate is maximal at pHs 6 to 7 but decreases only slowly at lower or higher pHs; (10) no exchange of radioactive aldehyde product into reactant alcohol occurs under several different conditions tried. The results constitute the first direct evidence for trivalent copper [Cu(III)] in an enzymic reaction. They also clarify the role of superoxide to a greater extent than has been possible with most other enzymic reactions. An overall mechanism consistent with all the data involves oxidation of the alcohol to the aldehyde by the Cu (III) enzyme with concomitant formation of a Cu (I) enzyme. Reoxidation of this species to the Cu(III) form by O2 apparently occurs with a Cu (II) superoxide species as a fleeting intermediate. About once in several thousand turnovers superoxide leaks out from the catalytic cycle to form the inactive Cu (II) enzyme which can be converted to the catalytically active Cu (III) enzyme with one-electron oxidants. The probable role of superoxide in most oxidase and oxygenase reactions and the likelihood of Cu (III) involvement in other copper enzymes are briefly discussed. © 1978, American Chemical Society. All rights reserved.