Galactose Oxidase as a Model for Reactivity at a Copper Superoxide Center

Abstract

The mononuclear copper enzyme, galactose oxidase, has been investigated under steady-state conditions via O 2 -consumption assays using 1-O-methyl-α-D-galactopyranoside as the sugar substrate to produce an aldehyde at the C-6 position. The rate-determining step of the oxidative halfreaction was probed through the measurement of substrate and solvent deuterium and O-18 isotope effects on k cat /K m (O 2 ). The reaction conforms to a ping-pong mechanism with the kinetic parameters for the reductive half, k cat /K m (S) = 8.3 × 10 3 M −1 s −1 at 10°C and pH 7.0, comparing favorably to literature values. The oxidative half-reaction yielded a value of k cat /K m (O 2 ) = 2.5 × 10 6 M −1 s −1 . A substrate deuterium isotope effect of 32 was measured for the k cat /K m (S), while a smaller, but significant value of 1.6-1.9 was observed on k cat /K m (O 2 ). O-18 isotope effects of 1.0185 with either protiated or deuterated sugar, together with the absence of any solvent isotope effect, lead to the conclusion that hydrogen atom transfer from reduced cofactor to a Cu(II)-superoxo intermediate is fully rate-determining for k cat /K m (O 2 ). The measured O-18 isotope effects provide corroborative evidence for the reactive superoxo species in the dopamine β-monooxygenase/peptidylglycine α-hydroxylating monooxygenase family, as well as providing a frame of reference for copper-superoxo reactivity. The combination of solvent and substrate deuterium isotope effects rules out solvent deuterium exchange into reduced enzyme as the origin of the relatively small substrate deuterium isotope effect on k cat /K m (O 2 ). These data indicate fundamental differences in the hydrogen transfer step from the carbon of substrate vs. the oxygen of reduced cofactor during the reductive and oxidative half reactions of galactose oxidase.

Structural Snapshots from the Oxidative Half-reaction of a Copper Amine Oxidase

“…However, the existence of an outer-sphere electron transfer mechanism in either copperor cobalt-containing CAOs has been recently challenged in pea seedling CAO and AGAO based on 18 O KIE studies and density functional theory (15,16), coupled with the smaller than expected 18 O KIE observed in HPAO-1 and bovine serum amine oxidase that are more in line with inner-sphere electron transfer (18,24). However, it should be noted that a larger 18 O KIE than observed in CAO is seen in the Cu(I) inner-sphere electron transfer activation of O 2 in galactose oxidase, indicating caution is warranted when linking 18 O KIE magnitude to the O 2 activation mechanism (52). The possibility of a Co(III)/Co(II) couple being in operation rather than the unlikely Co(II)/Co(I) couple cannot be discounted, and recent competitive 18 O KIE and deuterium KIE studies in AGAO coupled with density functional theory are consistent with an identical inner-sphere oxidative half-reaction mechanism for both the copper and cobalt forms of the enzyme (15).…”

Lewis Acid-Induced Change from Four- to Two-Electron Reduction of Dioxygen Catalyzed by Copper Complexes Using Scandium Triflate

“…1 For example, in the oxidation of their substrates, galactose oxidases 2 and amine oxidases 3 effect the two-electron reduction of O 2 to hydrogen peroxide 4 whereas multicopper oxidases (MCO’s) 5 and heme-copper oxidases (HCO’s) 6 facilitate the four-electron reduction of O 2 to H 2 O. The catalytic four-electron reduction of O 2 with synthetic copper complexes as well as other metal complexes has merited special attention because of not only the mechanistic interest in relation to MCO’s but also in development of a fuel cell technology and their application using earth-abundant metals such as iron, cobalt and copper.…”

Stepwise Protonation and Electron-Transfer Reduction of a Primary Copper–Dioxygen Adduct

“…10 This is followed by reduction-protonation by an H-atom donor, a substrate or proton-electron sources to give a cupric-hydroperoxo product ( B ). Further protonation could liberate hydrogen peroxide, such as occurs in the so-called T2 oxidases, galactose oxidase (GO) 11 or copper amine oxidase (CAO), 12 Figure 1.…”