Ligand interactions with galactose oxidase: mechanistic insights

Abstract

Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme.

The Stacking Tryptophan of Galactose Oxidase: A Second-Coordination Sphere Residue that Has Profound Effects on Tyrosyl Radical Behavior and Enzyme Catalysis,

“…The terminal nitrogen atom of N 3 − hydrogen bonds (3.25 Å) to W290-N ε1 , which may influence azide binding to the wild type enzyme. Bond distances to the equatorial and axial tyrosines (Table 3) are consistent with an increase of the Cu(II)-Y495 bond in the azide complex, as previously observed (43). Only small variations in the coordination geometry are evident in the various structures, and these are mostly below the significance level at the current resolution.…”

“…Loss of the ~800 nm band in the azide complexes of the oxidized proteins may reflect protonation (and dissociation from copper) of Y495 induced by azide binding (Figure 5), as detailed previously (43). The band at 500 nm may be assigned, in part, as a π → π* radical transition, indicative of the persistence of the tyrosyl radical in the W290 variants upon anion binding (68).…”

Room temperature aerobic oxidation of alcohols using CuBr2 with TEMPO and a tetradentate polymer based pyridyl-imine ligand

“…The result shows the specificity of our CuBr 2 /L1/TEMPO catalytic system towards the primary alcohol because 100% of the benzyl alcohol was converted into benzaldehyde, whereas 1-phenylethanol remained unreacted within 4 h. This chemoselectivity is consistent with one of the typical characteristics of the natural copper protein galactose oxidase (GOase) and can be used for the selective oxidation of different alcohols [57][58][59][60][61]. Also, this potentially offers some insight into the reaction mechanism in this system and the increased yields seen for L1-L6 compared with L7.…”

Identification of Catalytic Residues in Glyoxal Oxidase by Targeted Mutagenesis

“…In particular, one of the tyrosines (Tyr 272 ) is cross-linked to a cysteinyl residue, forming a Tyr-Cys dimer site (13) that has been identified in spectroscopic (14,15) and modeling (16 -18) studies as the radical redox site in the enzyme. The second tyrosine (Tyr 495 ) is bound axially to the active site metal ion, giving rise to distinctive features in the electronic spectra of the enzyme in the resting (Tyr ON ) state and this residue has been shown to be displaced (producing a Tyr OFF complex) when anions replace water in the inner sphere of the metal complex (19). This displacement is coupled to a protonation event that implies that Tyr 495 can serve as a general base, capable of activating bound substrate by proton abstraction.…”

“…Conservative mutagenesis of the "axial" tyrosine to phenylalanine (Y495F) (22,24) permanently converts the enzyme to a Tyr OFF form (19) lacking the spectroscopic signatures of axial tyrosinate coordination and virtually eliminates catalytic activity. Elimination of the cysteinyl residue that is involved in the Tyr-Cys redox cofactor of the mature protein by C228G mutagenesis (23) likewise profoundly alters both spectroscopic and catalytic properties of the copper center.…”