Cross-link formation of the cysteine 228-tyrosine 272 catalytic cofactor of galactose oxidase does not require dioxygen

Abstract

Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redox-active cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions. Reactions of premat-GO with Cu(II) were investigated using optical, EPR, and resonance Raman spectroscopy, SDS-PAGE, and X-ray crystallography. Premat-GO reacted anaerobically with excess Cu(II) to efficiently form the thioether bond but not the Y272 radical. A potential C228-copper coordinated intermediate (lambda max = 406 nm) in the processing reaction, which had not yet formed the C228-Y272 cross-link, was identified from the absorption spectrum. A copper-thiolate protein complex, with copper coordinated to C228, H496, and H581, was also observed in a 3 min anaerobic soak by X-ray crystallography, whereas a 24 h soak revealed the C228-Y272 thioether bond. In solution, addition of oxygenated buffer to premat-GO preincubated with excess Cu(II) generated the Y272 radical state. On the basis of these data, a mechanism for the formation of the C228-Y272 bond and tyrosyl radical generation is proposed. The 406 nm complex is demonstrated to be a catalytically competent processing intermediate under anaerobic conditions. We propose a potential mechanism which is in common with aerobic processing by Cu(II) until the step at which the second electron acceptor is required.

Figure 1

(A) Active site of mature galactose oxidase showing the C228–Y272 thioether bond (PDB entry 1GOG). (B) Schematic representation of unprocessed and mature galactose oxidase protein forms. (C) SDS–PAGE of unprocessed and mature galactose oxidase protein forms. The anomalous SDS–PAGE mobility of mature galactose oxidase (65.5 KDa) results from the presence of the covalent C228–Y272 cross-link.

Figure 2

Aerobic biogenesis of premat-GO. (A) Addition of a 3.5-fold excess of Cu(II) (175 μm) to premat-GO (50.4 μm) in 50 mM Pipes and 0.1 M NaCl (pH 6.8) at 25 °C recorded at 2.5 min intervals. The spectrum of premat-GO prior to copper addition is also shown. (B) Appearance of the 445 nm absorbance band of newly formed oxidized mature GO (k₄₄₅ = 0.21 ± 0.03 min^-1) fitted to a single exponential. The inset shows a 7% Tris-acetate SDS–PAGE analysis of formation of the C228–Y272 bond. (C) CD spectrum of the oxidized mature GO produced following treatment with an aerobic excess of Cu(II). The CD spectrum of copper-free premat-GO is also shown.

Figure 3

Formation of the anaerobic Cu(II)–premat-GO complex. Cu(II) (0.8 equiv, 104 μM) was added under anaerobic conditions to premat-GO (130 μm) in 50 mM Pipes (pH 6.8) at 25 °C. The inset shows the formation of the absorption band at 406 nm vs time.

Figure 4

Copper–thiolate protein complex. (A) CD spectrum of the anaerobic Cu(II)–premat-GO complex (thick line) and copper-free premat-GO (thin line). (B) Resonance Raman spectrum of the anaerobic Cu(II)–premat-GO complex.

Figure 5

Anaerobic reaction of premat-GO (10 μm) in 50 mM Pipes (pH 6.8) with Cu(II) (10 μM) over time. Under these conditions, a peak at 406 nm appears (A) followed by its decay (B). The time course for the 406 nm absorption band is shown in panel C, where the inset shows a 8% Tris-HCl SDS–PAGE analysis of formation of the C228–Y272 bond over the time course of 0.5–20 h.

Figure 6

Formation of oxidized galactose oxidase following the addition of dioxygen-saturated buffer to the bleached protein form. (A) Cu(II) (190 μm) was added in a single aliquot to premat-GO (53 μm) in 50 mM Pipes buffer (pH 6.8) with spectra recorded at 3, 5, 7, 9, 11, 12, and 13 min. The inset shows the formation of the cross-link as analyzed by 7% Tris-actate SDS–PAGE, sampled at 1, 3, 5, 10, and 30 min. (B) An equal volume of oxygenated buffer was added to the bleached protein form with spectra recorded at 1, 3, 5, 7, 9, 11, 13, and 14 min.

Figure 7

Effect of EDTA and Cu(I) on premat-GO processing analyzed by SDS–PAGE. (A) Migration of protein treated with no EDTA (0), an equimolar amount of EDTA (1), or a 3.5-fold molar excess of EDTA (3.5) prior to addition of a 3.5-fold molar excess of Cu(II). (B) Samples following a 5 min exposure to a stoichiometric amount of Cu(I) under anaerobic (−O₂) or aerobic (+O₂) conditions. (C) Samples of premat-GO incubated with a stoichiometric amount of Cu(I) anaerobically were removed for SDS–PAGE analysis at 2, 10, 60, 180, and 1200 min.

Figure 8

Crystal structures of the active site of GO processing forms. (A) Premat-GO prepared and crystallized under copper-free conditions, showing the final 2F_o – F_c map contoured at 1.0σ. (B) After a 3 min Cu(II) anaerobic soak, the F_{o(2 mM copper soak)} – F_{o(premat copper free)} map contoured at 4σ indicates coordination of copper to the active site histidines. A negative peak on the C228 sulfur and a positive peak adjacent to the copper are consistent with rotation of C228 to coordinate to the incoming copper. The structure shown is the premat-GO with a modeled copper ion (green). Note that W290 is not well ordered and is not shown. (C) After a 24 h Cu(II) anaerobic soak, the final F_o – F_c map contoured at 1.0σ shows the thioether bond clearly formed and with W290 now in a stacking interaction with the C228–Y272 feature. (D) Fully processed mature GO (PDB entry 1GOG) for comparison with panel C.

scheme 1