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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.

SUBMITTER: Rogers MS

PROVIDER: S-EPMC2762401 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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

Rogers Melanie S MS Hurtado-Guerrero Ramón R Firbank Susan J SJ Halcrow Malcolm A MA Dooley David M DM Phillips Simon E V SE Knowles Peter F PF McPherson Michael J MJ

Biochemistry 20080905 39

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 ...[more]

PMID: 18771294

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Project description:Heme-copper oxidases have a crucial role in the energy transduction mechanism, catalyzing the reduction of dioxygen to water. The reduction of dioxygen takes place at the binuclear center, which contains heme a3 and CuB. The X-ray crystal structures have revealed that the C6' of tyrosine 244 (bovine heart numbering) is cross-linked to a nitrogen of histidine 240, a ligand to CuB. The role of the cross-linked tyrosine at the active site still remains unclear. In order to provide insight into the function of the cross-linked tyrosine, we have investigated the spectroscopic and electrochemical properties of chemical analogues of the CuB-His-Tyr site. The analogues, a tridentate histidine-phenol cross-linked ether ligand and the corresponding Cu-containing complex, were previously synthesized in our laboratory (White, K.; et al. Chem. Commun. 2007, 3252-3254). Spectrophotometric titrations of the ligand and the Cu-complex indicate a pKa of the phenolic proton of 8.8 and 7.7, respectively. These results are consistent with the cross-linked tyrosine playing a proton delivery role at the cytochrome c oxidase active site. The presence of the phenoxyl radical was investigated at low temperature using electron paramagnetic resonance (EPR) and Fourier transform infrared (FT-IR) difference spectroscopy. UV photolysis of the ligand, without bound copper, generated a narrow g=2.0047 signal, attributed to the phenoxyl radial. EPR spectra recorded before and after UV photolysis of the Cu-complex showed a g=2 signal characteristic of oxidized copper, suggesting that the copper is not spin-coupled to the phenoxyl radical. An EPR signal from the phenoxyl radical was not observed in the Cu-complex, either due to spin relaxation of the two unpaired electrons or to masking of the narrow phenoxyl radical signal by the strong copper contribution. Stable isotope (13C) labeling of the phenol ring (C1') Cu-complex, combined with photoinduced difference FT-IR spectroscopy, revealed bands at 1485 and 1483 cm(-1) in the 12C-minus-13C-isotope-edited spectra of the ligand and Cu-complex, respectively. These bands are attributed to the radical v7a stretching frequency and are shifted to 1468 and 1472 cm(-1), respectively, with 13C1' labeling. These results show that a radical is generated in both the ligand and the Cu-complex and support the unambiguous assignment of a vibrational band to the phenoxyl radical v7a stretching mode. These data are discussed with respect to a possible role of the cross-linked tyrosine radical in cytochrome c oxidase.

Dataset Information

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

Publications

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

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