New insights into the catalytic active-site structure of multicopper oxidases

Abstract

Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of enzymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO from Escherichia coli and laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X-ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O2 to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O2-reduction process are discussed.

Structure and function of Aspergillus niger laccase McoG

“…Furthermore, the distances between the copper ions inside the trinuclear cluster (Table S1) are indicative of a reduction of the metal ions induced by the electrons liberated during the X-ray data collection at a synchrotron radiation source as already reported for other laccase structures [42][43][44][45][46]. We therefore refined a peroxide ion in this position.…”

A novel resting form of the trinuclear copper center in the double mutant of a multicopper oxidase, CueO, Cys500Ser/Glu506Ala

“…Since T2Cu and two N ligands are almost linearly arranged, the geometry of T2Cu does not significantly deviate from planer structures [3,7]. Two T3Cu signals with small A II values (15.0 x 10 -3 and 15.6 x 10 -3 cm -1 ) have been reported for a bilirubin oxidase mutant, His94Val, in which a His ligand to T2Cu was substituted by Val [15].…”

“…However, other resting TNC structures related with the reaction mechanism have also been discovered in an increased number of MCOs and mutants [1 and references therein, 3,4].…”

“…One of T3Cu (T3Cua) is in the d- interaction distance with Trp139 (~4 Å). A His ligand to another T3Cu (T3Cub) is hydrogen bonded with Asp112, one of key amino acids to form water molecules [3,7]. This unequivalence between T3Cus might have become more peculiar due to the loss in magnetic interaction.…”

“…The total EPR-detectable Cu(II) was 2.2 per a protein molecule. This fact excludes possibilities that the rhombic Cu-EPR signal came from Cu 2+ ions incorporated into the vacant T1Cu site and from T2Cu with a strong distortion.Cu 2+ ion has never been incorporated into the T1Cu site, of which Cys ligand is changed to Ser [2,5-9].Since T2Cu and two N ligands are almost linearly arranged, the geometry of T2Cu does not significantly deviate from planer structures [3,7]. Two T3Cu signals with small A II values (15.0 x 10 -3 and 15.6 x 10 -3 cm -1 ) have been reported for a bilirubin oxidase mutant, His94Val, in which a His ligand to T2Cu was substituted by Val [15].…”