Mechanistic Insights into Cu(I) Biogenesis of Galactose Oxidase

11/02/2020

Copper (Cu) active sites in metalloproteins, such as the secretory fungal enzyme galactose oxidase (GO), play essential roles in a wide range of biological processes. One of the most important is O2 activation, which mostly involves Cu(I) active sites. While a variety of spectroscopic methods have been utilized to provide insight into Cu(II) sites, Cu(I) sites are often considered to be “spectroscopically silent” due to their d10 closed-shell nature.

A study by Solomon et al. defines the Cu(I) frontier molecular orbital (FMO) that enables this O2 activation and demonstrates the considerable potential of Kβ X-ray emission spectroscopy (XES) in probing the FMO and key bonding interactions in “spectroscopically silent” Cu(I) active sites.

GO catalyzes the oxidation of primary alcohols to aldehydes. Some evidence suggests that GO may also be capable of the subsequent oxidation of the aldehyde to carboxylic acids. This and other research has led to the proposal that the physiological role of GO is the generation of H2O2, perhaps as a defense against pathogenic organisms. GO is also an important component in electrochemical biosensors of galactose that are used for various biotechnology applications. It is therefore critical to understand the mechanism of Cu(I) biogenesis.

Solomon et al. used Kβ XES on SSRL’s beam line 6-2 to characterize a series of Cu(I) compounds with varying biologically relevant ligand systems and compared the results to that of Cu(I) in GO to elucidate the detailed bonding interactions occurring between the metal site and the protein-based ligands. The studies were coupled with density functional theory (DFT) calculations to evaluate the sensitivity of the spectral features to the ligand environment.

The study reveals that the Cu(I) site in preprocessed GO (GOpre) is a three-coordinate environment with a 1Tyr/2His structural model. This is consistent with previously reported EXAFS measurements by the same research group. The frontier molecular orbital of Cu(I)-GOpre has mainly Cu 3d(x2−y2) character and dominantly contributes to the ∼8976 eV feature of the spectrum. This orbital is important since it has a lobe oriented along the open coordination position and functions to overlap with O during activation of O2. Kβ VtC XES of Cu(I)-GOpre shows that the highest-energy spectral feature originates from the redox-active orbital with Cu 3d character.

Together with characterization of the model data, Kβ XES provides a unique opportunity to probe the interaction between Cu(I) and O for O2 activation that is not accessible with other spectroscopic techniques. In this regard, Kβ XES is expected to give important insight into the reactivity of all dioxygen activating Cu(I) metalloprotein systems (i.e., not limited to GO) and relate the geometric structural assignment (obtained from crystallography or EXAFS methods) to unique electronic properties required for small-molecule activation in metalloproteins.

References

Lim, H., et al. 2020. “Kβ X‐ray Emission Spectroscopy as a Probe of Cu(I) Sites: Application to the Cu(I) Site in Preprocessed Galactose Oxidase,” Inorganic Chemistry 59(22), 16567–581. [DOI:10.1021/acs.inorgchem.0c02495]