The role of positive charged residue in the proton-transfer mechanism of two-domain laccase from Streptomyces griseoflavus Ac-993

Abstract

Multi-copper oxidases are capable of coupling the one-electron oxidation of four substrate equivalents to the four-electron reduction of dioxygen to two molecules of water. This process takes place at the trinuclear copper center of the enzymes. Previously, the main catalytic stages for three-domain (3D) laccases have been identified. However, for bacterial small two-domain (2D) laccases several questions remain to be answered. One of them is the nature of the protonation events upon the reductive cleavage of dioxygen to water. In 3D laccases, acidic residues play a key role in the protonation mechanisms. In this study, the role of the Arg240 residue, located within the T2 tunnel of 2D laccase from Streptomyces griseoflavus Ac-993, was investigated. X-ray structural analysis and kinetic characterization of two mutants, R240A and R240H, have provided support for a role of this residue in the protonation events.

Communicated by Ramaswamy H. Sarma

Graphical Abstract

Keywords:

• Two-domain laccases
• crystal structures
• T2/T3 copper site
• tunnels
• proton transfer

Acknowledgments

We would particularly like to acknowledge Manfred S. Weiss for his assistance during the experiment at the BESSY II.

Author contributions

Conceptualization: A.G. and S.T.; data curation: A.G. and S.T.; formal analysis: A.G.; funding acquisition: A.G. and S.T.; investigation: I.K. and S.T.; methodology: A.G., I.K., P.O., A.M. and S.T.; project administration: A.G. and S.T.; resources: A.G., P.O. and P.T.; supervision: A.G. and S.T.; validation: A.G.; visualization: A.G. and I.K.; A.G. and S.T. wrote the paper with input from all authors.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Russian Foundation for Basic Research under Grant # 18-04-00270a, 19-34-90121. Paulo Oliveira acknowledges financial support from Fundação para a Ciência e a Tecnologia (FCT Investigator grant IF/00256/2015).