Raman spectro-electrochemistry of O2-reducing S.Coelicolor laccase (SLAC): Exploring electron transfer pathways beyond the biocatalyst's active site

• Applicant: Professorin Dr. Patrycja Kielb
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 556062949

The scientific proposal aims to investigate the oxygen reduction reaction (ORR) in multi-metallic Cu-containing metalloenzyme, on the example of Streptomyces coelicolor laccase with a particular focus on electron transfer pathways beyond enzyme’s active site. Metalloenzymes evolved mechanisms to efficiently couple electron flow from external donors to their active sites, ensuring proper O2 reduction and minimizing ROS formation. A critical aspect of this process is kinetically well-balanced delivery of four electrons and protons to produce water molecules. To ensure the constant availability of electrons, redox chains composed of Tyr and Trp residues are hypothesized to act as electron reservoirs, helping to maintain the balance of electron delivery and prevent oxidative damage. However, the involvement of Tyr/Tp pathway during catalysis, specifically their thermodynamic and kinetic properties are unknown. In this project, we aim to understand the role and regulation of Tyr/Trp chains in SLAC during O2 reduction and their impact on catalysis. The specific objectives are: i) to unravel the mechanistic details of radical formation via ET pathways during O2 turnover in situ; ii) To understand the factors influencing the thermodynamics and kinetics of radical transfer activation, and iii) to explore the possibility of fine-tuning radical transfer through structural modifications that alter the redox and kinetic properties of redox-active residues. To achieve these goals, the project will use a combination of Raman spectroscopy, stopped-flow methodology, and electrochemistry. SLAC variants will be produced using protein expression, purification, and engineering techniques. The spectroscopic approach will monitor the vibrational fingerprints of aromatic radicals and metal active sites during catalysis under electrochemical control. Resonance Raman (RR) enhancement will allow selective monitoring of metal cofactors, oxygen-bound intermediates, and Tyr and Trp radicals. Stopped-flow methodology will enable time-resolved trapping of intermediates, and roughened electrodes will facilitate the control of applied electrochemical potential and enhance the Raman signal through the surface-enhanced Raman (SER) phenomenon. As a consequence, the detailed understanding of thermodynamics and kinetics of radical formation via Tyr/Trp pathway will pave the way for illuminating the detailed concept of coupled electron transfer reactions with substrate conversion in metalloenzymes.

• DFG Programme: Research Grants
• Major Instrumentation: Freeze quench stopped-flow
• Instrumentation Group: 1860 Spezielle Spektrographen und Spektrometer