Understanding Lignin Oxidation by a Two-Domain Multicopper Oxidase from Cellvibrio japonicus.

Bacterial two-domain multicopper oxidases (2dMCOs) represent a structurally distinct class of trimeric multicopper oxidases. Differing considerably from well-characterized monomeric three-domain laccases, type B 2dMCOs have a T1 active site positioned in a tunnel in the trimer center. The biochemical properties and roles in lignin conversion of 2dMCOs remain poorly understood. Here, we present a comprehensive biochemical characterization of a type B 2dMCO from Cellvibrio japonicus (CjMCO) and discuss links between its structural organization and activity. The T1 copper of CjMCO had a redox potential of 537 mV, but the turnover number (0.4 s-1) was ∼1000-fold lower than high-turnover fungal laccases. Stopped-flow UV-vis spectroscopy indicated that this low turnover likely reflects slow reoxidation of the enzyme by O2, which has not been previously reported for laccases. Despite the steric constraints imposed by its trimeric structure, CjMCO oxidized the lignin model compound guaiacylglycerol-β-guaiacyl ether, resulting in both oxidative coupling and bond cleavage, and CjMCO was able to act on oligomeric birch organosolv lignin, promoting net oxidative polymerization. Interestingly, 2,6-dimethoxyphenol oxidation kinetics and the product profile for guaiacylglycerol-β-guaiacyl ether oxidation by CjMCO were influenced by pH, buffer composition, and ionic strength, suggesting a potential strategy for tailoring product profiles. Together, these findings demonstrated that CjMCO functions as a laccase and oxidizes phenolic lignin moieties, but its slow rates and trimeric architecture indicate that it is unlikely to efficiently degrade lignin polymers in vivo. This study expands the current understanding of bacterial laccase diversity and provides a foundation for exploring other physiological roles of type B 2dMCOs beyond lignin degradation.