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

High Resolution Image Download MS PowerPoint Slide 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 ( Cj MCO) and discuss links between its structural organization and activity. The T1 copper of Cj MCO 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 O 2, which has not been previously reported for laccases. Despite the steric constraints imposed by its trimeric structure, Cj MCO oxidized the lignin model compound guaiacylglycerol-β-guaiacyl ether, resulting in both oxidative coupling and bond cleavage, and Cj MCO 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 Cj MCO were influenced by pH, buffer composition, and ionic strength, suggesting a potential strategy for tailoring product profiles. Together, these findings demonstrated that Cj MCO 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.