Spectroelectrochemical investigation of the glyoxal oxidase activation mechanism

Glyoxal oxidase (GLOX) is an extracellular source of H₂O₂ in white-rot secretomes, where it acts in concert with peroxidases to degrade lignin. It has been reported that GLOX requires activation prior to catalytic turnover and that a peroxidase system can fulfill this task. In this study, we verify that an oxidation product of horseradish peroxidase, the radical cation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), is an activator for GLOX. A spectroelectrochemical cell was used to generate the activating radical species, to continuously measure its concentration, and to simultaneously measure the catalytic activity of GLOX based on its O₂ consumption. The results show that GLOX can undergo multiple catalytic turnovers upon activation and that activity increases with the activator concentration. However, we also found that the ABTS cation radical can serve as an electron acceptor which becomes visible in the absence of O₂. Furthermore, GLOX activity is highly restrained by the naturally occurring, low O₂ concentration. We conclude that GLOX is indeed an auxiliary enzyme for H₂O₂ production in white-rot secretomes. Its turnover rate is strongly regulated by the availability of O₂ and the radical generating activity of peroxidases present in the secretome, which acts as a feedback loop for GLOX activity.