KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against HO-induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert HO to O and HO only if a PxED is present in the reaction mixture. Stopped-flow spectroscopy results indicated rapid initial rates of HO disproportionation slowing concomitantly with the accumulation of ferryl-like heme states. These states very slowly returned to resting ( ferric) enzyme, indicating that they represented catalase-inactive intermediates. We also show that an active-site tryptophan, Trp-321, participates in off-pathway electron transfer. A W321F variant in which the proximal tryptophan was replaced with a non-oxidizable phenylalanine exhibited higher catalase activity and less accumulation of off-pathway heme intermediates. Finally, rapid freeze-quench EPR experiments indicated that both WT and W321F KatG produce the same methionine-tyrosine-tryptophan (MYW) cofactor radical intermediate at the earliest reaction time points and that Trp-321 is the preferred site of off-catalase protein oxidation in the native enzyme. Of note, PxEDs did not affect the formation of the MYW cofactor radical but could reduce non-productive protein-based radical species that accumulate during reaction with HO Our results suggest that catalase-inactive intermediates accumulate because of off-mechanism oxidation, primarily of Trp-321, and PxEDs stimulate KatG catalase activity by preventing the accumulation of inactive intermediates.

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This paper describes the development of a fully remote upper-class biochemistry lab course. The sudden change to online teaching in the middle of spring semester 2020 had a primarily negative impact on laboratory teaching. These effects were mitigated because the students had done many of the basic hands-on procedures before the switch. A true "at-home" biochemistry lab module was implemented in the fall semester of 2020 to ensure students could have a hands-on lab experience in a remote setting despite the remaining COVID-19 restrictions placed upon universities. The module covered several fundamental concepts and techniques found in a first semester biochemistry lab sequence: extraction and purification of a protein from a sample, and further analysis of the protein. Tyrosinase was isolated and purified from a banana extract followed by kinetic analysis of the enzyme. A key component to the module is an LED light board that, in combination with a cell-phone app, made a simple at-home colorimeter. The module was implemented in three sections of a first semester biochemistry lab course (81 students total) in the fall of 2020, and components of it have been used periodically since. Some of the procedures are now being implemented into normal in-lab sessions. An assessment in terms of a student survey showed that most of the students were able to adapt to this format and felt that their learning was not impeded.

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