Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme
Year: 2017
Type: article
Abstract: KatG is a bifunctional, heme-dependent enzyme in the frontline defense of numerous bacterial and fungal pathogens against H 2 O 2 -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 H 2 O 2 to O 2 and H 2 O only if a PxED is present in the reaction mixture.Stopped-flow spectroscopy results indicated rapid initial rates of H 2 O 2 disproportionation slowing concomitantly with the accumulation of ferryl-like heme states.These states very slowly returned to resting (i.e.ferric) enzyme, indicating that they represented catalase-inactive intermediates.We also show that an active-site tryptophan, Trp-321, participates in offpathway 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 nonproductive protein-based radical species that accumulate during reaction with H 2 O 2 .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.
Source: Journal of Biological Chemistry
Language: English
Subfield: Inorganic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W2763538797