Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme

Abstract

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.

Phenolics decontamination of olive mill wastewater using onion solid by-products homogenate

“…Such removal will induce a decrease in the color intensity. Upon initiation of the reaction using H 2 O 2 and peroxidase, reaction mixtures changed color, indicating the formation of phenolic polymers [40]. Treatment was carried out for three hours, after which a significant decrease in the color intensity was observed by increasing the concentration of hydrogen peroxide.…”

Functional and protective hole hopping in metalloenzymes

“…In the KatG from Mycobacterium tuberculosis ( Mt KatG), this radical is generated by hole transfer from a porphyrin radical formed in the reaction of the ferric enzyme with H 2 O 2 . 88 In the absence of peroxidase substrates, Mt KatG loses its catalase activity after about 20 000 turnovers, owing to formation of off-pathway protein radicals, with Trp321 foremost among them. Peroxidase substrates can reduce these off-pathway radicals as well as the resulting CII species to restore catalase activity.…”

“…Mt KatG, like CCP and DyPs, is rich in oxidizable amino acids (Trp, 4.2%; Tyr, 3.7%; Met, 3.2%; Cys, 0.5%). 88 In the absence of peroxidase substrates, these residues serve as sacrificial electron donors to extend enzyme survival and catalase activity. 88 …”

“… 88 In the absence of peroxidase substrates, these residues serve as sacrificial electron donors to extend enzyme survival and catalase activity. 88 …”

Novel molecular insights into the anti‐oxidative stress response and structure–function of a salt‐inducible cyanobacterial Mn‐catalase

“…The commonly found catalases in prokaryotes are the typical haem catalases and the bifunctional catalase‐peroxidases (KatGs), both of which contain haem (Borges, Frazão, Miranda, Carrondo, & Romão, ; Njuma et al, ). However, another class of catalases that lack haem, called as non‐haem catalase or pseudocatalase, is present exclusively in prokaryotes and archaea (Andrews, ; Zámocký, Gasselhuber, Furtmüller, & Obinger, ).…”