Resolving the Paradoxical Nature of a Bifunctional Enzyme: Pathways and Regulation of Intramolecular Electron Transfer In KatG
Type of DegreePhD Dissertation
DepartmentChemistry and Biochemistry
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Catalase-peroxidases (KatGs) are heme-dependent enzymes that use a single active site to perform two activities by protein-based radical dependent mechanisms. As a catalase, in its first step, it reduces H2O2 to water and the ferric enzyme is converted to a high valent ferryl-porphyrin π cation radical intermediate, compound I (i.e., FeIV=O[porphyrin]•+). Thereafter, compound I is proposed to undergo an intramolecular electron transfer where the porphyrin radical is reduced by a unique methionine tyrosine tryptophan (MYW) adduct cofactor to form compound I* (i.e., FeIV=O[MYW]•+). Compound I* then reacts with a second equivalent of H2O2 producing an oxyperoxidase or compound III-like species referred to as a compound III* (i.e., FeIII-O2•-[MYW]•+). Finally, another intramolecular electron transfer from the FeIII-O2•- heme to the MYW adduct radical occurs forming the ferric heme, O2, H2O and a closed shell MYW adduct. In contrast, for the peroxidase cycle, upon the formation of compound I, the enzyme is reduced in two sequential one electron steps by exogenous electron donors, first to form a ferryl intermediate known as compound II (i.e., FeIV=O) and then to the ferric enzyme, generating two equivalents of donor radical. For the nearly 30 years since its initial discovery, the nature of the interplay between these two activities in KatG has not been understood. All evidence including the pH-dependence of its two activities and the properties of site specific variants has suggested mutual antagonism between both activities. However, our lab recently observed stimulation of KatG catalase activity by peroxidatic electron donors at conditions that coincide with host-defense mechanisms like the neutrophil oxidative burst (i.e., pH 5.0, large [H2O2]). Hence, evidence is also accumulating that KatG uses a peroxidase scaffold, a unique Met-Tyr-Trp covalent adduct, and even assistance from peroxidatic electron donors (PxEDs) to perform its dominant catalase activity. The synergistic effect expands the conditions for KatG as an efficient catalase. This is especially useful for plant and animal pathogens which encounter enormous amounts of H2O2 produced by the immune responses of their hosts. This is particularly important for Mycobacterium tuberculosis, a known intracellular pathogen most abundant in host neutrophils and macrophages. To this hostile environment, M. tuberculosis brings KatG as its only catalase active enzyme. The purpose of this dissertation is to elucidate the role of PxEDs in this novel mechanism of H2O2 decomposition by KatG. We have observed that the return of KatG ferric state at the conclusion of H2O2 consumption is not catalytically competent to account for the rate of catalatic H2O2 decomposition. These data suggested that catalase inactive intermediates accumulated during turnover. We hypothesized inactive intermediates might accumulate because of off-pathway protein oxidation events and PxEDs prevent the accumulation of these inactive enzyme forms. We also hypothesized that the proximal tryptophan (W321 in M. tuberculosis KatG) was a prominent conduit for off-pathway electron transfer. In order to evaluate these hypotheses, we produced the W321F KatG variant to make this site unoxidizable and compared its properties with wild-type KatG. At neutral pH where catalase activity is optimal, the catalase activity of KatG and its W321F variant are nearly identical. At lower pH where the stimulatory effect of PxEDs is maximal (pH 5.0), the unassisted catalase activity of W321F exceeded that of the wild-type enzyme. By stopped-flow, both proteins showed identical initial rates of H2O2 decomposition, and compound III-like species (i.e., FeIII-O2•-) dominated the heme spectrum at these early reaction times. As turnover progressed, wild-type KatG more rapidly lost activity than W321F and transitioned to a mixture of heme states at the conclusion of H2O2 consumption. Conversely, W321F sustained higher rates of H2O2 consumption and transitioned to a compound I-like species (i.e., FeIV=O [porphyrin]•+) at the time H2O2 consumption ceased. Thereafter, the W321F KatG ferric state returned an order of magnitude more rapidly than the wild-type enzyme. PxEDs stimulated catalatic H2O2 consumption by both proteins as well as the return of the ferric state after H2O2 depletion. However, the effect was more pronounced in both respects for wild-type than W321F KatG. Samples of both proteins freeze-quenched 10 ms after reaction with H2O2 produced a narrow-doublet radical species as detected by electron paramagnetic resonance (EPR) spectroscopy. This signal is consistent with a radical centered on the MYW covalent adduct of KatG. Reactions of wild-type KatG quenched at the time H2O2 consumption ceased (6 s), produced a singlet EPR spectrum with clear evidence of exchange-coupling due most likely to the heme center. This is consistent with a radical centered on the proximal tryptophan. At 1 and 5 min after H2O2 depletion, protein-based singlet radical species were still detected, albeit at lesser intensity and with much diminished exchange-coupling. These data are indicative of radical migration away from the KatG active site with time. Although W321F KatG showed a protein radical signal at the time of H2O2 deletion (1.6 s), no exchange-coupling was evident. In addition, the intensity and persistence of radical species at all subsequent reaction times were substantially diminished compared to the wild-type enzyme. Inclusion of PxED in reactions of wild-type and W321F KatG with H2O2 produced only the narrow-doublet signal corresponding to the MYW adduct radical detected at 10 ms. Little or no protein-based radical was observed at H2O2 depletion or any time thereafter. All these data supported our hypotheses that PxEDs stimulate KatG catalase activity by rescuing inactive intermediates that result from off-pathway protein oxidation events starting with oxidation of the proximal tryptophan. The pH-dependence of the stimulatory effect of PxEDs is striking not only because of the physiological implications of a low-pH catalase activity, but also because of known structural adjustments in KatG that are also pH-dependent. Without a PxED, KatG shows poor catalase activity at pH 5.0. This is attributed at least in part to the position of the enzyme’s so-called arginine switch (R418 in M. tuberculosis KatG). At pH 8.5, the R418 guanidinium side chain is oriented toward the phenolate oxygen of the MYW adduct, and at pH 4.5, it is oriented to the protein’s solvent accessible surface. At pH 6.5, there are equal populations of both conformational states and this corresponds to KatGs optimum unassisted catalase activity. To investigate the participation of R418 in the mechanism of PxED-stimulated catalase activity, we evaluated R418K and R418A KatG. In most respects, R418K was indistinguishable from wild-type KatG. In contrast, R418A KatG showed a diminished kcat/KM (7.4 × 10 3 M-1s-1) for catalase activity at pH 7.0. Activity increased appreciably at pH 5.0 with a kcat/KM of 5.2 × 10 5 M-1s-1. PxEDs were able to stimulate R418A KatG to an extent similar to wild-type KatG, albeit with much more PxEDs required for its catalase restoration. Similarly, R418A KatG produced more PxED radicals to H2O2 consumed also supports its need for more PxED for its rescue mechanism. By rapid-freeze quench EPR, R418A KatG produced a MYW narrow-doublet radical at 10 ms after its reaction with H2O2. Over time at its point of H2O2 depletion (i.e., 20 s), R418A transitioned to an exchange-coupled proximal tryptophanyl radical. More so, remote protein-based radicals persisted throughout turnover even at later times of its reaction with H2O2. The less broad and less intense radicals observed are consistent with radical migration from the active site. When PxEDs were included in the reactions, R418A only formed the narrow-doublet radical at 10 ms. Strikingly, the intensity of the narrow-doublet radical produced by R418A KatG when PxED was added was two-fold greater than that seen for either wild-type or W321F KatG. This suggested that R418A KatG recycle more non-MYW adduct radicals back to the active MYW adduct radical. As with wild-type KatG and W321F KatG, little or no other radical was detected at the point of H2O2 depletion and thereafter. All these data suggest that R418 influences KatG activity, inactivation, and restoration mechanisms. In conclusion, at early reaction times corresponding to the most rapid rates of H2O2 decomposition, all KatG proteins formed a putative FeIII-O2•-[MYW]•+species. Wild-type KatG and R418A KatG transitioned to an exchange-coupled proximal tryptophanyl radical intermediate (FeIV=O [W321•+]) at their points of H2O2 depletion. This species slowly reverts to the KatG ferric resting state. In contrast, W321F formed an uncoupled radical which rapidly returns to its ferric state. These data suggest that KatG proximal tryptophan is the first residue that gets oxidized for KatG inactivation. PxEDs reduce all non-MYW radicals but leave the MYW adduct radical untouched thereby stimulating KatG catalase activity. We propose that PxEDs restores catalase active states simultaneous with optimizing catalatic H2O2 degradation. This thesis enlightens us on KatG intramolecular electron transfer and protein-based radicals which relates the enzymes catalase and peroxidase mechanisms of H2O2 detoxification. As such, we also propose that the proximal tryptophan facilitates the mutual synergism of both the catalase and peroxidase activities of KatG. Until the present time, the identity and role of KatG physiological electron donor has been a conundrum in the field. The synergism observed in this dissertation may provide some insights into the characteristics of the unknown physiological electron donor. This will carry important ramifications for those organisms which utilize KatG to detoxify the threat posed by H2O2, most importantly for organisms like M. tuberculosis whose only catalase active enzyme is KatG.
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