Crystal Structure of Leishmania major Peroxidase and Characterization of the Compound I Tryptophan Radical

Jasion, Victoria S.; Polanco, Julio; Meharenna, Yergalem T.; Li, Huiying; Poulos, T.L.

doi:10.1074/jbc.m111.230524

Cited by 33 publications

(67 citation statements)

References 39 publications

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“…Overall, these results strongly support the participation of a Trp 233 -derived radical in the generation of the compound I-like product. Detailed inspection of the heme microenvironment in hybrid type A and CcP peroxidases revealed the presence of a Cys residue (Cys 222 for T. cruzi enzyme) located near Trp 233 (25,34); this Cys residue is absent in the APx family (23). It has been proposed that the sulfur atom of this Cys residue favors stabilization of the LmP compound I-like product.…”

Section: Resultsmentioning

confidence: 99%

“…The resting enzyme exhibited a Soret peak at 409 nm that shifted to 420 nm after reaction with H 2 O 2 , with the appearance of two humps at 539 and 560 nm. These changes in the optical spectra are indicative of the generation of a compound I-like product (25,26,28) (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

“…LmP can use both Asc (as monofunctional APx) and Cc (as monofunctional CcP) as electron donors (25). H 2 O 2 reacts with the heme generating the oxoferryl (Fe IV = O ) and the porphyrin-centered radical (P •+ ) forming the classical compound I species.…”

Section: Significancementioning

confidence: 99%

“…Finally, using either Asc or Cc as the reducing substrate, the enzyme recovers the resting state (Fe-III-P). Classical compound I formation in these hybrid type A peroxidases was established by direct electron paramagnetic resonance (EPR) analysis (25) and/or by spectroscopic studies using a W208F mutant (25,26). To date, LmP represents the only trypanosomatid hybrid type A peroxidase characterized as using Asc and Cc as electron donors, with its activity implicated in parasite differentiation and oxidative stress protection (27).…”

Section: Significancementioning

confidence: 99%

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Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Hugo

Martínez

Trujillo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H 2 O 2 (k = 2.9 × 10 7 M −1 ·s −1 ) and catalytically decomposes H 2 O 2 using Cc as the reducing substrate with higher efficiency than Asc (k cat /K m = 2.1 × 10 5 versus 3.5 × 10 4 M −1 ·s −1 , respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H 2 O 2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp 233•+ ). Mutation of Trp 233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp 233•+ , a Cys 222 -derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H 2 O 2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp 233 and Cys 222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.Trypanosoma cruzi | heme peroxidase | oxidants | virulence | kinetics

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Hugo

Martínez

Trujillo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Sample homogeneity was verified with 12-15% SDS/PAGE. LmP was expressed and purified as previously described (4,5).…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of the Leishmania major peroxidase–cytochrome c complex

Jasion

Doukov

Pineda

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The causative agent of leishmaniasis is the protozoan parasite Leishmania major . Part of the host protective mechanism is the production of reactive oxygen species including hydrogen peroxide. In response, L. major produces a peroxidase, L. major peroxidase (LmP), that helps to protect the parasite from oxidative stress. LmP is a heme peroxidase that catalyzes the peroxidation of mitochondrial cytochrome c . We have determined the crystal structure of LmP in a complex with its substrate, L. major cytochrome c (LmCytc) to 1.84 Å, and compared the structure to its close homolog, the yeast cytochrome c peroxidase–cytochrome c complex. The binding interface between LmP and LmCytc has one strong and one weak ionic interaction that the yeast system lacks. The differences between the steady-state kinetics correlate well with the Lm redox pair being more dependent on ionic interactions, whereas the yeast redox pair depends more on nonpolar interactions. Mutagenesis studies confirm that the ion pairs at the intermolecular interface are important to both k cat and K M . Despite these differences, the electron transfer path, with respect to the distance between hemes, along the polypeptide chain is exactly the same in both redox systems. A potentially important difference, however, is the side chains involved. LmP has more polar groups (Asp and His) along the pathway compared with the nonpolar groups (Leu and Ala) in the yeast system, and as a result, the electrostatic environment along the presumed electron transfer path is substantially different.

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Cytochrome c Peroxidase

Chreifi

Poulos

2017

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Cytochrome c peroxidase (CcP), produced by Saccharomyces cerevisiae , is a heme peroxidase that catalyzes the reduction of H 2 O 2 to water using electrons from two molecules of ferrocytochrome c . CcP has played a unique role in the early studies of enzymology and biological electron transfer and in our understanding of how heme‐containing enzymes stabilize higher oxidation states. Recently, a heme peroxidase from the parasitic protozoan Leishmania major (LmP) was also confirmed to be a CcP. This article briefly describes our current knowledge of CcP (and LmP) biochemistry and structural biology.

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Crystal Structure of Leishmania major Peroxidase and Characterization of the Compound I Tryptophan Radical

Abstract: The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX

Cited by 33 publications

References 39 publications

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Crystal structure of the Leishmania major peroxidase–cytochrome c complex

Cytochrome c Peroxidase

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