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Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate

  • Samuel L. Freeman
    Affiliations
    School of Chemistry, University of Bristol, Bristol, United Kingdom
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  • Vera Skafar
    Affiliations
    Departamento de Bioquímica, Facultad of Medicina, Universidad de la República, Montevideo, Uruguay

    Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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  • Hanna Kwon
    Affiliations
    Department of Molecular and Cell Biology and Leicester Institute of Structural and Chemical Biology, University of Leicester, Leicester, United Kingdom
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  • Alistair J. Fielding
    Affiliations
    Centre for Natural Products Discovery, School of Pharmacy and Biomolecular Sciences, Liverpool John Moore University, Liverpool, United Kingdom
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  • Peter C.E. Moody
    Affiliations
    Department of Molecular and Cell Biology and Leicester Institute of Structural and Chemical Biology, University of Leicester, Leicester, United Kingdom
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  • Alejandra Martínez
    Affiliations
    Departamento de Bioquímica, Facultad of Medicina, Universidad de la República, Montevideo, Uruguay

    Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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  • Federico M. Issoglio
    Affiliations
    CONICET-Universidad de Buenos Aires, Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Buenos Aires, Argentina

    Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Oeiras, Portugal
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  • Lucas Inchausti
    Affiliations
    Laboratorio de Bioinformática, Departamento de Genómica, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay

    Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
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  • Pablo Smircich
    Affiliations
    Laboratorio de Bioinformática, Departamento de Genómica, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay

    Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
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  • Ari Zeida
    Affiliations
    Departamento de Bioquímica, Facultad of Medicina, Universidad de la República, Montevideo, Uruguay

    Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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  • Lucía Piacenza
    Affiliations
    Departamento de Bioquímica, Facultad of Medicina, Universidad de la República, Montevideo, Uruguay

    Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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  • Rafael Radi
    Correspondence
    For correspondence: Emma L. Raven; Rafael Radi
    Affiliations
    Departamento de Bioquímica, Facultad of Medicina, Universidad de la República, Montevideo, Uruguay

    Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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  • Emma L. Raven
    Correspondence
    For correspondence: Emma L. Raven; Rafael Radi
    Affiliations
    School of Chemistry, University of Bristol, Bristol, United Kingdom
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Open AccessPublished:June 27, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102204
      The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

      Keywords

      Abbreviations:

      APX (ascorbate peroxidase), CcP (cytochrome c peroxidase), MD (molecular dynamics)
      Chagas disease is named after the Brazilian physician Carlos Chagas, who first described it in 1909; its formal name is American trypanosomiasis. It most seriously affects isolated rural and indigenous communities with low economic development and little access to healthcare. The disease was once mainly confined to the American continent, principally Latin America, but the distribution of Chagas disease is expanding into Europe and North America and is becoming a public health issue at nonendemic sites (
      • Bonney K.M.
      Chagas disease in the 21st century: a public health success or an emerging threat?.
      ,
      • Bern C.
      • Kjos S.
      • Yabsley M.J.
      • Montgomery S.P.
      Trypanosoma cruzi and Chagas' disease in the United States.
      ). The protozoan parasite Trypanosoma cruzi is the causative agent of the disease. It is estimated that around 10 million people across the whole of Latin America are infected with T. cruzi, causing ca. 20,000 deaths per annum. T. cruzi strains are heterogeneous, exhibiting a high degree of biochemical and genetic variability; this means that disease outcomes vary from asymptomatic during the course of infection to fatal in other cases (
      • Luquetti A.O.
      • Miles M.A.
      • Rassi A.
      • de Rezende J.M.
      • de Souza A.A.
      • Povoa M.M.
      • et al.
      Trypanosoma cruzi: zymodemes associated with acute and chronic Chagas' disease in central Brazil.
      ).
      In order to survive and proliferate in the host cells, the T. cruzi parasite needs antioxidant defense systems (
      • Piacenza L.
      • Peluffo G.
      • Alvarez M.N.
      • Martinez A.
      • Radi R.
      Trypanosoma cruzi antioxidant enzymes as virulence factors in Chagas disease.
      ,
      • Piacenza L.
      • Trujillo M.
      • Radi R.
      Reactive species and pathogen antioxidant networks during phagocytosis.
      ) to cope against the cytotoxic effects of reactive oxygen and nitrogen species (
      • Martinez A.
      • Prolo C.
      • Estrada D.
      • Rios N.
      • Alvarez M.N.
      • Pineyro M.D.
      • et al.
      Cytosolic Fe-superoxide dismutase safeguards Trypanosoma cruzi from macrophage-derived superoxide radical.
      ,
      • Estrada D.
      • Specker G.
      • Martinez A.
      • Dias P.P.
      • Hissa B.
      • Andrade L.O.
      • et al.
      Cardiomyocyte diffusible redox mediators control trypanosoma cruzi infection: role of parasite mitochondrial iron superoxide dismutase.
      ,
      • Alvarez M.N.
      • Peluffo G.
      • Piacenza L.
      • Radi R.
      Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity.
      ). One of these is a heme-containing peroxidase (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ,
      • Wilkinson S.R.
      • Obado S.O.
      • Mauricio I.L.
      • Kelly J.M.
      Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum.
      ). The enzyme is located in the endoplasmic reticulum and mitochondria in all parasite stages and is additionally located at the plasma membrane in the infective parasite stages (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ). Originally designated as an ascorbate peroxidase (APX) (TcAPx (
      • Wilkinson S.R.
      • Obado S.O.
      • Mauricio I.L.
      • Kelly J.M.
      Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum.
      )), this T. cruzi enzyme has high sequence identity to members of the class I peroxidase family. The class I peroxidases include APX and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). APX, CcP, and LmP are all heme-dependent enzymes that scavenge, by reduction, hydrogen peroxide (H2O2) in cells and use ascorbate or cytochrome c as the reducing substrate (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ,
      • Sharp K.H.
      • Moody P.C.E.
      • Raven E.L.
      Defining substrate specificity in haem peroxidases.
      ,
      • Raven E.L.
      Understanding functional diversity and substrate specificity in haem peroxidases: what can we learn from ascorbate peroxidase?.
      ,
      • Raven E.L.
      Peroxidase-catalysed oxidation of ascorbate: structural, spectroscopic and mechanistic correlations in ascorbate peroxidase.
      ,
      • Adak S.
      • Datta A.K.
      Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: a novel role of the transmembrane domain.
      ). Like LmP (
      • Jasion V.S.
      • Doukov T.
      • Pineda S.H.
      • Li H.
      • Poulos T.L.
      Crystal structure of the Leishmania major peroxidase-cytochrome c complex.
      ,
      • Jasion V.S.
      • Polanco J.A.
      • Meharenna Y.T.
      • Li H.
      • Poulos T.L.
      Crystal structure of Leishmania major peroxidase and characterization of the compound I tryptophan radical.
      ), TcAPx was later found to have both ascorbate and cytochrome c activity (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ) and was thus renamed as TcAPx-CcP. The parasite’s ability to utilize both ascorbate (which is endogenously synthesized from the host organism (
      • Wilkinson S.R.
      • Prathalingam S.R.
      • Taylor M.C.
      • Horn D.
      • Kelly J.M.
      Vitamin C biosynthesis in trypanosomes: a role for the glycosome.
      )) and cytochrome c as electron sources may be an evolutionary adaptation in these parasites, which have been present in some form since prehistoric times.
      A lack of structural information for TcAPx-CcP has hindered the understanding of key enzymological aspects. Here, we present a crystal structure for TcAPx-CcP that, together with functional spectroscopic data, allows full rationalization of the substrate specificity and reactivity of this important enzyme target.

      Results

      Structure of the TcAPx-CcP enzyme

      The structure of ferric TcAPx-CcP has been solved to 2.0 Å, Figure 1A; a comparison with the structures of related peroxidases is shown in Figure 1, AD. Data and refinement statistics are shown in Table 1. The TcAPx-CcP enzyme is similar in its overall structure to the related LmP enzyme with which TcAPx-CcP shares 57% sequence identity. The structure comprises of 10 α-helical bundles, which is consistent with the characteristics of other peroxidase structures. TcAPx-CcP also features a limited amount of ordered β-structure (located approximately between Gly250-Asn270), which has until now only been identified in CcP and to a slightly lesser extent LmP (
      • Jasion V.S.
      • Polanco J.A.
      • Meharenna Y.T.
      • Li H.
      • Poulos T.L.
      Crystal structure of Leishmania major peroxidase and characterization of the compound I tryptophan radical.
      ). The active site of TcAPx-CcP comprises Trp92 in the distal pocket, along with distal histidine (His93) and arginine (Arg89) residues. On the proximal side there is a typical peroxidase His217-Asp278-Trp233 proximal triad, as observed in yeast CcP (Fig. 2) and in soybean APX (sAPX, His163-Asp208-Trp179, Fig. S2). There are two molecules in the asymmetric unit for both the wild type and mutant structures, each one exhibiting different electron density at the distal heme position, with one appearing to bind an oxygen molecule and another a water molecule approximately 2.3 Å above the iron (not shown). There are two metal cations, Figure 1A, most likely sodium from the crystallization conditions, located at ≈13 and ≈16 Å from the heme center near the solvent accessible outer edge of the structure.
      Figure thumbnail gr1
      Figure 1Crystal structures of TcAPx-CcP and related heme peroxidases. A, TcAPx-CcP (PDB 7OPT). B, sAPX with ascorbate bound (1OAF). C, CcP (1ZBY). D, LmP (3RIV). Active site residues are shown as red sticks. Sodium atoms are shown as purple spheres, calcium as green spheres, and potassium as orange spheres. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase; PDB, Protein Data Bank.
      Table 1Data collection and refinement statistics for TcAPx-CcP (PDB code 7OPT) and W233F (7OQR)
      Data collectionTcAPx-CcPW233F
      Resolution (Å)62.02–2.02 (2.07–2.02)29.28–1.76 (1.80–1.76)
      Total measured reflections987,7801,516,631
      Unique reflections50,314 (3670)76,005 (4170)
      Completeness (%)99.7 (99.1)99.8 (97.3)
      Redundancy19.6 (20.5)20 (19.6)
      I/σ (I)19.0 (2.4)22.5 (4.1)
      Unit cell dimensions (Å)a = 71.6 b = 71.6 and c = 253.371.6, 71.6, 254.6
      Space groupP3121P3121
      Rmerge0.08 (1.4)0.08 (0.74)
      Refinement
      Rwork/Rfree0.16/0.230.12/0.16
      r.m.s.d. bond (Å)/angle (°)0.01/1.80.01/1.9
      B-factor analysis (Å2)
      Protein4927
      Water5036
      Ramachandran analysis
      Most favored (%)94.9497.58
      Allowed (%)5.062.05
      Outliers Disallowed (%)0.00.37
      Values in parenthesis are for high resolution shells.
      Figure thumbnail gr2
      Figure 2The active site heme environment in TcAPx-CcP (in green) superimposed with CcP (cyan). Active site residues are shown as red sticks and labeled for TcAPx-CcP with the equivalent residue in CcP in parentheses. TcAPx-CcP features the C222 residue (in yellow), whereas T180 is found in CcP (see ). An equivalent of C222 is also found in LmP (C197). CcP, cytochrome c peroxidase.

      Nature of the compound I intermediate

      In CcP, reaction of the enzyme with H2O2 leads to formation of a protein radical located on Trp191 (
      • Sivaraja M.
      • Goodin D.B.
      • Smith M.
      • Hoffman B.M.
      Identification by endor of Trp191 as the free-radical site in cytochrome c peroxidase compound ES.
      ). This same Trp residue (Trp179) is not used in sAPX (
      • Lad L.
      • Mewies M.
      • Basran J.
      • Scrutton N.S.
      • Raven E.L.
      Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.
      ,
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      ,
      • Patterson W.R.
      • Poulos T.L.
      • Goodin D.B.
      Identification of a porphyrin pi-cation-radical in ascorbate peroxidase compound-I.
      ), despite the CcP and sAPX enzymes being essentially identical in the region of the proximal pocket. The different substrate specificities of the two enzymes—with ascorbate binding at the γ-heme edge in sAPX (
      • Sharp K.H.
      • Mewies M.
      • Moody P.C.
      • Raven E.L.
      Crystal structure of the ascorbate peroxidase-ascorbate complex.
      ) and cytochrome c in CcP instead binding on an election transfer pathway that includes Trp191 (
      • Pelletier H.
      • Kraut J.
      Crystal-structure of a complex between electron-transfer partners, cytochrome c peroxidase and cytochrome c.
      )—is probably in part responsible for the difference in reactivity of the Trp residues between the two enzymes. The presence of a potassium cation on the proximal side of the heme in sAPX might also destabilize radical formation on Trp179 in APX (
      • Bonagura C.A.
      • Bhaskar B.
      • Sundaramoorthy M.
      • Poulos T.L.
      Conversion of an engineered potassium-binding site into a calcium-selective site in cytochrome c peroxidase.
      ,
      • Bonagura C.A.
      • Sundaramoorthy M.
      • Bhaskar B.
      • Poulos T.L.
      The effects of an engineered cation site on the structure, activity, and EPR properties of cytochrome c peroxidase.
      ,
      • Bonagura C.A.
      • Sundaramoorthy M.
      • Pappa H.S.
      • Patterson W.R.
      • Poulos T.L.
      An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan.
      ), although we do not observe K+ cations in all of our sAPX structures (and there is no metal cation at the equivalent position in the TcAPx-CcP structure, as above).
      Electron paramagnetic resonance (EPR) was used to assess whether TcAPx-CcP uses the corresponding Trp233 radical. EPR spectra taken at 4 K for TcAPx-CcP on reaction with H2O2 are consistent with the formation of a [Fe(IV)=O Trp•+] radical in compound I, with a concomitant reduction of the high spin iron heme, g = 6, signal (Fig. 3Ai- ii). The radical was centered at g = 2 and showed a pronounced broad linewidth only present at the lowest temperatures with a narrower radical signal observed at 70 K (Fig. 3B(i)). This is consistent with the compound I intermediate containing an exchange coupled porphyrin-tryptophanyl radical, [Fe(IV)=O Por/Trp•+] at 4 K (
      • Sivaraja M.
      • Goodin D.B.
      • Smith M.
      • Hoffman B.M.
      Identification by endor of Trp191 as the free-radical site in cytochrome c peroxidase compound ES.
      ,
      • Houseman A.L.P.
      • Doan P.E.
      • Goodin D.B.
      • Hoffman B.M.
      Comprehensive explanation of the anomalous epr-spectra of wild-type and mutant cytochrome-C peroxidase compound-Es.
      ). It is also consistent with the absorption spectra of TcAPx-CcP on reaction with H2O2 (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ), which show peaks (539 and 560 nm) close to those observed for CcP (
      • Casadei C.M.
      • Gumiero A.
      • Metcalfe C.L.
      • Murphy E.J.
      • Basran J.
      • Concilio M.G.
      • et al.
      Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase.
      ,
      • Pond A.E.
      • Bruce G.S.
      • English A.M.
      • Sono M.
      • Dawson J.H.
      Spectroscopic study of the compound ES and the oxoferryl compound II states of cytochrome c peroxidase: comparison with the compound II of horseradish peroxidase.
      ) and different from those for sAPX (which forms a porphyrin pi-cation radical instead (
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      ,
      • Patterson W.R.
      • Poulos T.L.
      • Goodin D.B.
      Identification of a porphyrin pi-cation-radical in ascorbate peroxidase compound-I.
      ,
      • Kwon H.
      • Basran J.
      • Casadei C.M.
      • Fielding A.J.
      • Schrader T.E.
      • Ostermann A.
      • et al.
      Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme.
      )). Similar EPR spectra were reported (
      • Jasion V.S.
      • Polanco J.A.
      • Meharenna Y.T.
      • Li H.
      • Poulos T.L.
      Crystal structure of Leishmania major peroxidase and characterization of the compound I tryptophan radical.
      ) for the exchange coupled porphyrin-tryptophanyl radical in LmP. This broadening was clearly absent in parallel experiments at 4 K on the W233F variant (Fig. 3Aiii-iv). A structure of W233F, Fig. S4 and Table 1, shows no changes in the heme active site. In the case of the W233F variant, a narrow radical was observed at 4 K (Fig. 3A(iii-iv), which persisted at 70 K (Fig. 3B(ii)) with a g value of 2.0048. This is similar to the W191F variant of CcP that forms a tyrosyl radical (
      • Ivancich A.
      • Dorlet P.
      • Goodin D.B.
      • Un S.
      Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.
      ).
      Figure thumbnail gr3
      Figure 3Low-temperature EPR spectra of TcAPx-CcP. A, 9-GHz CW EPR spectra of (i) ferric TcAPx-CcP, (ii) compound I of TcAPx-CcP formed by reaction of the ferric sample with H2O2 for 10 s, (iii) ferric W233F, (iv) ferric W233F mixed with H2O2 for 10 s, (v) ferric C222A, and (vi) C222A mixed with H2O2 for 10 s. Spectra were recorded at 4.3 K, 4 G modulation amplitude, 1 mW microwave power, 100 kHz modulation frequency, two scans. B, (i) The same sample of compound I of TcAPx-CcP as in (A)(ii), (ii) The same sample as in (A)(iv), (iii) The same sample as in (A)(vi). Spectra in (B) were recorded at 70 K, 1 G modulation amplitude, 0.2 mW microwave power, 100 kHz modulation frequency, 100 scans. (ii) and (iii) have been multiplied by a factor of 6 to allow comparison. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase.
      Formation of a cysteinyl radical has previously been proposed for TcAPx-CcP (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ). The EPR spectra of TcAPx-CcP on reaction with H2O2 at 70 K (Fig. 3B(i)) shows resonances with g values centered at 2.0041, again consistent with the formation of amino acid radical/s (
      • Jeschke G.
      EPR techniques for studying radical enzymes.
      ). Cysteinyl radicals are known to have an axially symmetric g-tensor with gx values of 2.16 to 2.5 (
      • Jeschke G.
      EPR techniques for studying radical enzymes.
      ,
      • Kolberg M.
      • Bleifuss G.
      • Sjoberg B.M.
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      Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.
      ,
      • Kolberg M.
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      Protein thiyl radicals directly observed by EPR spectroscopy.
      ); we do not clearly observe this feature, although the gx line is known to be weak (
      • Kolberg M.
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      • Sjoberg B.M.
      • Graslund A.
      • Lubitz W.
      • Lendzian F.
      • et al.
      Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.
      ,
      • Kolberg M.
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      • Graslund A.
      • Sjoberg B.M.
      • Lubitz W.
      • Lendzian F.
      • et al.
      Protein thiyl radicals directly observed by EPR spectroscopy.
      ). The spectral features at g = 2.0041 span a width of ∼12.5 mT, which is consistent with that observed previously for cysteinyl radicals (
      • Kolberg M.
      • Bleifuss G.
      • Sjoberg B.M.
      • Graslund A.
      • Lubitz W.
      • Lendzian F.
      • et al.
      Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.
      ,
      • Kolberg M.
      • Bleifuss G.
      • Graslund A.
      • Sjoberg B.M.
      • Lubitz W.
      • Lendzian F.
      • et al.
      Protein thiyl radicals directly observed by EPR spectroscopy.
      ). This is not typical of isolated tyrosyl and tryptophanyl radicals, which have narrower spectral widths (
      • Ivancich A.
      • Dorlet P.
      • Goodin D.B.
      • Un S.
      Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.
      ,
      • Bleifuss G.
      • Kolberg M.
      • Potsch S.
      • Hofbauer W.
      • Bittl R.
      • Lubitz W.
      • et al.
      Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz.
      ). When the EPR experiments were repeated for the C222A variant (Fig. 3A(v-vi)), a signal with g value of 2.0048 was observed at 4 K and persisted at 70 K (Fig. 3B(iii)), consistent with either tyrosine (
      • Gerfen G.J.
      • Bellew B.F.
      • Un S.
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      • Griffin R.G.
      • et al.
      High-frequency (139.5 GHz) EPR spectroscopy of the tyrosyl radical in Escherichia coli ribonucleotide reductase.
      ) or tryptophan (
      • Ivancich A.
      • Dorlet P.
      • Goodin D.B.
      • Un S.
      Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.
      ,
      • Bleifuss G.
      • Kolberg M.
      • Potsch S.
      • Hofbauer W.
      • Bittl R.
      • Lubitz W.
      • et al.
      Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz.
      ) radicals. Formation of amino acid radicals observed at higher temperatures is often observed in off-pathway processes in heme proteins (
      • Fielding A.J.
      • Brodhun F.
      • Koch C.
      • Pievo R.
      • Denysenkov V.
      • Feussner I.
      • et al.
      Multifrequency electron paramagnetic resonance characterization of PpoA, a CYP450 fusion protein that catalyzes fatty acid dioxygenation.
      ,
      • Fielding A.J.
      • Singh R.
      • Boscolo B.
      • Loewen P.C.
      • Ghibaudi E.M.
      • Ivancich A.
      Intramolecular electron transfer versus substrate oxidation in lactoperoxidase: investigation of radical intermediates by stopped-flow absorption spectrophotometry and (9-285 GHz) electron paramagnetic resonance spectroscopy.
      ). The radical/s generated from C222A mutant were notably narrower (Fig. 3B(iii)) than that found for TcAPx-CcP. We tentatively interpret the spectrum shown in Figure 3B(i) as evidence of cysteinyl radical formation in TcAPx-CcP, which is consistent with the previous assignment using spin trapping assays (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ). Further multifrequency EPR work would be needed to unambiguously identify these radical species. Corresponding changes in absorbance spectra for the C222A mutant are shown in Fig. S5.

      Substrate specificity

      In sAPX, the ascorbate substrate is hydrogen bonded to Arg172 at the γ-heme edge (
      • Sharp K.H.
      • Mewies M.
      • Moody P.C.
      • Raven E.L.
      Crystal structure of the ascorbate peroxidase-ascorbate complex.
      ). No equivalent Arg residue is present in TcAPx-CcP, and this residue is instead replaced with Asn226, which is fully conserved in every T. cruzi genome evaluated (Figs. 4 and S2). Interestingly, this position is occupied by tyrosine or phenylalanine in Leishmania related species (Fig. 4). The ordered water molecules found in this region in sAPX are not present in TcAPx-CcP (not shown). Arg172 in sAPX is essential for ascorbate activity (
      • Macdonald I.K.
      • Badyal S.K.
      • Ghamsari L.
      • Moody P.C.
      • Raven E.L.
      Interaction of ascorbate peroxidase with substrates: a mechanistic and structural analysis.
      ). Lys30, which also interacts with the bound ascorbate in the sAPX-ascorbate complex, is also missing in TcAPx-CcP and is replaced with Glu79 and Asp80 (see also later). In CcP, an ascorbate-binding site can be engineered into the enzyme (
      • Murphy E.J.
      • Metcalfe C.L.
      • Basran J.
      • Moody P.C.
      • Raven E.L.
      Engineering the substrate specificity and reactivity of a heme protein: creation of an ascorbate binding site in cytochrome c peroxidase.
      ) by inclusion of an Arg residue at the appropriate location (N184R mutation in CcP, Figs. 4 and S2). Ascorbate binding and catalytic activity is weak (kcat = 1.5 s−1; KM = 1.7 mΜ) but detectable in this N184R variant of CcP (
      • Murphy E.J.
      • Metcalfe C.L.
      • Basran J.
      • Moody P.C.
      • Raven E.L.
      Engineering the substrate specificity and reactivity of a heme protein: creation of an ascorbate binding site in cytochrome c peroxidase.
      ), when compared to wild type sAPX (kcat = 272 s−1; KM = 389 μM; kcat/KM = 0.69 μM−1s−1 (
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      )). The equivalent N226R mutation in TcAPx-CcP (equivalent to N184R in CcP) is viable in terms of protein expression and protein stability but showed very weak heme binding. It was therefore not possible to measure ascorbate activity for this variant. This indicates that building an ascorbate binding at this site is not viable, even in the presence of the required Arg residue. Attempts to crystallize TcAPx-CcP in complex with ascorbate were unsuccessful.
      Figure thumbnail gr4
      Figure 4Comparison of the structures of the γ-heme edge of TcAPx-CcP (green), sAPX (yellow), and yeast CcP (cyan). Ascorbate bound to sAPX is shown as yellow sticks. The important R172 ascorbate-binding residue in APX is shown superimposed with the equivalent residues (N226, N184) from TcAPx-CcP and CcP, respectively. The relevant sequence in this region is also shown, along with sequence logos obtained from Trypanosoma cruzi and Leishmania spp. alignments (see also and ). The extended loop which might interfere with ascorbate binding (
      • Sharp K.H.
      • Mewies M.
      • Moody P.C.
      • Raven E.L.
      Crystal structure of the ascorbate peroxidase-ascorbate complex.
      ) is clearly visible in CcP but missing in TcAPx-CcP. Hydrogen bonds and their distances between R172 of sAPX and ascorbate are indicated with yellow dashes. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase.
      To better understand the interaction of ascorbate with TcAPx-CcP, we performed molecular dynamics (MD) simulations of both the sAPX–ascorbate and TcAPx-CcP–ascorbate complexes. The initial ascorbate pose in the TcAPx-CcP active site was assumed from the sAPX–ascorbate crystal structure (
      • Sharp K.H.
      • Mewies M.
      • Moody P.C.
      • Raven E.L.
      Crystal structure of the ascorbate peroxidase-ascorbate complex.
      ) and then minimized prior to MD simulations (see Experimental procedures). An initial inspection of ascorbate dynamical behavior in both active sites showed significant differences between these complexes; while the interactions displayed by ascorbate with sAPX are specific and long-lived, leading to one preferential ascorbate conformation, in the case of the TcAPx-CcP–ascorbate complex, those interactions are not as strong and a myriad of ascorbate orientations were observed (Fig. 5A). Using the MM-GBSA formalism, we estimated ascorbate binding free energies (ΔGbind) for both cases, which shows a significantly lower ascorbate ΔGbind for the sAPX case (Fig. 5B), that is, stronger substrate interaction with the enzyme. Furthermore, a residue basis decomposition of the estimated ΔGbind values indicates a major role for Arg172 in stabilizing the sAPX–ascorbate complex, explaining a very significant portion of the observed difference in ΔGbind values (Fig. 5B). Lys237, likely to be the most important residue in stabilizing the TcAPx-CcP–ascorbate interaction (Fig. 5B), is fully conserved both in T. cruzi and Leishmania spp. (see Fig. S3).
      Figure thumbnail gr5
      Figure 5Interaction of ascorbate at the active site of APX evaluated by MD simulation. A, superimposition of the conformations adopted by ascorbate at the active site of sAPX (blue cartoon, up) and TcAPx-CcP (black cartoon, down). B, ascorbate binding free energy (ΔGbind, kcal/mol) estimation using the MM-GBSA formalism, along with a residue basis decomposition of the free energy. Only residues contributing more than 0.5 kcal/mol to the calculated binding free energy are shown. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase; MD, molecular dymamics.

      Complex formation with cytochrome c

      Cytochrome c has a cluster of positively charged lysine residues on the surface, which facilitates its binding to the overall negatively charged surfaces of CcP (
      • Pearl N.M.
      • Jacobson T.
      • Meyen C.
      • Clementz A.G.
      • Ok E.Y.
      • Choi E.
      • et al.
      Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: evidence for a single, catalytically active, cytochrome c binding domain.
      ,
      • Leesch V.W.
      • Bujons J.
      • Mauk A.G.
      • Hoffman B.M.
      Cytochrome c peroxidase cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis.
      ), Figure 6A. We were not able to obtain a crystal structure of TcAPx-CcP in complex with cytochrome c, but we carried out an analysis of the electrostatic surface charges of TcAPx-CcP, CcP, APX, and LmP, Figure 6. The surface electrostatics of TcAPx-CcP more closely resemble those of CcP than they do sAPX, Figure 6B, with TcAPx-CcP showing a broad negatively charged region on the surface where cytochrome c would be expected to bind. The similarity is even more striking when comparing TcAPx-CcP to LmP, Figure 6B; LmP has been categorized as a CcP-like enzyme (
      • Jasion V.S.
      • Poulos T.L.
      Leishmania major peroxidase is a cytochrome c peroxidase.
      ).
      Figure thumbnail gr6
      Figure 6Comparison of electron transfer pathways and surface electrostatics. A, alignment of the structures of TcAPx-CcP (faded green) and CcP (faded cyan). The residues involved in the delivery of electrons from cytochrome c in CcP (Trp191, Gly192, Ala193, and Ala194, in green) overlay well with an equivalent electron pathway in TcAPx-CcP (Trp233, Thr234, His235, and Asp236, in green). The heme group is shown for both proteins. The residues involved in binding of cytochrome c in CcP (Asp34, Glu35 (
      • Pelletier H.
      • Kraut J.
      Crystal-structure of a complex between electron-transfer partners, cytochrome c peroxidase and cytochrome c.
      )) overlay well in the TcAPx-CcP structure (Glu79 and Asp80, respectively). B, electrostatic surface representation of TcAPx-CcP (±5 kT), CcP, sAPX, and LmP, obtained using the APBS software (
      • Jurrus E.
      • Engel D.
      • Star K.
      • Monson K.
      • Brandi J.
      • Felberg L.E.
      • et al.
      Improvements to the APBS biomolecular solvation software suite.
      ). The predicted cyt c binding surface is represented in red where the overall charge is strongly electronegative. This electronegative area is substantially less prominent in sAPX; sAPX does not bind cyt c. The residues in TcAPx-CcP which are expected to be responsible for the electron transfer path are shown as dark blue sticks (W233, T234, H235, and D236). The equivalent residues (W191, G192, A193, and A194) in CcP are shown as cyan sticks in (B). See for a sequence alignment highlighting these residues. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase.

      Discussion

      The denomination of the TcAPx-CcP enzyme is derived from its relationship to two other peroxidase enzymes with which TcAPx-CcP shares sequence similarities: APX and CcP. APX and CcP have different substrate binding properties—APX binds to and sources its electrons from a small molecule, ascorbate, whereas CcP transiently interacts with another protein reductase, cytochrome c. Both APX and CcP function through a mechanism that involves formation of a high-valent (oxidized) heme species, known as compound I, as shown below (P = peroxidase):
      P + H2O2 → Compound I +H2O


      Compound I + HS → Compound II + S


      Compound II + HS → P + S + H2O


      A crucial distinction is that CcP uses a highly atypical protein-based radical (located on Trp191 (
      • Sivaraja M.
      • Goodin D.B.
      • Smith M.
      • Hoffman B.M.
      Identification by endor of Trp191 as the free-radical site in cytochrome c peroxidase compound ES.
      )) in its compound I, rather than the usual porphyrin-π-cation radical used in APX (
      • Lad L.
      • Mewies M.
      • Basran J.
      • Scrutton N.S.
      • Raven E.L.
      Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.
      ,
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      ,
      • Patterson W.R.
      • Poulos T.L.
      • Goodin D.B.
      Identification of a porphyrin pi-cation-radical in ascorbate peroxidase compound-I.
      ) and all other peroxidases (
      • Moody P.C.E.
      • Raven E.L.
      The nature and reactivity of ferryl heme in compounds I and II.
      ). While APX and CcP are structurally very similar, with a well-conserved fold, there are notable differences in the substrate-binding regions that help to account for the different substrate-binding behaviors (
      • Sharp K.H.
      • Mewies M.
      • Moody P.C.
      • Raven E.L.
      Crystal structure of the ascorbate peroxidase-ascorbate complex.
      ,
      • Macdonald I.K.
      • Badyal S.K.
      • Ghamsari L.
      • Moody P.C.
      • Raven E.L.
      Interaction of ascorbate peroxidase with substrates: a mechanistic and structural analysis.
      ). In contrast, the lack of structural information for TcAPx-CcP has made interpretation of its reactivity more difficult.
      The information presented in this article now substantially clarify the properties and catalytic behavior of the TcAPx-CcP enzyme. The active site of TcAPx-CcP closely resembles that of APX, CcP, and LmP. The low temperature EPR spectra support the formation of a reactive compound I intermediate with an exchange-coupled [Fe(IV)=O Trp233•+] radical. If the presence of a cation on the proximal side of the heme has a role in destabilizing radical formation on Trp (
      • Bonagura C.A.
      • Bhaskar B.
      • Sundaramoorthy M.
      • Poulos T.L.
      Conversion of an engineered potassium-binding site into a calcium-selective site in cytochrome c peroxidase.
      ,
      • Bonagura C.A.
      • Sundaramoorthy M.
      • Bhaskar B.
      • Poulos T.L.
      The effects of an engineered cation site on the structure, activity, and EPR properties of cytochrome c peroxidase.
      ,
      • Bonagura C.A.
      • Sundaramoorthy M.
      • Pappa H.S.
      • Patterson W.R.
      • Poulos T.L.
      An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan.
      ), then the absence of such a cation in the TcAPx-CcP structure is logical. All the EPR spectra at 70 K showed evidence of the generation of amino acid radicals on reaction of TcAPx-CcP with H2O2. The spectral features in the case of the wild type TcAPx-CcP enzyme were consistent with the generation of a cysteinyl radical, as suggested previously (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ).
      Based on the sequence and the new structural and MD simulation information, TcAPx-CcP would not be expected to be highly competent for ascorbate binding at the γ-heme edge because, like CcP (
      • Murphy E.J.
      • Metcalfe C.L.
      • Basran J.
      • Moody P.C.
      • Raven E.L.
      Engineering the substrate specificity and reactivity of a heme protein: creation of an ascorbate binding site in cytochrome c peroxidase.
      ), it lacks the key Arg residue (Arg172 in APX) that is required for ascorbate binding (
      • Macdonald I.K.
      • Badyal S.K.
      • Ghamsari L.
      • Moody P.C.
      • Raven E.L.
      Interaction of ascorbate peroxidase with substrates: a mechanistic and structural analysis.
      ), Figures 4 and 5. It also lacks Lys30, which is replaced by a pair of negatively charged residues (Glu79, Asp80) as found in CcP. However, TcAPx-CcP also lacks the extended loop that is present in CcP in the γ-heme–binding region, and this loop probably prevents ascorbate binding in CcP (
      • Sharp K.H.
      • Mewies M.
      • Moody P.C.
      • Raven E.L.
      Crystal structure of the ascorbate peroxidase-ascorbate complex.
      ). This might work in favor of ascorbate binding at this location, but we were not able to detect ascorbate at this site in the structure. While it is possible to re-engineer ascorbate activity into CcP by introduction of an Arg in the correct location (N184R mutation (
      • Murphy E.J.
      • Metcalfe C.L.
      • Basran J.
      • Moody P.C.
      • Raven E.L.
      Engineering the substrate specificity and reactivity of a heme protein: creation of an ascorbate binding site in cytochrome c peroxidase.
      )), this was not successful for in TcAPx-CcP (N226R). On this basis, it is difficult to conclude that TcAPx-CcP is a bona fide APX. The higher catalytic efficiency of TcAPx-CcP for cytochrome c (kcat/KM = 2.1 × 105 M−1s−1) than for ascorbate (kcat/KM = 3.5 × 104 M−1s−1, Table 2) (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ) supports this conclusion. Ascorbate activity for sAPX is around 10-fold higher (kcat/KM = 6.9 × 105 M−1s−1 (
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      )) than that for TcAPx-CcP; CcP is itself a very poor APX (kcat/KM = 1.1 × 103 M−1s−1 for ascorbate oxidation (
      • Murphy E.J.
      • Metcalfe C.L.
      • Basran J.
      • Moody P.C.
      • Raven E.L.
      Engineering the substrate specificity and reactivity of a heme protein: creation of an ascorbate binding site in cytochrome c peroxidase.
      )), Table 2.
      Table 2Summary of steady state data for ascorbate oxidation for various peroxidases
      EnzymeAscorbateCytochrome cRef
      kcat (s−1)KM (μM)kcat/KM (M−1s−1)kcat (s−1)KM (μM)kcat/KM (M−1s−1)
      TcAPx-CcP
      The W233F variant of TcAPX-CcP has no measurable activity for cytochrome c and exhibits a 10-fold decrease in ascorbate activity compared to the wild type enzyme (9).
      0.0671903.5 × 1046.1292.1 × 105(
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      )
      APX
      APXs have no measurable activity for cytochrome c (12, 64).
      2723896.9 × 105---(
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      )
      CcP0.837101.1 × 103150931.6 × 106(
      • Murphy E.J.
      • Metcalfe C.L.
      • Basran J.
      • Moody P.C.
      • Raven E.L.
      Engineering the substrate specificity and reactivity of a heme protein: creation of an ascorbate binding site in cytochrome c peroxidase.
      )
      LmP---170082 × 108(
      • Jasion V.S.
      • Polanco J.A.
      • Meharenna Y.T.
      • Li H.
      • Poulos T.L.
      Crystal structure of Leishmania major peroxidase and characterization of the compound I tryptophan radical.
      )
      a The W233F variant of TcAPX-CcP has no measurable activity for cytochrome c and exhibits a 10-fold decrease in ascorbate activity compared to the wild type enzyme (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ).
      b APXs have no measurable activity for cytochrome c (
      • Raven E.L.
      Understanding functional diversity and substrate specificity in haem peroxidases: what can we learn from ascorbate peroxidase?.
      ,
      • Dalton D.A.
      Ascorbate peroxidase.
      ).
      The known electron transfer pathway from cytochrome c in the CcP/c complex (
      • Pelletier H.
      • Kraut J.
      Crystal-structure of a complex between electron-transfer partners, cytochrome c peroxidase and cytochrome c.
      ) is replicated in TcAPx-CcP, Figure 6A. Whilst we were unable to obtain a structure for the TcAPx-CcP-cytochrome c, a comparison of surface electrostatics, Figure 6B, is compelling in its similarity to both CcP and LmP, the latter of which it shares a 57% sequence identity (Fig. S2). TcAPx-CcP contains the residues equivalent to Asp34 and Glu35 in CcP (Glu79 and Asp80, Fig, S2), which align well in the structure, Figure 6. These residues, along with Asp37 and Glu290 in CcP (which are absent in TcAPx-CcP, Fig. S2), are important for cytochrome c binding (
      • Pelletier H.
      • Kraut J.
      Crystal-structure of a complex between electron-transfer partners, cytochrome c peroxidase and cytochrome c.
      ) and indeed are entirely absent in sAPX.
      The structural information and bioinformatic analysis provided herein are consistent with an assignment of TcAPx-CcP as a credible cytochrome c peroxidase but a poorer APX and in agreement with other conclusions for LmP (
      • Jasion V.S.
      • Doukov T.
      • Pineda S.H.
      • Li H.
      • Poulos T.L.
      Crystal structure of the Leishmania major peroxidase-cytochrome c complex.
      ,
      • Jasion V.S.
      • Polanco J.A.
      • Meharenna Y.T.
      • Li H.
      • Poulos T.L.
      Crystal structure of Leishmania major peroxidase and characterization of the compound I tryptophan radical.
      ). The steady state kinetic information, when compared across all three enzymes (TcAPx-CcP, APX, and CcP) and LmP, bears this out. Having said that, while ascorbate activity is evidently weaker in TcAPx-CcP, it would not be surprising for a CcP-like enzyme to exhibit some level of ascorbate-dependent activity given the structural similarities of the CcP, APX, and TcAPx-CcP enzymes. Peroxidases are not fussy enzymes in terms of their substrate specificity—they can usually oxidize several different substrates and can bind the same substrate at several different locations (
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      ,
      • Murphy E.J.
      • Metcalfe C.L.
      • Nnamchi C.
      • Moody P.C.
      • Raven E.L.
      Crystal structure of guaiacol and phenol bound to a heme peroxidase.
      ). Even APXs, while presumably designed to favor ascorbate binding, might bind ascorbate at other (unknown) locations (
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      ) (and can oxidize numerous other substrates too). This could well be the case here for TcAPx-CcP, with ascorbate binding at one of several nonspecific locations (at, or remote from, the γ-heme edge) and thus retaining some ascorbate activity (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ). This would be beneficial to the parasite at the plasma membrane in infective extracellular trypomastigotes and intracellular amastigotes (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ), as millimolar concentrations of ascorbate are known to be present in the mammalian extracellular milieu or host cell cytoplasm (
      • Clark D.
      • Albrecht M.
      • Arevalo J.
      Ascorbate variations and dehydroascorbate reductase activity in trypanosoma cruzi epimastigotes and trypomastigotes.
      ,
      • Levine M.
      • Conry-Cantilena C.
      • Wang Y.
      • Welch R.W.
      • Washko P.W.
      • Dhariwal K.R.
      • et al.
      Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance.
      ). Thus, TcAPX can be considered kinetically, evolutionarily, and functionally as a hybrid peroxidase. While is clear that it is less finely tuned for oxidation of ascorbate in comparison to sAPX, it still exhibits reasonable KM and kcat/KM (see comparative values in Table 2). Thus, TcAPX likely binds and utilizes ascorbate opportunistically and in an unoptimized manner in host cells as an evolutionary adaptation.

      Experimental procedures

      Solutions

      All assays, unless specified, were performed in potassium phosphate buffer 50 mM pH 7.4. The concentrations of TcAPx-CcP and H2O2 were spectrophotometrically determined at 409 nm (101 mM−1 cm−1) and 240 nm (39.4 M−1 cm−1 (
      • Nelson D.P.
      • Kiesow L.A.
      Enthalpy of decomposition of hydrogen-peroxide by catalase at 25 degrees C (with molar extinction coefficients of H2O2 solutions in Uv).
      )), respectively.

      Expression and purification

      The plasmid for expression of TcAPx-CcP in Escherichia coli was provided by Shane Wilkinson, Queen Mary University, UK (
      • Wilkinson S.R.
      • Obado S.O.
      • Mauricio I.L.
      • Kelly J.M.
      Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum.
      ) in the pTrcHis expression vector. Purification was carried out as previously described (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ,
      • Wilkinson S.R.
      • Obado S.O.
      • Mauricio I.L.
      • Kelly J.M.
      Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum.
      ) except that δ-aminolevulinic acid (0.5 mM) was added at the point of inducing expression with IPTG (0.8 mM) in order to improve the incorporation of heme into the protein. Terrific broth growth medium was used for all expressions. Purity of protein preparations was assessed by a 12.5 % SDS-PAGE gel and the relative height of the 409 nm Soret peak (ε409 nm = 101 mM−1 cm−1) to the overall protein content peak according to the 280 nm signal (ε280 nm = 56.9 mM−1 cm−1). Typically, after a nickel affinity column, a range of fractions of varying purity (Fig. S1) were obtained. For kinetics experiments, only the purest samples were used (as judged by SDS-gel and the ASoret/A280 ratio). For crystallography experiments, an additional purification step was carried out using a Superdex 75 gel filtration column on an FPLC instrument. For all experiments, the protein concentration was determined spectrophotometrically using the 409 nm Soret peak. Percentage heme incorporation in purified samples was calculated from the A409/A280 ratio, with the holoenzyme representing typically ca 60%. The concentration as determined from the 409 nm signal representing holoprotein was used exclusively in all subsequent experiments.
      Site-directed variants (C222A and N226R) were produced according to the KLD enzyme mix protocol (New England Biolabs). Mutations were confirmed by DNA sequencing by Eurofins Genomics using pTrcHis forward and reverse standard primers. The purification protocol for the variants was the same as for the WT protein except for N226R, which required the addition of free heme before gel filtration to increase the proportion of holoprotein in the sample.

      Crystallography

      TcAPx-CcP was purified as described previously and was concentrated to 9 mg/ml. Initial crystals of both WT and the W233F variant were obtained using an Art Robbins Phoenix/Gryphon crystallography robot and Molecular Dimensions screens. The best crystals were grown in 2 μl of reservoir solution (2.0 M ammonium sulfate, 0.1 M sodium acetate pH 4.6) and an equal amount of protein solution using the hanging drop method. Irregularly sized, hexagonal prism–shaped crystals grew to their maximum size within a week at 18 °C. Crystals of holoprotein were soaked in a cryoprotectant solution consisting of the reservoir solution plus 25% glycerol and then flash frozen in liquid nitrogen prior to data collection.
      Data collection was carried out at the Diamond Light Source I03. Data were indexed using iMOSFLM and then scaled and merged using AIMLESS as part of the CCP4 suite. The protein crystallized with two molecules per asymmetric unit and belongs to the P 31 2 2 trigonal space group with unit cell dimensions of a = 71.6 Å, b = 71.6 Å, and c = 253.3 Å. The crystal was found to be twinned and was refined as such. Data collection and refinements statistics are shown in Table 1. The structure was determined by molecular replacement using the LmP structure (Protein Data Bank 3RIV) as the search model in Phaser (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ) and refined with REFMAC (
      • Vagin A.A.
      • Steiner R.A.
      • Lebedev A.A.
      • Potterton L.
      • McNicholas S.
      • Long F.
      • et al.
      REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use.
      ) and COOT (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of coot.
      ). The average B factor for the WT TcAPx-CcP is significantly higher than for the W233F variant (50 vs 26), which is indicative of relatively higher mobility in WT. Attempts to crystallize TcAPx-CcP in complex with either ascorbate or cytochrome c were unsuccessful.

      EPR

      Samples (75–100 μM, 50 mM sodium phosphate buffer, pH 7.4) were prepared by mixing an equal volume of hydrogen peroxide with TcAPx-CcP and reacting for 10 s before being flash frozen into 4 mm quartz EPR tubes. Continuous-wave EPR spectra at X band (9 GHz) were recorded using a Bruker EMX spectrometer. The spectrometer was equipped with a super high sensitivity probe head and a liquid helium cryostat (Oxford Instruments). Typical X-band spectra were recorded under nonsaturating conditions at 4 and 70 K to ascertain unique species. EPR parameters are stated on the Figure legends. g-values were calibrated against a 2,2-diphenyl-1-picrylhydrazyl standard.

      Bioinformatic data retrieval and analysis

      Amino acid sequences of T. cruzi CL Brener Esmeraldo–like TcCLB.503745.30 (328 aa) were obtained from TriTrypDB version 56 (
      • Altschul S.F.
      • Gish W.
      • Miller W.
      • Myers E.W.
      • Lipman D.J.
      Basic local alignment search tool.
      ) and used as query for a BLASTp search (
      • Aslett M.
      • Aurrecoechea C.
      • Berriman M.
      • Brestelli J.
      • Brunk B.P.
      • Carrington M.
      • et al.
      TriTrypDB: a functional genomic resource for the trypanosomatidae.
      ) in T. cruzi and Leishmania species. Seventy-two APX sequences were obtained with e-values > 8,00E-06: 58 sequences from 18 Leishmania species (Leishmania enriettii, Leishmania ghana, Leishmania namibia, Leishmania aethiopica, Leishmania amazonensis, Leishmania arabica, Leishmania donovani, Leishmania gerbilli, Leishmania infantum, L. major, Leishmania martiniquensis, Leishmania tarantolae, Leishmania mexicana, Leishmania braziliensis, Leishmania turanica, Leishmania tropica, Leishmania orientalis, and Leishmania panamensis) and 16 sequences from nine T. cruzi strains (T. cruzi Brazil A4, T. cruzi CL Brener [Esmeraldo Like and Non-Esmeraldo Like haplotypes], T. cruzi Dm28c, T. cruzi marinkellei, T. cruzi CL, T. cruzi G, T. cruzi TCC, T. cruzi Sylvio, and T. cruzi Y). In addition, BLASTp was performed against Glycine max and Saccharomyces cerevisiae to get homologous sequences from those species (IDs Q43758 from UniProt and AJS52974.1 from the NCBI database, respectively). First 73 amino acids from the S. cerevisiae CcP sequence, corresponding to mitochondrial signal peptide, were removed to perform the subsequent analysis.
      Multiple sequence alignment was performed using MUSCLE (
      • Edgar R.C.
      Muscle: a multiple sequence alignment method with reduced time and space complexity.
      ). Alignments were manually curated removing partial sequences. Finally, sequence logos (
      • Schneider T.D.
      • Stephens R.M.
      Sequence logos - a new way to display consensus sequences.
      ) were made (that is, graphical representations of the sequence conservation of amino acids) for T. cruzi and Leishmania species alignments, using the web-based application WebLogo (
      • Crooks G.E.
      • Hon G.
      • Chandonia J.M.
      • Brenner S.E.
      WebLogo: a sequence logo generator.
      ). Briefly, the sequence conservation at a particular position Rseq is defined as the difference between the maximum possible entropy (Smax) and the entropy of the observed symbol distribution (Sobs):
      Rseq=SmaxSobs=log2N(n=1Npnlog2pn),


      where pn is the observed frequency of symbol n at a particular position and N is the number of distinct symbols for the given sequence type (20 for protein). Consequently, the maximum sequence conservation per site is log2 (
      • Lad L.
      • Mewies M.
      • Raven E.L.
      Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.
      )≈4 bits (
      • Schneider T.D.
      • Stephens R.M.
      Sequence logos - a new way to display consensus sequences.
      ).

      Classical MD and binding free energy estimations

      MD simulations of both sAPX and TcAPx-CcP dimeric structures were performed, using as starting models the sAPX (1OAF (
      • Sharp K.H.
      • Mewies M.
      • Moody P.C.
      • Raven E.L.
      Crystal structure of the ascorbate peroxidase-ascorbate complex.
      )) and TcAPx-CcP (7OPT, this work) structures, respectively. The concomitant ascorbate complexes (sAPX–ascorbate and TcAPx-CcP–ascorbate) were simulated. The initial ascorbate pose in the TcAPx-CcP active site was generated by a structural alignment of the sAPX–ascorbate structure and that of TcAPx-CcP, followed by an energy minimization of the ascorbate moiety. These four systems assumed the heme to be in the oxyferryl state and were subjected to the same MD protocol. Briefly, systems were solvated using a default method, with an octahedral box of 12 Å in radius with TIP3P water molecules (
      • Jorgensen W.L.
      • Chandrasekhar J.
      • Madura J.D.
      • Impey R.W.
      • Klein M.L.
      Comparison of simple potential functions for simulating liquid water.
      ). Protein residue parameters correspond to the parm14SB Amber force field (
      • Maier J.A.
      • Martinez C.
      • Kasavajhala K.
      • Wickstrom L.
      • Hauser K.E.
      • Simmerling C.
      ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB.
      ), oxyferryl-heme parameters correspond to the previously developed ones (
      • Hugo M.
      • Martinez A.
      • Trujillo M.
      • Estrada D.
      • Mastrogiovanni M.
      • Linares E.
      • et al.
      Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).
      ,
      • Capece L.
      • Lewis-Ballester A.
      • Marti M.A.
      • Estrin D.A.
      • Yeh S.R.
      Molecular basis for the substrate stereoselectivity in tryptophan dioxygenase.
      ), and ascorbate parameters were developed by a standard procedure: partial charges were computed using the restricted electrostatic potential recipe and density functional theory based electronic structure calculations with the Perdew-Burke-Ernzerhof functional and dzvp basis set. Equilibrium distances and angles, as well as force constants, were computed using the same methods and the basis set used for computed charges. All simulations were performed using periodic boundary conditions with a 10 Å cutoff and particle mesh Ewald summation method for treating the electrostatic interactions. The hydrogen bond lengths were kept at their equilibrium distance by using the SHAKE algorithm, while temperature and pressure were kept constant with a Langevin thermostat and barostat, respectively, as implemented in the AMBER program (
      • Case D.A.
      • Ben-Shalom I.Y.
      • Brozell S.R.
      • Cerutti D.S.
      • Cheatham T.E.
      • I
      • et al.
      Amber 2018.
      ). In every case, the system was optimized in 1000 steps (10 with steep gradient and the rest with conjugate gradient). Then, it was slowly heated from 0 K to 300 K for 20 ps at constant pressure, with Berendsen thermostat, and pressure was equilibrated at 1 bar for 5 ps. After these two steps, a 10 ns MD long simulation at constant temperature (300 K) and constant volume was performed. Afterward, 500 ns trajectories in which a “wall-like” restraint was applied, biasing carboxylic groups from heme and ascorbate species distance to be less than 5 Å was performed. Binding free energy calculations were performed at the molecular mechanics/generalized Born and surface area level (
      • Rastelli G.
      • Del Rio A.
      • Degliesposti G.
      • Sgobba M.
      Fast and accurate predictions of binding free energies using MM-PBSA and MM-GBSA.
      ), selecting 500 representative equally spaced structures from each trajectory for analysis. The residue basis free energy decomposition among the closest 25 protein residues from ascorbate was calculated. All dynamics visualizations and molecular drawings were performed with VMD 1.9.1 (
      • Humphrey W.
      • Dalke A.
      • Schulten K.
      Vmd: visual molecular dynamics.
      ).

      Data availability

      Atomic coordinates have been deposited in the Protein Data Bank (PDB ID codes 7OPT [TcAPx-CcP] and 7OQR [W233F TcAPx-CcP]).

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Author contributions

      L. P., R. R., and E. L. R. conceptualization; S. L. F., H. K., A. J. F., and L. P. methodology; F. M. I., L. I., A. Z., and P. S. software; H. K. validation; S. L. F., V. S., H. K., A. J. F., A. M., F. M. I., A. Z., L. I., P. S., and L. P. investigation; A. J. F. resources; S. L. F., A. J. F., P. C. M. E., A. Z., R. R., and E. L. R. writing–original draft; S. L. F. visualization; R. R. and E. L. R. supervision; R. R. and E. L. R. project administration; P. C. M. E., R. R., and E. L. R. funding acquisition.

      Funding and additional information

      We thank the Royal Society for funding (grant IC170118 to R. R. and E. L. R., and RSWF∖R3∖183003 to P. C. E. M.), the National Engineering and Physical Sciences Research Council EPR service and facility for instrument and EI_2020 and CSIC Grupos_2018 to R. R.), and Diamond Light Source for beamtime (proposal MX23269). Additional funding was provided by Programa de Desarrollo de Ciencias Básicas (PEDECIBA, Uruguay). V. S. was partially funded by a graduate student fellowship from Universidad de la República.

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