RESUMEN
Heme enzymes are ubiquitous in biology and catalyze a vast array of biological redox processes. The formation of high valent ferryl intermediates of the heme iron (known as Compounds I and Compound II) is implicated for a number of catalytic heme enzymes, but these species are formed only transiently and thus have proved somewhat elusive. In consequence, there has been conflicting evidence as to the nature of these ferryl intermediates in a number of different heme enzymes, in particular the precise nature of the bond between the heme iron and the bound oxygen atom. In this work, we present high resolution crystal structures of both Compound I and Compound II intermediates in two different heme peroxidase enzymes, cytochrome c peroxidase and ascorbate peroxidase, allowing direct and accurate comparison of the bonding interactions in the different intermediates. A consistent picture emerges across all structures, showing lengthening of the ferryl oxygen bond (and presumed protonation) on reduction of Compound I to Compound II. These data clarify long standing inconsistencies on the nature of the ferryl heme species in these intermediates.
Asunto(s)
Hemo/química , Hemoglobinas/química , Hierro/química , Oxihemoglobinas/química , Ascorbato Peroxidasas , Cristalografía por Rayos X , Citocromo-c Peroxidasa/metabolismo , Hemo/metabolismo , Hemoglobinas/metabolismo , Hierro/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Oxihemoglobinas/metabolismo , Peroxidasas/metabolismo , Estructura Terciaria de Proteína , Protones , EstereoisomerismoRESUMEN
Indoleamine 2,3-dioxygenase catalyzes the O(2)-dependent oxidation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O(2) and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O(2) binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E(m)(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E(m)(0) (that increases in the presence of L-Trp) and the rate constant for O(2) binding. This means that the initial formation of ferric superoxide (Fe(3+)-O(2)(â¢-)) from Fe(2+)-O(2) becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E(m)(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.
Asunto(s)
Bioquímica/métodos , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Catálisis , Química Farmacéutica/métodos , Hemo/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Cinética , Quinurenina/análogos & derivados , Quinurenina/química , Mutagénesis Sitio-Dirigida , Oxígeno/química , Unión Proteica , Especificidad por Sustrato , Termodinámica , Triptófano/químicaRESUMEN
As members of the family of heme-dependent enzymes, the heme dioxygenases are differentiated by virtue of their ability to catalyze the oxidation of l-tryptophan to N-formylkynurenine, the first and rate-limiting step in tryptophan catabolism. In the past several years, there have been a number of important developments that have meant that established proposals for the reaction mechanism in the heme dioxygenases have required reassessment. This focused review presents a summary of these recent advances, written from a structural and mechanistic perspective. It attempts to present answers to some of the long-standing questions, to highlight as yet unresolved issues, and to explore the similarities and differences of other well-known catalytic heme enzymes such as the cytochromes P450, NO synthase, and peroxidases.
Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/análogos & derivados , Triptófano Oxigenasa/metabolismo , Triptófano/metabolismo , Animales , Biocatálisis , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Quinurenina/química , Quinurenina/metabolismo , Modelos Moleculares , Estructura Molecular , Estructura Terciaria de Proteína , Triptófano/química , Triptófano Oxigenasa/químicaRESUMEN
We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path. Kinetic constants for H(2)O(2)-dependent enzyme oxidation to Compound I, k(1), and subsequent reduction of Compound II, k(3), were determined in steady-state assays by variation of both H(2)O(2) and ascorbate concentrations. A high value of the SKIE for wild type APX ((D)k(3) = 4.9) as well as a clear nonlinear dependence on the deuterium composition of the solvent in proton inventory experiments suggest the simultaneous participation of several protons in the transition state for proton transfer. The full SKIE and the proton inventory data were modeled by applying Gross-Butler-Swain-Kresge theory to a proton path inferred from the known structure of APX. The model has been tested by constructing and determining the X-ray structures of the R38K and R38A variants and accounts for their observed SKIEs. This work confirms APX uses two arginine residues in the proton path. Thus, Arg38 and Arg172 have dual roles, both in the formation of the ferryl species and binding of ascorbate respectively and to facilitate proton transfer between the two.
Asunto(s)
Ascorbato Peroxidasas/metabolismo , Hemo/metabolismo , Protones , Ascorbato Peroxidasas/química , Cristalografía por Rayos X , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Glycine max/enzimologíaRESUMEN
Heme dioxygenases catalyze the oxidation of L-tryptophan to N-formylkynurenine (NFK), the first and rate-limiting step in tryptophan catabolism. Although recent progress has been made on early stages in the mechanism, there is currently no experimental data on the mechanism of product (NFK) formation. In this work, we have used mass spectrometry to examine product formation in a number of dioxygenases. In addition to NFK formation (m/z = 237), the data identify a species (m/z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during O(2)-driven turnover. The fragmentation pattern for this m/z = 221 species is consistent with a cyclic amino acetal structure; independent chemical synthesis of the 3a-hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. Labeling experiments with (18)O(2) confirm the origin of the oxygen atom as arising from O(2)-dependent turnover. These data suggest that the dioxygenases use a ring-opening mechanism during NFK formation, rather than Criegee or dioxetane mechanisms as previously proposed.
Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/análogos & derivados , Triptófano Oxigenasa/metabolismo , Hemo/metabolismo , Humanos , Quinurenina/metabolismo , Espectrometría de Masas , Oxígeno/metabolismo , Xanthomonas campestris/enzimologíaRESUMEN
The interactions of heme peroxidase enzymes with their substrates have been studied for many years, but only in the last decade or so has structural information begun to appear. This review looks at crystal structures for a number of heme peroxidases in complex with a number of (mainly organic) substrates. It examines the nature and location of the binding interaction, and explores functional similarities and differences across the family.
Asunto(s)
Hemo/química , Peroxidasas/química , Peroxidasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Hemo/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Especificidad por SustratoRESUMEN
Ascorbate peroxidase (APX), cytochrome c peroxidase (CcP), and the catalase-peroxidases (KatG) share very similar active site structures and are distinguished from other peroxidases by the presence of a distal tryptophan residue. In KatG, this distal tryptophan forms a covalent link to an adjacent tyrosine residue, which in turn links to a methionine residue. We have previously shown [ Pipirou, Z. et al. ( 2007 ) Biochemistry 46 , 2174 - 2180 ] that reaction of APX with peroxide leads, over long time scales, to formation of a covalent link with the distal tryptophan (Trp41) in a mechanism that proceeds through initial formation of a compound I species bearing a porphyrin pi-cation radical followed by radical formation on Trp41, as implicated in the KatG enzymes. Formation of such a covalent link in CcP has never been reported, and we proposed that this could be because compound I in CcP uses Trp191 instead of a porphyrin pi-cation radical. To test this, we have examined the reactivity of the W191F variant of CcP with H(2)O(2), in which formation of a porphyrin pi-cation radical occurs. We show, using electronic spectroscopy, HPLC, and mass spectroscopy, that in W191F partial formation of a covalent link from Trp51 to the heme is observed, as in APX. Radical formation on Trp51, as seen for KatG and APX, is implicated; this is supported by QM/MM calculations. Collectively, the data show that all three members of the class I heme peroxidases can support radical formation on the distal tryptophan and that the reactivity of this radical can be controlled either by the protein structure or by the nature of the compound I intermediate.
Asunto(s)
Citocromo-c Peroxidasa/química , Hemo/química , Peróxidos/química , Triptófano/química , Cromatografía Líquida de Alta Presión , Citocromo-c Peroxidasa/metabolismo , Estructura Molecular , Oxidantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The heme peroxidase and heme oxygenase enzymes share a common heme prosthetic group but catalyze fundamentally different reactions, the first being H(2)O(2)-dependent oxidation of substrate using an oxidized Compound I intermediate, and the second O(2)-dependent degradation of heme. It has been proposed that these enzymes utilize a common reaction intermediate, a ferric hydroperoxide species, that sits at a crossroads in the mechanism and beyond which there are two mutually exclusive mechanistic pathways. Here, we present evidence to support this proposal in a heme peroxidase. Hence, we describe kinetic data for a variant of ascorbate peroxidase (W41A) which reacts slowly with tert-butyl hydroperoxide and does not form the usual peroxidase Compound I intermediate; instead, structural data show that a product is formed in which the heme has been cleaved at the alpha-meso position, analogous to the heme oxygenase mechanism. We interpret this to mean that the Compound I (peroxidase) pathway is shut down, so that instead the reaction intermediate diverts through the alternative (heme oxygenase) route. A mechanism for formation of the product is proposed and discussed in the light of what is known about the heme oxygenase reaction mechanism.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/química , Hemo Oxigenasa (Desciclizante)/metabolismo , Peroxidasas/química , Peroxidasas/metabolismo , Ascorbato Peroxidasas , Ácido Aspártico/genética , Cristalización , Cristalografía por Rayos X , Variación Genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Peroxidasas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Glycine max/enzimología , Glycine max/genética , Triptófano/genética , terc-Butilhidroperóxido/química , terc-Butilhidroperóxido/metabolismoRESUMEN
Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme enzymes that catalyze the O(2)-dependent oxidation of L-tryptophan to N-formyl-kynurenine. Previous proposals for the mechanism of this reaction have suggested that deprotonation of the indole NH group, either by an active-site base or by oxygen bound to the heme iron, as the initial step. In this work, we have examined the activity of 1-Me-L-Trp with three different heme dioxygenases and their site-directed variants. We find, in contrast to previous work, that 1-Me-L-Trp is a substrate for the heme dioxygenase enzymes. These observations suggest that deprotonation of the indole N(1) is not essential for catalysis, and an alternative reaction mechanism, based on the known chemistry of indoles, is presented.
Asunto(s)
Química Orgánica/métodos , Dioxigenasas/química , Hemo/química , Catálisis , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Indoles/química , Cinética , Quinurenina/química , Modelos Químicos , Mutagénesis Sitio-Dirigida , Oxígeno/química , Protones , Triptófano/química , Triptófano Oxigenasa/químicaRESUMEN
Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-alpha antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-alpha dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-alpha therapy. These findings place IDO(+) DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.
Asunto(s)
Células Dendríticas/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Listeria monocytogenes/crecimiento & desarrollo , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Complejo CD3/análisis , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Células Dendríticas/citología , Células Dendríticas/microbiología , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/genética , Granuloma/genética , Granuloma/metabolismo , Granuloma/microbiología , Humanos , Immunoblotting , Inmunohistoquímica , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Antígeno Lewis X/análisis , Listeriosis/genética , Listeriosis/metabolismo , Listeriosis/microbiología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas S100/análisis , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The family of haem dioxygenases catalyse the initial oxidative cleavage of L-tryptophan to N-formylkynurenine, which is the first, rate-limiting, step in the L-kynurenine pathway. In the present paper, we discuss and compare structure and function across the family of haem dioxygenases by focusing on TDO (tryptophan 2,3-dioxygenase) and IDO (indoleamine 2,3-dioxygenase), including a review of recent structural information for both enzymes. The present paper describes how the recent development of recombinant expression systems has informed our more detailed understanding of the substrate binding, catalytic activity and mechanistic properties of these haem dioxygenases.
Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Triptófano Oxigenasa/metabolismo , Triptófano/metabolismo , Sitios de Unión , Catálisis , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Triptófano Oxigenasa/químicaRESUMEN
The binding of substrates to heme enzymes has been widely assumed to occur at the so-called delta-heme edge. Recently, however, a number of examples have appeared in which substrate binding at an alternative site, the gamma-heme edge, is also possible. In previous work [Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307], we showed that binding of ascorbate to ascorbate peroxidase occurred at the gamma-heme edge. Here, we show that the closely related cytochrome c peroxidase enzyme can duplicate the substrate binding properties of ascorbate peroxidase through the introduction of relatively modest structural changes at Tyr36 and Asn184. Hence, crystallographic data for the Y36A/N184R/W191F triple variant of cytochrome c peroxidase shows ascorbate bound to the gamma-heme edge, with hydrogen bonds to the heme propionate and Arg184. In parallel mechanistic studies in variants incorporating the W191F mutation, we show that a transient porphyrin pi-cation radical in Compound I of cytochrome c peroxidase, analogous to that observed in ascorbate peroxidase, is competent for ascorbate oxidation but that under steady state conditions this intermediate decays too rapidly to sustain efficient turnover of ascorbate. The results are discussed in terms of our more general understanding of substrate oxidation across other heme proteins, and the emerging role of the heme propionates at the gamma-heme edge.
Asunto(s)
Ácido Ascórbico/metabolismo , Citocromo-c Peroxidasa/metabolismo , Ingeniería de Proteínas , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Citocromo-c Peroxidasa/genética , Hemoproteínas , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Previous work [Metcalfe, Ott, Patel, Singh, Mistry, Goff and Raven (2004) J. Am. Chem. Soc. 126, 16242-16248] has shown that the introduction of a methionine residue (S160M variant) close to the 2-vinyl group of the haem in ascorbate peroxidase leads to the formation of a covalent haem-methionine linkage under oxidative conditions (i.e. on reaction with H2O2). In the present study, spectroscopic, HPLC and mass spectrometric evidence is presented to show that covalent attachment of the haem to an engineered cysteine residue can also occur in the S160C variant, but, in this case, under reducing conditions analogous to those used in the formation of covalent links in cytochrome c. The data add an extra dimension to our understanding of haem to protein covalent bond formation because they show that different types of covalent attachment (one requiring an oxidative mechanism, the other a reductive pathway) are both accessible within same protein architecture.
Asunto(s)
Hemo/metabolismo , Peroxidasas/metabolismo , Secuencia de Aminoácidos , Ascorbato Peroxidasas , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Peroxidasas/química , Peroxidasas/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H+ ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction.
Asunto(s)
Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Enterobacter cloacae/química , Difracción de Neutrones/métodos , Oxidorreductasas/química , Proteínas/química , Hidrógeno/química , Modelos Moleculares , Oxidación-Reducción , Peroxidasas/química , Análisis Espectral/métodosRESUMEN
We have previously demonstrated (Badyal et al., J. Biol. Chem., 2006, 281, 24512) that removal of the active site tryptophan (Trp41) in ascorbate peroxidase increases the conformational mobility of the distal histidine residue (His42) and that His42 coordinates to the iron in the oxidised W41A enzyme to give a 6-coordinate, low-spin peroxidase. In this work, we probe the conformational flexibility of the active site in more detail. We examine whether other residues (Cys, Tyr, Met) can also ligate to the heme at position 42; we find that introduction of other ligating amino acids created a cavity in the heme pocket, but that formation of 6-coordinate heme is not observed. In addition, we examine the role of Asn-71, which hydrogen bonds to His42 and tethers the distal histidine in the active site pocket; we find that removal of this hydrogen bond increases the proportion of low-spin heme. We suggest that, in addition to its well-known role in facilitating the reaction with peroxide, His42 also plays a role in defining the shape and folding of the active site pocket.
Asunto(s)
Ascorbato Peroxidasas/química , Ascorbato Peroxidasas/metabolismo , Dominio Catalítico , Hemo/metabolismo , Ascorbato Peroxidasas/genética , Histidina , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Glycine max/enzimologíaRESUMEN
Heme iron is often used in biology for activation of oxygen. The mechanisms of oxygen activation by heme-containing monooxygenases (the cytochrome P450s) are well known, and involve formation of a Compound I species, but information on the heme-containing dioxygenase enzymes involved in tryptophan oxidation lags far behind. In this review, we gather together information emerging recently from structural, mechanistic, spectroscopic, and computational approaches on the heme dioxygenase enzymes involved in tryptophan oxidation. We explore the subtleties that differentiate various heme enzymes from each other, and use this to piece together a developing picture for oxygen activation in this particular class of heme-containing dioxygenases.
Asunto(s)
Dioxigenasas/metabolismo , Hemo/metabolismo , Biocatálisis , Dioxigenasas/química , Dioxigenasas/clasificación , Hemo/química , Humanos , Oxidación-Reducción , Especificidad por Sustrato , Triptófano/química , Triptófano/metabolismoRESUMEN
The involvement of the metallic element iron in various biological systems is well known. In many cases, iron is employed in the form of a heme group and the family of proteins and catalytic enzymes that contain heme is well documented (e.g. the globins, cytochromes, and P450s). For many of these proteins, there is a great deal of information available in terms of structures, catalytic mechanism and function. This has led to a collective view that the main role of heme in biological systems is as a prosthetic group, binding to individual proteins and thereby conferring upon them particular functional properties. It is now becoming clear that this description represents only a part of a much more complex involvement of heme in biology and that other roles, for example in regulation and sensing, have been overlooked. This mini-review focuses on one such emerging role: the regulatory role of heme in neurons.
Asunto(s)
Hemo/metabolismo , Neuronas/metabolismo , Animales , Hemo/análisis , Hemo/genética , Humanos , Hierro/metabolismo , Modelos Moleculares , Neuronas/citologíaRESUMEN
The potential of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium for biocatalysis and biotechnological application is widely acknowledged. The catalytic and structural analysis of the Ala82Phe mutant of P450 BM3 has shown that filling a hydrophobic pocket near the active site improved the binding of small molecules, such as indole (see Huang et al., J. Mol. Biol., 2007, 373, 633) and styrene. In this paper, additional mutations at Thr438 are shown to decrease the binding of and catalytic activity towards laurate, whereas they significantly increased the stereo-specificity of styrene epoxidation. Production of R-styrene oxide with 48% and 64% e.e., respectively, was achieved by the Ala82Phe-Thr438Leu and Ala82Phe-Thr438Phe mutants. These structure-based mutants of P450 BM3 illustrate the promise of rational design of synthetically useful biocatalysts for regio- and stereo- specific mono-oxygenation reactions.
Asunto(s)
Bacillus megaterium/enzimología , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos Epoxi/metabolismo , Mutagénesis Sitio-Dirigida , NADPH-Ferrihemoproteína Reductasa/metabolismo , Estireno/metabolismo , Bacillus megaterium/química , Bacillus megaterium/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Microbiología Industrial , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/genética , Unión Proteica , Conformación ProteicaRESUMEN
Isoniazid (INH, isonicotinic acid hydrazine) is one of only two therapeutic agents effective in treating tuberculosis. This prodrug is activated by the heme enzyme catalase peroxidase (KatG) endogenous to Mycobacterium tuberculosis but the mechanism of activation is poorly understood, in part because the binding interaction has not been properly established. The class I peroxidases ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP) have active site structures very similar to KatG and are also capable of activating isoniazid. We report here the first crystal structures of complexes of isoniazid bound to APX and CcP. These are the first structures of isoniazid bound to any activating enzymes. The structures show that isoniazid binds close to the delta-heme edge in both APX and CcP, although the precise binding orientation varies slightly in the two cases. A second binding site for INH is found in APX at the gamma-heme edge close to the established ascorbate binding site, indicating that the gamma-heme edge can also support the binding of aromatic substrates. We also show that in an active site mutant of soybean APX (W41A) INH can bind directly to the heme iron to become an inhibitor and in a different mode when the distal histidine is replaced by alanine (H42A). These structures provide the first unambiguous evidence for the location of the isoniazid binding site in the class I peroxidases and provide rationalization of isoniazid resistance in naturally occurring KatG mutant strains of M. tuberculosis.
Asunto(s)
Antituberculosos/química , Proteínas Bacterianas/química , Catalasa/química , Hemo/química , Isoniazida/química , Mycobacterium tuberculosis/enzimología , Profármacos/química , Antituberculosos/metabolismo , Ascorbato Peroxidasas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Catalasa/genética , Catalasa/metabolismo , Cristalografía por Rayos X , Citocromo-c Peroxidasa/química , Citocromo-c Peroxidasa/genética , Citocromo-c Peroxidasa/metabolismo , Farmacorresistencia Bacteriana/genética , Hemo/genética , Hemo/metabolismo , Isoniazida/metabolismo , Mutación , Mycobacterium tuberculosis/genética , Peroxidasas/química , Peroxidasas/genética , Peroxidasas/metabolismo , Profármacos/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína , Homología Estructural de Proteína , Tuberculosis/tratamiento farmacológico , Tuberculosis/enzimología , Tuberculosis/genéticaRESUMEN
We have previously shown [Badyal, S. K., et al. (2006) J. Biol. Chem. 281, 24512-24520] that the distal histidine (His42) in the W41A variant of ascorbate peroxidase binds to the heme iron in the ferric form of the protein but that binding of the substrate triggers a conformational change in which His42 dissociates from the heme. In this work, we show that this conformational rearrangement also occurs upon reduction of the heme iron. Thus, we present X-ray crystallographic data to show that reduction of the heme leads to dissociation of His42 from the iron in the ferrous form of W41A; spectroscopic and ligand binding data support this observation. Structural evidence indicates that heme reduction occurs through formation of a reduced, bis-histidine-ligated species that subsequently decays by dissociation of His42 from the heme. Collectively, the data provide clear evidence that conformational movement within the same heme active site can be controlled by both ligand binding and metal oxidation state. These observations are consistent with emerging data on other, more complex regulatory and sensing heme proteins, and the data are discussed in the context of our developing views in this area.