RESUMEN
The multifunctional cytochrome P450 17A1 (CYP17A1) plays a crucial role in human steroid hormone synthesis (UniProtKBâP05093). It first carries out standard monooxygenase chemistry, converting pregnenolone (PREG) and progesterone (PROG) into 17OH-PREG and 17OH-PROG, utilizing a "Compound I" to initiate hydrogen abstraction and radical recombination in the classic "oxygen rebound" mechanism. Additionally, these hydroxylated products also serve as substrates in a second oxidative cycle which cleaves the 17-20 carbon-carbon bond to form dehydroepiandrosterone and androstenedione, which are key precursors in the generation of powerful androgens and estrogens. Interestingly, in humans, with 17OH-PREG, this so-called lyase reaction is more efficient than with 17OH-PROG, based on Kcat/Km values. In the present work, the asparagine residue at 202 position was replaced by serine, an alteration which can affect substrate orientation and control substrate preference for the lyase reaction. First, we report studies of solvent isotope effects for the N202S CYP17A1 mutant in the presence of 17OH-PREG and 17OH-PROG, which suggest that the ferric peroxo species is the predominant catalytically active intermediate in the lyase step. This conclusion is further supported by employing a combination of cryoradiolysis and resonance Raman techniques to successfully trap and structurally characterize the key reaction intermediates, including the peroxo, the hydroperoxo, and the crucial peroxo-hemiketal intermediate. Collectively, these studies show that the mutation causes active site structural changes that alter the H-bonding interactions with the key Fe-O-O fragment and the degree of protonation of the reactive ferric peroxo intermediate, thereby impacting lyase efficiency.
Asunto(s)
Asparagina , Esteroide 17-alfa-Hidroxilasa , Androstenodiona , Dominio Catalítico , Humanos , Pregnenolona/química , Progesterona/química , Esteroide 17-alfa-Hidroxilasa/químicaRESUMEN
Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.
Asunto(s)
Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/ultraestructura , Esteroides/metabolismo , Andrógenos/biosíntesis , Andrógenos/metabolismo , Androstenodiona/metabolismo , Dominio Catalítico , Deshidroepiandrosterona/metabolismo , Humanos , Enlace de Hidrógeno , Hidroxilación , Mutación , Polimorfismo de Nucleótido Simple/genética , Pregnenolona/metabolismo , Progesterona/metabolismo , Espectrometría Raman/métodos , Esteroide 17-alfa-Hidroxilasa/metabolismo , Esteroides/biosíntesis , Especificidad por Sustrato , Translocación GenéticaRESUMEN
Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O2 dissociation kinetics, identifying roles for these residues in modulating DcpG gas sensing. In addition, the resonance Raman spectra of the DcpG wild type and heme-edge mutants revealed that the mutations alter the heme electrostatic environment, vinyl group conformations, and spin state population. Using small-angle X-ray scattering and negative stain electron microscopy, the heme-edge mutations were demonstrated to cause changes to the protein conformation, which resulted in altered signaling transduction and enzyme kinetics. These findings provide insights into molecular interactions that regulate DcpG gas sensing as well as mechanisms that have evolved to control multidomain bacterial signaling proteins.
Asunto(s)
Proteínas Bacterianas/química , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/química , Hemo/química , Hemoproteínas/química , Paenibacillus/química , Hidrolasas Diéster Fosfóricas/química , Liasas de Fósforo-Oxígeno/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , GMP Cíclico/química , GMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Hemo/metabolismo , Hemoproteínas/genética , Hemoproteínas/metabolismo , Cinética , Modelos Moleculares , Oxígeno/química , Oxígeno/metabolismo , Paenibacillus/enzimología , Paenibacillus/genética , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Electricidad Estática , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome b5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b5 on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b5 (Mn-Cytb5) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn-b5, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb5. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.
Asunto(s)
Citocromos b5/química , Esteroide 17-alfa-Hidroxilasa/metabolismo , Regulación Alostérica , Biocatálisis , Dominio Catalítico , Citocromos b5/metabolismo , Humanos , Pregnenolona/química , Pregnenolona/metabolismo , Esteroide 17-alfa-Hidroxilasa/química , Especificidad por SustratoRESUMEN
Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C17 -C20 bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-Op -Ot fragment, facilitating peroxo- attack on the C20 carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction.
Asunto(s)
Liasas/genética , Liasas/metabolismo , Protones , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Humanos , Liasas/química , Pregnenolona , Progesterona , Esteroide 17-alfa-Hidroxilasa/químicaRESUMEN
A novel family of bacterial hemoproteins named NosP has been discovered recently; its members are proposed to function as nitric oxide (NO) responsive proteins involved in bacterial group behaviors such as quorum sensing and biofilm growth and dispersal. Currently, little is known about molecular activation mechanisms in NosP. Here, functional studies were performed utilizing the distinct spectroscopic characteristics associated with the NosP heme cofactor. NosPs from Pseudomonas aeruginosa ( Pa), Vibrio cholerae ( Vc), and Legionella pneumophila ( Lpg) were studied in their ferrous unligated forms as well as their ferrous CO, ferrous NO, and ferric CN adducts. The resonance Raman (rR) data collected on the ferric forms strongly support the existence of a distorted heme cofactor, which is a common feature in NO sensors. The ferrous spectra exhibit a 213 cm-1 feature, which is assigned to the Fe-Nhis stretching mode. The Fe-C and C-O frequencies in the spectra of ferrous CO NosP complexes are inversely correlated with relatively similar frequencies, consistent with a proximal histidine ligand and a relatively hydrophobic environment. The rR spectra obtained for isotopically labeled ferrous NO adducts provide evidence of formation of a 5-coordinate NO complex, resulting from proximal Fe-Nhis cleavage, which is believed to play a role in biological heme-NO signal transduction. Additionally, we found that of the three NosPs studied, Lpg NosP contains the most electropositive ligand binding pocket, while Pa NosP has the most electronegative ligand binding pocket. This pattern is also observed in the measured heme reduction potentials for these three proteins, which may indicate distinct functions for each.
Asunto(s)
Hemoproteínas/química , Hemoproteínas/metabolismo , Hierro/metabolismo , Legionella pneumophila/enzimología , Óxido Nítrico/metabolismo , Pseudomonas aeruginosa/enzimología , Vibrio cholerae/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Percepción de QuorumRESUMEN
CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon-carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different Kd and kcat values when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme's native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially decreasing the rate of formation of dehydroepiandrosterone from OHPREG. In a series of resonance Raman experiments, it was observed that, in contrast with the case for the wild-type protein, in the mutant the 17α alcohol of OHPROG tends to form a H-bond with the proximal rather than terminal oxygen of the oxy-ferrous complex. When OHPREG was a substrate, the mutant enzyme was found to have a H-bonding interaction with the proximal oxygen that is substantially weaker than that of the wild type. These results demonstrate that a single-point mutation in the active site pocket of CYP17A1, even when far from the heme, has profound effects on steroidogenic selectivity in androgen biosynthesis.
Asunto(s)
17-alfa-Hidroxipregnenolona/metabolismo , 17-alfa-Hidroxiprogesterona/metabolismo , Androstenodiona/biosíntesis , Deshidroepiandrosterona/biosíntesis , Esteroide 17-alfa-Hidroxilasa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Catálisis , Dominio Catalítico , Secuencia Conservada , Genes Sintéticos , Humanos , Enlace de Hidrógeno , Mamíferos/genética , Modelos Moleculares , Mutación Missense , Mutación Puntual , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/genética , Especificidad por SustratoRESUMEN
The multifunctional enzyme, cytochrome P450 (CYP17A1), plays a crucial role in the production of androgens, catalyzing two key reactions on pregnenolone (PREG) and progesterone (PROG), the first being a 17-hydroxylation to generate 17-OH PREG and 17-OH PROG, with roughly equal efficiencies. The second is a C-C bond scission or "lyase" reaction in which the C17-C20 bond is cleaved, leading to the eventual production of powerful androgens, whose involvement in the proliferation of prostate cancer has generated intense interest in developing inhibitors of CYP17A1. For humans, the significance of the C-C bond cleavage of 17-OH PROG is lessened, because it is about 50 times less efficient than for 17-OH PREG in terms of kcat/Km. Recognizing the need to clarify relevant reaction mechanisms involved with such transformations, we first report studies of solvent isotope effects, results of which are consistent with a Compound I mediated PROG hydroxylase activity, yet exclude this intermediate as a participant in the formation of androstenedione (AD) via the lyase reaction. This finding is also supported by a combination of cryoreduction and resonance Raman spectroscopy that traps and structurally characterizes the key hemiketal reaction intermediates. Adding to a previous study of PREG and 17-OH PREG metabolism, the current work provides definitive evidence for a more facile protonation of the initially formed ferric peroxo-intermediate for 17-OH PROG-bound CYP17A1, compared to the complex with 17-OH PREG. Importantly, Raman characterization also reveals an H-bonding interaction with the terminal oxygen of the peroxo fragment, rather than with the proximal oxygen, as is present for 17-OH PREG. These factors would favor a diminished lyase activity of the sample with 17-OH PROG relative to the complex with 17-OH PREG, thereby providing a convincing structural explanation for the dramatic differences in activity for these lyase substrates in humans.
Asunto(s)
17-alfa-Hidroxiprogesterona/química , Liasas de Carbono-Carbono/química , Enzimas Multifuncionales/química , Esteroide 17-alfa-Hidroxilasa/química , Dominio Catalítico , Humanos , Enlace de Hidrógeno , Hidroxilación , Cinética , Oxidación-Reducción , Espectrometría Raman/métodosRESUMEN
Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C17-C20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.
Asunto(s)
17-alfa-Hidroxipregnenolona/metabolismo , Andrógenos/metabolismo , Deshidroepiandrosterona/metabolismo , Pregnenolona/metabolismo , Esteroide 17-alfa-Hidroxilasa/metabolismo , 17-alfa-Hidroxipregnenolona/química , Andrógenos/química , Biocatálisis , Vías Biosintéticas , Deshidroepiandrosterona/química , Humanos , Enlace de Hidrógeno , Hidroxilación , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Pregnenolona/química , Conformación Proteica , Espectrofotometría/métodos , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/genética , Especificidad por Sustrato , TemperaturaRESUMEN
The rate-limiting step in the steroid synthesis pathway is catalyzed by CYP11A1 through three sequential reactions. The first two steps involve hydroxylations at positions 22 and 20, generating 20(R),22(R)-dihydroxycholesterol (20R,22R-DiOHCH), with the third stage leading to a C20-C22 bond cleavage, forming pregnenolone. This work provides detailed information about the active site structure of CYP11A1 in the resting state and substrate-bound ferric forms as well as the CO-ligated adducts. In addition, high-quality resonance Raman spectra are reported for the dioxygen complexes, providing new insight into the status of Fe-O-O fragments encountered during the enzymatic cycle. Results show that the three natural substrates of CYP11A1 have quite different effects on the active site structure, including variations of spin state populations, reorientations of heme peripheral groups, and, most importantly, substrate-mediated distortions of Fe-CO and Fe-O2 fragments, as revealed by telltale shifts of the observed vibrational modes. Specifically, the vibrational mode patterns observed for the Fe-O-O fragments with the first and third substrates are consistent with H-bonding interactions with the terminal oxygen, a structural feature that tends to promote O-O bond cleavage to form the Compound I intermediate. Furthermore, such spectral data are acquired for complexes with the natural redox partner, adrenodoxin (Adx), revealing protein-protein-induced active site structural perturbations. While this work shows that Adx has an only weak effect on ferric and ferrous CO states, it has a relatively stronger impact on the Fe-O-O fragments of the functionally relevant oxy complexes.
Asunto(s)
Adrenodoxina/química , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Modelos Moleculares , Adrenodoxina/metabolismo , Dominio Catalítico , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Humanos , Estructura Cuaternaria de ProteínaRESUMEN
DGCR8 is the RNA-binding partner of the nuclease Drosha. Their complex (the "Microprocessor") is essential for processing of long, primary microRNAs (pri-miRNAs) in the nucleus. Binding of heme to DGCR8 is essential for pri-miRNA processing. On the basis of the split Soret ultraviolet-visible (UV-vis) spectrum of ferric DGCR8, bis-thiolate sulfur (cysteinate, Cys(-)) heme iron coordination of DGCR8 heme iron was proposed. We have characterized DGCR8 heme ligation using the Δ276 DGCR8 variant and combined electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), electron nuclear double resonance, resonance Raman, and electronic absorption spectroscopy. These studies indicate DGCR8 bis-Cys heme iron ligation, with conversion from bis-thiolate (Cys(-)/Cys(-)) axial coordination in ferric DGCR8 to bis-thiol (CysH/CysH) coordination in ferrous DGCR8. Pri-miRNA binding does not perturb ferric DGCR8's optical spectrum, consistent with the axial ligand environment being separated from the substrate-binding site. UV-vis absorption spectra of the Fe(II) and Fe(II)-CO forms indicate discrete species exhibiting peaks with absorption coefficients substantially larger than those for ferric DGCR8 and that previously reported for a ferrous form of DGCR8. Electron-nuclear double resonance spectroscopy data exclude histidine or water as axial ligands for ferric DGCR8 and favor bis-thiolate coordination in this form. UV-vis MCD and near-infrared MCD provide data consistent with this conclusion. UV-vis MCD data for ferrous DGCR8 reveal features consistent with bis-thiol heme iron coordination, and resonance Raman data for the ferrous-CO form are consistent with a thiol ligand trans to the CO. These studies support retention of DGCR8 cysteine coordination upon reduction, a conclusion distinct from those of previous studies of a different ferrous DGCR8 isoform.
Asunto(s)
Hemo/química , Hierro/química , Proteínas de Unión al ARN/química , Clonación Molecular , Humanos , Proteínas de Unión al ARN/genética , Análisis Espectral/métodosRESUMEN
Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second CC lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced â¼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal FeS vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b5 has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis.
Asunto(s)
Andrógenos/síntesis química , Citocromos b5/química , Esteroide 17-alfa-Hidroxilasa/química , Sitios de Unión , Activación Enzimática , Oxidación-Reducción , Unión ProteicaRESUMEN
The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this "stored" proton to the hydroperoxo fragment, it rapidly undergoes O-O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe-O-O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O-O bond-cleavage product, whose ν(FeâO) frequencies reflect substantially different FeâO bond strengths. In the process of conducting these studies, rR structural characterization of the dioxygen adduct of LPO, commonly called Compound III, has also been completed, demonstrating a substantial difference in the strengths of the Fe-O linkage of the Fe-O-O fragment under acidic and alkaline conditions, an effect most reasonably attributed to a corresponding weakening of the trans-axial histidyl imidazole linkage at lower pH. Collectively, these new results provide important insight into the impact of pH on the disposition of the key Fe-O-O and FeâO fragments of intermediates that arise in the enzymatic cycles of LPO, other mammalian peroxidases, and related proteins.
Asunto(s)
Dominio Catalítico , Hemo/química , Peróxido de Hidrógeno/química , Lactoperoxidasa/química , Oxígeno/química , Espectrometría Raman , Hemo/metabolismo , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Lactoperoxidasa/metabolismo , Estructura Molecular , Oxígeno/metabolismoRESUMEN
An important function of steroidogenic cytochromes P450 is the transformation of cholesterol to produce androgens, estrogens, and the corticosteroids. The activities of cytochrome P450c17 (CYP17) are essential in sex hormone biosynthesis, with severe developmental defects being a consequence of deficiency or mutations. The first reaction catalyzed by this multifunctional P450 is the 17α-hydroxylation of pregnenolone (PREG) to 17α-hydroxypregnenolone (17-OH PREG) and progesterone (PROG) to 17α-hydroxyprogesterone (17-OH PROG). The hydroxylated products then either are used for production of corticoids or undergo a second CYP17 catalyzed transformation, representing the first committed step of androgen formation. While the hydroxylation reactions are catalyzed by the well-known Compound I intermediate, the lyase reaction is believed to involve nucleophilic attack of the earlier peroxo- intermediate on the C20-carbonyl. Herein, resonance Raman (rR) spectroscopy reveals that substrate structure does not impact heme structure for this set of physiologically important substrates. On the other hand, rR spectra obtained here for the ferrous CO adducts with these four substrates show that substrates do interact differently with the Fe-C-O fragment, with large differences between the spectra obtained for the samples containing 17-OH PROG and 17-OH PREG, the latter providing evidence for the presence of two Fe-C-O conformers. Collectively, these results demonstrate that individual substrates can differentially impact the disposition of a heme-bound ligand, including dioxygen, altering the reactivity patterns in such a way as to promote preferred chemical conversions, thereby avoiding the profound functional consequences of unwanted side reactions.
Asunto(s)
Pregnenolona/metabolismo , Progesterona/metabolismo , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/metabolismo , Dominio Catalítico , Hemo/metabolismo , Humanos , Hidroxilación , Ligandos , Espectrometría Raman , Especificidad por SustratoRESUMEN
CYP19A1, or aromatase, a cytochrome P450 responsible for estrogen biosynthesis in humans, is an important therapeutic target for the treatment of breast cancer. There is still controversy surrounding the identity of reaction intermediate that catalyzes carbon-carbon scission in this key enzyme. Probing the oxy-complexes of CYP19A1 poised for hydroxylase and lyase chemistries using resonance Raman spectroscopy and drawing a comparison with CYP17A1, we have found no significant difference in the frequencies or isotopic shifts for these two steps in CYP19A1. Our experiments implicate the involvement of Compound I in the terminal lyase step of CYP19A1 catalysis.
Asunto(s)
Androstenodiona/metabolismo , Aromatasa/metabolismo , Liasas/metabolismo , Espectrometría Raman , Androstenodiona/química , Humanos , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismoRESUMEN
The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or ß hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe-histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or ß chains, prior to the quaternary R-T and T-R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe-His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem. 2002, 98, 149). Experimentally, the νFeHis evolution is faster for ß than for α chains, and pump-probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 µs for ß but not for α hemes, an interval previously shown to be the first step in the R-T transition. In the α chain νFeHis dropped sharply at 20 µs, the final step in the R-T transition. The time courses are fully consistent with recent computational mapping of the R-T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.
Asunto(s)
Biología Computacional/métodos , Hemo/química , Hemoglobina A/química , Espectrometría Raman/métodos , Globinas alfa/química , Globinas beta/química , Regulación Alostérica , Mesoporfirinas/química , Modelos Moleculares , Estructura Cuaternaria de ProteínaRESUMEN
When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Esteroide 17-alfa-Hidroxilasa , Esteroide 17-alfa-Hidroxilasa/metabolismo , Deshidroepiandrosterona/química , Deshidroepiandrosterona/metabolismo , 17-alfa-Hidroxiprogesterona/química , 17-alfa-Hidroxiprogesterona/metabolismo , 17-alfa-Hidroxipregnenolona/química , 17-alfa-Hidroxipregnenolona/metabolismo , Androstenodiona/química , Androstenodiona/metabolismo , Humanos , Liasas/metabolismo , Liasas/química , Rayos gamma , Especificidad por Sustrato , Oxígeno/químicaRESUMEN
Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental signals. Changes in oxygen levels can alter c-di-GMP signaling through a family of proteins termed globin coupled sensors (GCS) that contain diguanylate cyclase domains. Previous studies have found that GCS diguanylate cyclase activity is controlled by ligand binding to the heme within the globin domain, with oxygen binding resulting in the greatest increase in catalytic activity. Herein, we present evidence that heme-edge residues control O2-dependent signaling in PccGCS, a GCS protein from Pectobacterium carotovorum, by modulating heme distortion. Using enzyme kinetics, resonance Raman spectroscopy, small angle X-ray scattering, and multi-wavelength analytical ultracentrifugation, we have developed an integrated model of the full-length PccGCS tetramer and have identified conformational changes associated with ligand binding, heme conformation, and cyclase activity. Taken together, these studies provide new insights into the mechanism by which O2 binding modulates activity of diguanylate cyclase-containing GCS proteins.
Asunto(s)
Proteínas Bacterianas , Hemo , Pectobacterium carotovorum , Liasas de Fósforo-Oxígeno , Liasas de Fósforo-Oxígeno/metabolismo , Liasas de Fósforo-Oxígeno/química , Hemo/química , Hemo/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Pectobacterium carotovorum/enzimología , Conformación Proteica , Oxígeno/química , Oxígeno/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Proteínas de Escherichia coliRESUMEN
The human sterol 14α-demethylases (CYP51, CYP is an abbreviation for cytochrome P450) catalyze three-step oxidative removal of 14α-methyl group of lanosterol by first forming an alcohol, then an aldehyde, and finally conducting a CC bond cleavage reaction. This present study utilizes a combination of Resonance Raman spectroscopy and Nanodisc technology to probe the active site structure of CYP51 in the presence of its hydroxylase and lyase substrates. Ligand-binding induced partial low-to-high-spin conversion is observed by applying electronic absorption spectroscopy and Resonance Raman (RR) spectroscopy. This low degree of spin conversion of CYP51 is contributed by the retention of the water ligand coordinated to the heme iron as well as direct interaction between the hydroxyl group of lyase substrate and the iron center. No significant changes in active site structure are found between detergent-stabilized CYP51 and nanodisc-incorporated CYP51, nevertheless, it is demonstrated that nanodisc-incorporated assemblies provide much more well-defined active site RR spectroscopic responses, which induces a larger conversion from low-to-high-spin state in presence of the substrates. Moreover, a positive polar environment around the exogenous diatomic ligand is detected, providing insight into the mechanism of this essential CC bond cleavage reaction.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Espectrometría Raman , Humanos , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/metabolismo , Hierro , Ligandos , Oxidación-ReducciónRESUMEN
The Cytochrome P450 (CYP450) superfamily has been the subject of intense research for over six decades. Here the HU227 strain of E. coli, lacking the δ-aminolevulinic acid (δ-ALA) synthase gene, was employed, along with [5-13C] δ-ALA, in the heterologous expression of P450cam harboring a prosthetic group labeled with 13C at the four methine carbons (Cm) and pyrrole Cα positions. The product was utilized as a proof of principle strategy for defining and refining solution phase active site structure in cytochrome P450cam, providing proton-to-proton distances from 13CmH to protons on bound substrate or nearby amino acid residues, using short mixing time 2D or 3D NOESY-HMQC methods. The results reveal the interesting finding that 2D 13C-filtered NOESY-HMQC can be used to obtain distances between protons on labeled 13C to positions of protons nearby in the active site, confirming the utility of this NMR-based approach to probing active site structure under physiological conditions. Such 13C-heme-filtered NOE data complement X-ray crystallographic and T1-based NMR measurements; and, may also be of potentially significant utility in furnishing experimental distance constraints in validations of docking routines commonly employed for determining the relative affinities and binding orientations of drug candidates with CYP450s.