RESUMEN
Cytochromes P450 (P450s) metabolize a wide range of endogenous compounds and xenobiotics, such as pollutants, environmental compounds, and drug molecules. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. Cytochrome P450 3A4 (CYP3A4) metabolizes more drug molecules than all other isoforms combined. Here we report three crystal structures of CYP3A4: unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. The structures revealed a surprisingly small active site, with little conformational change associated with the binding of either compound. An unexpected peripheral binding site is identified, located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Metirapona/metabolismo , Progesterona/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Citocromo P-450 CYP3A , Hemo/química , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Agua/metabolismoRESUMEN
Escherichia coli cytochrome c nitrite reductase is a homodimeric enzyme whose 10 heme centres range in reduction potential from ca. -30 to -320 mV. Protein film voltammetry (PFV) was performed to assess how the reactivity of the enzyme towards a number of small molecules was influenced by heme oxidation state. The experimental approach provided a high-resolution description of activity across the electrochemical potential domain by virtue of the fact that the enzyme sample was under the precise potential control of an electrode at all times. The current potential profiles displayed by nitrite reductase revealed that heme oxidation state has a profound, and often unanticipated, effect on the interactions with substrate molecules, nitrite and hydroxylamine, as well as the inhibitor, cyanide. Thus, PFV provides a powerful route to define redox-triggered events in this complex multi-centred redox enzyme.
Asunto(s)
Cianuros/química , Citocromos a1/análisis , Citocromos a1/química , Citocromos c1/análisis , Citocromos c1/química , Electroquímica/métodos , Hemo/química , Hidroxilamina/química , Nitrato Reductasas/análisis , Nitrato Reductasas/química , Nitritos/química , Materiales Biocompatibles Revestidos/análisis , Materiales Biocompatibles Revestidos/química , Citocromos a1/antagonistas & inhibidores , Citocromos c1/antagonistas & inhibidores , Activación Enzimática , Inhibidores Enzimáticos/química , Estabilidad de Enzimas , Enzimas Inmovilizadas/análisis , Enzimas Inmovilizadas/antagonistas & inhibidores , Enzimas Inmovilizadas/química , Escherichia coli/enzimología , Nitrato Reductasas/antagonistas & inhibidores , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug-drug interactions.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Warfarina/metabolismo , Anticoagulantes/química , Anticoagulantes/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Sitios de Unión , Cristalografía por Rayos X , Citocromo P-450 CYP2C9 , Humanos , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína , Warfarina/químicaRESUMEN
The cytochrome c nitrite reductases perform a key step in the biological nitrogen cycle by catalyzing the six-electron reduction of nitrite to ammonium. Graphite electrodes painted with Escherichia coli cytochrome c nitrite reductase and placed in solutions containing nitrite (pH 7) exhibit large catalytic reduction currents during cyclic voltammetry at potentials below 0 V. These catalytic currents were not observed in the absence of cytochrome c nitrite reductase and were shown to originate from an enzyme film engaged in direct electron exchange with the electrode. The catalytic current-potential profiles observed on progression from substrate-limited to enzyme-limited nitrite reduction revealed a fingerprint of catalytic behavior distinct from that observed during hydroxylamine reduction, the latter being an alternative substrate for the enzyme that is reduced to ammonium in a two electron process. Cytochrome c nitrite reductase clearly interacts differently with these two substrates. However, similar features underlie the development of the voltammetric response with increasing nitrite or hydroxylamine concentration. These features are consistent with coordinated two-electron reduction of the active site and suggest that the mechanisms for reduction of both substrates are underpinned by common rate-defining processes.
Asunto(s)
Citocromos a1 , Citocromos c1 , Hidroxilamina/metabolismo , Nitrato Reductasas/metabolismo , Nitritos/metabolismo , Sitios de Unión , Catálisis , Escherichia coli/enzimología , Cinética , Oxidación-ReducciónRESUMEN
The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner. The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand. Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water. Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples. Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV. A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3). A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2. The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8. Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV. Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E. coli NrfA.