RESUMEN
The crystal structure of Escherichia coli nitrate reductase A (NarGHI) in complex with pentachlorophenol has been determined to 2.0 A of resolution. We have shown that pentachlorophenol is a potent inhibitor of quinol:nitrate oxidoreductase activity and that it also perturbs the EPR spectrum of one of the hemes located in the membrane anchoring subunit (NarI). This new structural information together with site-directed mutagenesis data, biochemical analyses, and molecular modeling provide the first molecular characterization of a quinol binding and oxidation site (Q-site) in NarGHI. A possible proton conduction pathway linked to electron transfer reactions has also been defined, providing fundamental atomic details of ubiquinol oxidation by NarGHI at the bacterial membrane.
Asunto(s)
Escherichia coli/enzimología , Nitrato Reductasas/química , Ubiquinona/análogos & derivados , Sitios de Unión , Membrana Celular/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/metabolismo , Hemo/química , Histidina/química , Hidroxiquinolinas/química , Cinética , Lisina/química , Modelos Químicos , Modelos Moleculares , Mutación , Naftoles/química , Nitrato-Reductasa , Oxidorreductasas/química , Oxígeno/química , Pentaclorofenol/química , Plásmidos/metabolismo , Unión Proteica , Protones , Terpenos/química , Ubiquinona/químicaRESUMEN
We have used EPR spectroscopy, redox potentiometry, and protein crystallography to characterize the [4Fe-4S] cluster (FS0) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). FS0 is clearly visible in the crystal structure of NarGHI [Bertero, M. G., et al. (2003) Nat. Struct. Biol. 10, 681-687] but has novel coordination comprising one His residue and three Cys residues. At low temperatures (<15 K), reduced NarGHI exhibits a previously unobserved EPR signal comprising peaks at g = 5.023 and g = 5.556. We have assigned these features to a [4Fe-4S](+) cluster with an S = (3)/(2) ground state, with the g = 5.023 and g = 5.556 peaks corresponding to subpopulations exhibiting DeltaS = (1)/(2) and DeltaS = (3)/(2) transitions, respectively. Both peaks exhibit midpoint potentials of approximately -55 mV at pH 8.0 and are eliminated in the EPR spectrum of apomolybdo-NarGHI. The structure of apomolybdo-NarGHI reveals that FS0 is still present but that there is significant conformational disorder in a segment of residues that includes one of the Cys ligands. On the basis of these observations, we have assigned the high-spin EPR features of reduced NarGHI to FS0.
Asunto(s)
Dominio Catalítico , Proteínas de Escherichia coli/química , Proteínas Hierro-Azufre/química , Nitrato Reductasas/química , Subunidades de Proteína/química , Secuencia de Aminoácidos , Apoproteínas/química , Coenzimas/química , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón/métodos , Nucleótidos de Guanina/química , Datos de Secuencia Molecular , Molibdeno/química , Nitrato-Reductasa , Oxidación-Reducción , Potenciometría , Pterinas/químicaRESUMEN
The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative. As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations. This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force. However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out. This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate. Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential. The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site. The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E. coli.
Asunto(s)
Proteínas de Escherichia coli/química , Nitrato Reductasas/química , Nitratos/química , Azidas/química , Sitios de Unión , Catálisis , Cloruros/química , Citoplasma/enzimología , Transporte de Electrón , Inhibidores Enzimáticos/química , Concentración de Iones de Hidrógeno , Molibdeno/química , Nitrato-Reductasa , Nitratos/antagonistas & inhibidores , Nitritos/química , Oxidación-Reducción , Potenciometría , Tiocianatos/químicaRESUMEN
The facultative anaerobe Escherichia coli is able to assemble specific respiratory chains by synthesis of appropriate dehydrogenases and reductases in response to the availability of specific substrates. Under anaerobic conditions in the presence of nitrate, E. coli synthesizes the cytoplasmic membrane-bound quinol-nitrate oxidoreductase (nitrate reductase A; NarGHI), which reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force. We present here the crystal structure of NarGHI at a resolution of 1.9 A. The NarGHI structure identifies the number, coordination scheme and environment of the redox-active prosthetic groups, a unique coordination of the molybdenum atom, the first structural evidence for the role of an open bicyclic form of the molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor in the catalytic mechanism and a novel fold of the membrane anchor subunit. Our findings provide fundamental molecular details for understanding the mechanism of proton-motive force generation by a redox loop.