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1.
J Biol Chem ; 281(4): 2170-6, 2006 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-16286471

RESUMEN

Understanding when and how metal cofactor insertion occurs into a multisubunit metalloenzyme is of fundamental importance. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJ-assisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. Here, we have shown that the NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interfering with its membrane anchoring and another one being involved in molybdenum cofactor insertion. The presence of the two NarJ-binding sites within NarG is required to ensure productive formation of active nitrate reductase. Our findings supported the view that enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Chaperonas Moleculares/química , Molibdeno/química , Nitrato-Reductasa/química , Nitratos/química , Sitios de Unión , Técnicas Biosensibles , Escherichia coli/metabolismo , Eliminación de Gen , Modelos Biológicos , Modelos Moleculares , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Fracciones Subcelulares , Thermus/enzimología , Técnicas del Sistema de Dos Híbridos
2.
J Bacteriol ; 187(12): 3990-6, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15937161

RESUMEN

The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.


Asunto(s)
Nitrato Reductasas/metabolismo , Thermus thermophilus/enzimología , Proteínas Bacterianas/fisiología , Membrana Celular/enzimología , Citocromos b/fisiología , Citocromos c/fisiología , Activación Enzimática , Expresión Génica , Mutagénesis Insercional , Nitrato-Reductasa , Operón
3.
Biochemistry ; 44(4): 1300-8, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15667223

RESUMEN

Nitrate reductase A (NRA, NarGHI) is expressed in Escherichia coli by growing the bacterium in anaerobic conditions in the presence of nitrate. This enzyme reduces nitrate to nitrite and uses menaquinol (or ubiquinol) as the electron donor. The location of quinones in the enzyme, their number, and their role in the electron transfer mechanism are still controversial. In this work, we have investigated the spectroscopic and thermodynamic properties of a semiquinone (SQ) in membrane samples of overexpressed E. coli nitrate reductase poised in appropriate redox conditions. This semiquinone is highly stabilized with respect to free semiquinone. The g-values determined from the numerical simulation of its Q-band (35 GHz) EPR spectrum are equal to 2.0061, 2.0051, 2.0023. The midpoint potential of the Q/QH(2) couple is about -100 mV, and the SQ stability constant is about 100 at pH 7.5. The semiquinone EPR signal disappears completely upon addition of the quinol binding site inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). A semiquinone radical could also be stabilized in preparations where only the NarI membrane subunit is overexpressed in the absence of the NarGH catalytic dimer. Its thermodynamic and spectroscopic properties show only slight variations with those of the wild-type enzyme. The X-band continuous wave (cw) electron nuclear double resonance (ENDOR) spectra of the radicals display similar proton hyperfine coupling patterns in NarGHI and in NarI, showing that they arise from the same semiquinone species bound to a single site located in the NarI membrane subunit. These results are discussed with regard to the location and the potential function of quinones in the enzyme.


Asunto(s)
Benzoquinonas/química , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Escherichia coli/química , Nitrato Reductasas/química , Subunidades de Proteína/química , Benzoquinonas/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Estabilidad de Enzimas , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Hidroxiquinolinas/química , Nitrato-Reductasa , Nitrato Reductasas/biosíntesis , Nitrato Reductasas/genética , Oxidación-Reducción , Unión Proteica/genética , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Termodinámica
4.
J Biol Chem ; 280(15): 14836-43, 2005 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-15615728

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/química
5.
J Biol Chem ; 279(44): 45369-78, 2004 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-15292214

RESUMEN

A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.


Asunto(s)
NADH Deshidrogenasa/fisiología , Nitratos/metabolismo , Thermus thermophilus/enzimología , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , Conjugación Genética , Transporte de Electrón , Datos de Secuencia Molecular , NADH Deshidrogenasa/química , NADH Deshidrogenasa/genética , Operón , Oxidación-Reducción , Transcripción Genética
6.
J Biol Chem ; 279(40): 41398-403, 2004 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15247236

RESUMEN

The maturation of Escherichia coli nitrate reductase A requires the incorporation of the Mo-(bis-MGD) cofactor to the apoprotein. For this process, the NarJ chaperone is strictly required. We report the first description of protein interactions between molybdenum cofactor biosynthetic proteins (MogA, MoeA, MobA, and MobB) and the aponitrate reductase (NarG) using a bacterial two-hybrid approach. Two conditions have to be satisfied to allow the visualization of the interactions, (i) the presence of an active and mature molybdenum cofactor and (ii) the presence of the NarJ chaperone and of the NarG structural partner subunit, NarH. Formation of tungsten-substituted cofactor prevents the interaction between NarG and the four biosynthetic proteins. Our results suggested that the final stages of molybdenum cofactor biosynthesis occur on a complex made up by MogA, MoeA, MobA, and MobB, which is also in charge with the delivery of the mature cofactor onto the aponitrate reductase A in a NarJ-assisted process.


Asunto(s)
Proteínas de Escherichia coli/biosíntesis , Molibdeno , Nitrato Reductasas/biosíntesis , Apoproteínas/metabolismo , Proteínas de Escherichia coli/metabolismo , Sustancias Macromoleculares , Chaperonas Moleculares/metabolismo , Nitrato-Reductasa , Subunidades de Proteína/metabolismo , Sulfurtransferasas/metabolismo , Transactivadores/metabolismo , Técnicas del Sistema de Dos Híbridos
7.
Biochemistry ; 43(18): 5324-33, 2004 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-15122898

RESUMEN

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/química
8.
Nat Struct Biol ; 10(9): 681-7, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12910261

RESUMEN

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.


Asunto(s)
Transporte de Electrón , Electrones , Nitrato Reductasas/química , Catálisis , Membrana Celular/metabolismo , Cristalografía por Rayos X , Citoplasma/enzimología , Escherichia coli/enzimología , Modelos Biológicos , Modelos Moleculares , Nitrato-Reductasa , Oxidación-Reducción , Estructura Terciaria de Proteína , Protones
9.
J Biol Chem ; 277(50): 48199-204, 2002 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-12372836

RESUMEN

The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin. Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli. Numerous interactions among these proteins have been identified. Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA. Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins.


Asunto(s)
Coenzimas , Escherichia coli/metabolismo , Metaloproteínas/biosíntesis , Escherichia coli/genética , Genes Bacterianos , Metaloproteínas/genética , Sondas Moleculares , Cofactores de Molibdeno , Mutación , Pteridinas
10.
Biochim Biophys Acta ; 1555(1-3): 54-9, 2002 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-12206891

RESUMEN

It is no surprise that the catalytic activity of electron-transport enzymes may be optimised at certain electrochemical potentials in ways that are analogous to observations of pH-rate optima. This property is observed clearly in experiments in which an enzyme is adsorbed on an electrode surface which can supply or receive electrons rapidly and in a highly controlled manner. In such a way, the rate of catalysis can be measured accurately as a function of the potential (driving force) that is applied. In this paper, we draw attention to a few examples in which this property has been observed in enzymes that are associated with membrane-bound respiratory chains, and we discuss its possible origins and implications for in vivo regulation.


Asunto(s)
Proteínas Hierro-Azufre , Oxidorreductasas/química , Animales , Catálisis , Bovinos , Electroquímica , Transporte de Electrón , Escherichia coli , Concentración de Iones de Hidrógeno , Mitocondrias/enzimología , Nitrato-Reductasa , Nitrato Reductasas , Oxidación-Reducción , Succinato Deshidrogenasa/química
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