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The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichiu coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range + 200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are + 60 mV and + 80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4SI1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4SI1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential ironsulfur clusters of the two enzymes can be proposed.The membrane-bound complex nitrate reductase of Escherichiu coli is the terminal enzyme of the respiratory chain when the bacterium is grown anaerobically in the presence of nitrate. Previous studies have shown that it is composed of three types of subunits and that it contains three kinds of metal centers. One is a molybdenum center, which is considered to be the catalytic site; in the Mo(V) valence state, this center gives a pH-dependent EPR spectrum [l]. The variations of its amplitude as a function of the applied potential follow a bellshaped curve, which has been attributed to the interplay of Mo(VI)/Mo(V) and Mo(V)/Mo(IV) redox couples [2]. Secondly, there are 6-type hemes, which have so far been detected by EPR only as Fe-NO species [l]. Lastly, there are several iron-sulfur centers which were studied by EPR clusters by magnetic circular dichroism [3]. Two redox potentials were measured for these centers at 80 20 mV and 50 f 20 mV [2], but EPR spectra recorded at different redox states were interpreted in terms of the enzyme containing four or five iron-sulfur centers [3]. Therefore, the over-all stoichiometry and the redox Correspondence to B. Guigliarelli,

Reduction of trimethylamine N-oxide (TMAO) in Escherichia coli involves the terminal molybdoreductase TorA, located in the periplasm, and the membrane anchored c type cytochrome TorC. In this study, the role of the TorD protein, encoded by the third gene of torCAD operon, is investigated. Construction of a mutant, in which the torD gene is interrupted, showed that the absence of TorD protein leads to a two times decrease of the final amount of TorA enzyme. However, specific activity and biochemical properties of TorA enzyme were similar to those of the enzyme produced in the wild type. Excess of TorD protein restores the normal level of TorA enzyme, and also, leads to the appearance of a new cytoplasmic form of TorA on SDS-polyacrylamide gel electrophoresis using gentle conditions. This probably indicates a new folding state of the cytoplasmic TorA protein when TorD is overexpressed. BIAcore techniques demonstrated direct specific interaction between the TorA and TorD proteins. This interaction was enhanced when TorA was previously unfolded by heating. Finally, as TorA is a molybdoenzyme, we demonstrated that TorD can interact with TorA before the molybdenum cofactor has been inserted. As TorD homologue encoding genes are found in various TMAO reductase loci, we propose that TorD is a chaperone protein specific for the TorA enzyme. It belongs to a family of TorD-like chaperones present in several bacteria, and, probably, involved in TMAO reductase folding.In Escherichia coli, the main anaerobic respiratory pathway responsible for reduction of trimethylamine N-oxyde (TMAO) 1 to TMA (trimethylamine) requires the products of the torCAD operon which, in anaerobiosis, is induced in the presence of TMAO or related compounds via the two-component regulatory system, TorS/TorR (1, 2).TMAO reductase (TorA), the terminal reductase of this system, is a 97-kDa molybdoprotein encoded by the torA gene (3, 4). Based on sequence homologies, TorA belongs to the Me 2 SO/ TMAO reductase family which includes the M 2 SO/TMAO reductases from Rhodobacter capsulatus and Rhodobacter sphaeroides (5) and TMAO reductase of Shewanella species.2 These enzymes share several properties: (i) they are all molybdoenzymes located in the periplasm of the bacterium and (ii) in each case, it has been proposed that a membrane anchored pentahemic c type cytochrome feeds electrons to the terminal enzyme (6, 7). In E. coli, this cytochrome, TorC, is encoded by torC, the first gene of the torCAD operon (4,8). While the role of the TorC and TorA proteins is well documented, the role of the third protein, TorD, predicted in the torCAD operon is not (4). However, the presence of TorD homologue proteins, not only in R. capsulatus and R. sphaeroides Tor systems (6, 7), but also in Shewanella species 2 is intriguing and suggests a similar role for this protein in these systems. As TorD contains two small hydrophobic segments, at its amino and carboxyl ends, respectively, it was proposed to be a membranous b type cytochrome involved in the electron transfer path...

Tellurite and selenate reductase activities were identified in extracts of Escherichia coli. These activities were detected on non-denaturing polyacrylamide gels using an in situ methyl viologen activity-staining technique. The activity bands produced from membrane-protein extracts had the same R, values as those of nitrate reductases (NRs) A and 2. Tellurite and selenate reductase activities were absent from membranes obtained from mutants deleted in NRs A and 2. Further evidence of the tellurite and selenate reductase activities of NR was demonstrated using rocket immunoelectrophoresis analysis, where the tellurite and selenate reductase activities corresponded to the precipitation arc of NR. Additionally, hypersensitivity to potassium tellurite was observed under aerobic growth conditions in nar mutants. The tac promoter expression of NR A resulted in elevated tellurite resistance. The data obtained also imply that a minimal threshold level of NR A is required to increase resistance. Under anaerobic growth conditions additional tellurite reductase activity was identified in the soluble fraction on non-denaturing gels. Nitrate reductase mutants were not hypersensitive under anaerobic conditions, possibly due to the presence of this additional reductase activity.

In the presence of nitrate, the major anaerobic respiratory pathway includes formate dehydrogenase (FDH-N) and nitrate reductase (NAR-A), which catalyze formate oxidation coupled to nitrate reduction. Two aerobically expressed isoenzymes, FDH-Z and NAR-Z, have been recently characterized. Enzymatic analysis of plasmid subclones carrying min 88 of the Escherichia coli chromosome was consistent with the location of the fdo locus encoding FDH-Z between the fdhD and fdhE genes which are necessary for the formation of both formate dehydrogenases. The fdo locus produced three proteins (107, 34, and 22 kDa) with sizes similar to those of the subunits of the purified FDH-N. In support to their structural role, these polypeptides were recognized by antibodies specific to FDH-N. Expression of a chromosomal fdo-uidA operon fusion was induced threefold by aerobic growth and about twofold by anaerobic growth in the presence of nitrate. However, it was independent of the two global regulatory proteins FNR and ArcA, which control genes for anaerobic and aerobic functions, respectively, and of the nitrate response regulator protein NARL. In contrast, a mutation affecting either the nucleoid-associated H-NS protein or the CRP protein abolished the aerobic expression. A possible role for FDH-Z during the transition from aerobic to anaerobic conditions was examined. Synthesis of FDH-Z was maximal at the end of the aerobic growth and remained stable after a shift to anaerobiosis, whereas FDH-N production developed only under anaerobiosis. Furthermore, in an fnr strain deprived of both FDH-N and NAR-A activities, aerobically expressed FDH-Z and NAR-Z enzymes were shown to reduce nitrate at the expense of formate under anaerobic conditions, suggesting that this pathway would allow the cell to respond quickly to anaerobiosis.Under anaerobic conditions and in the presence of nitrate, Escherichia coli synthesizes the major respiratory chain which, by coupling oxidation of formate to reduction of nitrate, provides the highest yield of energy available in the absence of oxygen. It is constituted by two membrane-bound protontranslocating enzymes, formate dehydrogenase N (FDH-N) and terminal nitrate reductase (NAR-A), which are linked by a quinone (46).The two corresponding fdnGHI (6) and narGHJI (8, 45) operons located at 32 and 27 min on the E. coli genetic map, respectively, have been cloned and sequenced. They encode the three ␣, ␤, and ␥ subunits of both enzymes. The ␣ subunits consist of molybdoproteins that have been proposed to contain the catalytic site. In addition, the ␣ subunit of FDH-N harbors a selenocysteine residue essential for the enzyme (5). The ␤ subunits are electron transfer units which accommodate four iron-sulfur centers, and the ␥ subunits specify the apoproteins of the b type cytochromes. Expression of the fdnGHI and narGHJI operons is induced during anaerobic growth in the presence of nitrate. It is subject to the dual control of the transcriptional activators FNR and NARL. Anaerobic induction is mediated by the g...

Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia coli. Both isoenzymes are composed of three structural subunits, alpha, beta, and gamma encoded by narG/narZ, narH/narY and narI/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, delta, which is not part of the final enzyme. A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the delta and gamma polypeptides in the formation and/or processing of the nitrate reductase. When only the alpha and beta polypeptides are produced, an (alpha beta) complex exists which is inactive and soluble. When the alpha, beta and delta polypeptides are produced, the (alpha beta) complex is active with artificial donors such as benzyl viologen but is soluble. When the alpha, beta and gamma polypeptides are produced, the (alpha beta) complex is inactive but partially binds the membrane. It was concluded that the gamma polypeptide is involved in the binding of the (alpha beta) complex to the membrane while the delta polypeptide is indispensable for the (alpha beta) nitrate reductase activity. The activation by the delta polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (alpha beta) complex was shown to contain the four iron-sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the delta polypeptide promotes the correct assembly of the alpha and beta subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the delta or gamma polypeptide encoded by one nar operon can be substituted successfully for by its respective counterpart from the other nar operon to give an active membrane bound heterologous nitrate reductase enzyme.

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Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid-protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme b D center and is responsible for structural adjustments of b D and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.bioenergetics | EPR spectroscopy | metalloprotein | molybdenum