The trimethylamine N-oxide (TMAO) respiratory system is subject to a strict positive control by the substrate. This property was exploited in the performance of miniMu replicon-mediated in vivo cloning of the promoter region of gene(s) positively regulated by TMAO. This region, located at 22 min on the chromosome, was shown to control the expression of a transcription unit composed of three open reading frames, designated torC, torA and torD, respectively. The presence of five putative c-type haem-binding sites within the TorC sequence, as well as the specific biochemical characterization, indicated that torC encodes a 43,300 Da c-type cytochrome. The second open reading frame, torA, was identified as the structural gene for TMAO reductase. A comparison of the predicted amino-terminal sequence of the torA gene product to that of the purified TMAO reductase indicated cleavage of a 39 amino acid signal peptide, which is in agreement with the periplasmic location of the enzyme. The predicted TorA protein contains the five molybdenum cofactor-binding motifs found in other molybdoproteins and displays extensive sequence homology with BisC and DmsA proteins. As expected, insertions in torA led to the loss of TMAO reductase. The 22,500 Da polypeptides encoded by the third open reading frame does not share any similarity with proteins listed in data banks.

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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,