Removal of the high-potential [4Fe-4S] center of the beta-subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutated enzymes.

Abstract

The beta-subunit of the nitrate reductase of Escherichia coli contains four groups of Cys residues (I-IV) which are thought to bind the single [3Fe-4S] center and the three [4Fe-4S] centers. The first or second Cys residue of group I was substituted by site-directed mutagenesis with Ala or Ser. Physiological, biochemical, and EPR studies were performed on the mutated enzymes. With small variations, the properties of these mutant enzymes do not differ from one another. They were found to be as abundant and as stably bound to the membrane as the native enzyme, provided the gamma-subunit was present. Although physiological activity was reduced, it was sufficient to allow growth on nitrate. The study of variations in EPR intensity as a function of the redox potential indicated that these enzymes only contained three iron-sulfur centers instead of the usual four in the native enzyme. Spectral EPR analysis showed that the [4Fe-4S] center of high redox potential (center 1, +80 mV) was missing. The loss of this center did not affect the stable integration of the other three centers. The data presented here are in total contrast to those we have reported for each of the other three centers (centers 2-4), the loss of which was detrimental to the integration of all centers and to the integration of the molybdenum cofactor (Augier et al., in press). Taken together, our results demonstrated that the first and second Cys residues of group I are the ligands of the [4Fe-4S] center (center 1, +80 mV) and that this center participates in electron transfer, but is dispensable. On the basis of these results, it is proposed that the [3Fe-4S] center (center 2, +60 mV) also plays a biological role and that in the native enzyme both high-potential centers, centers 1 and 2, contribute independently and in parallel to the electron transfer to the molybdenum cofactor.