REMOVAL OF THE HIGH-POTENTIAL [4FE-4S] CENTER OF THE BETA-SUBUNIT FROM ESCHERICHIA-COLI NITRATE REDUCTASE - PHYSIOLOGICAL, BIOCHEMICAL, ANDEPR CHARACTERIZATION OF SITE-DIRECTED MUTATED ENZYMES
V. Augier et al., REMOVAL OF THE HIGH-POTENTIAL [4FE-4S] CENTER OF THE BETA-SUBUNIT FROM ESCHERICHIA-COLI NITRATE REDUCTASE - PHYSIOLOGICAL, BIOCHEMICAL, ANDEPR CHARACTERIZATION OF SITE-DIRECTED MUTATED ENZYMES, Biochemistry, 32(19), 1993, pp. 5099-5108
The beta-subunit of the nitrate reductase of Escherichia coli contains
four groups of Cys residues (I-IV) which are thought to bind the sing
le [3Fe-4S] center and the three [4Fe-4S] centers. The first or second
Cys residue of group I was substituted by site-directed mutagenesis w
ith Ala or Ser. Physiological, biochemical, and EPR studies were perfo
rmed on the mutated enzymes. With small variations, the properties of
these mutant enzymes do not differ from one another. They were found t
o be as abundant and as stably bound to the membrane as the native enz
yme, provided the gamma-subunit was present. Although physiological ac
tivity was reduced, it was sufficient to allow growth on nitrate. The
study of variations in EPR intensity as a function of the redox potent
ial indicated that these enzymes only contained three iron-sulfur cent
ers instead of the usual four in the native enzyme. Spectral EPR analy
sis 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 stabl
e integration of the other three centers. The data presented here are
in total contrast to those we have reported for each of the other thre
e centers (centers 2-4), the loss of which was detrimental to the inte
gration of all centers and to the integration of the molybdenum cofact
or (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 participat
es in electron transfer, but is dispensable. On the basis of these res
ults, 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-potent
ial centers, centers 1 and 2, contribute independently and in parallel
to the electron transfer to the molybdenum cofactor.