Complete coordination of the four Fe-S centers of the beta subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential [4Fe-4S] clusters.
The beta subunit of the nitrate reductase A from Escherichia coli contains four groups of cysteine residues (I-IV) which are thought to bind the four iron-sulfur centers (1-4) of the enzyme. The fourth Cys residue of each group was replaced by Ala by site-directed mutagenesis, which led to the C26A, C196A, C227A, and C263A mutants. Physiological and biochemical effects of the mutations were investigated on both the membrane-bound and the soluble forms of the enzyme. In addition, detailed redox titrations of the mutants were monitored by EPR spectroscopy. The C196A and C227A mutations resulted in the full loss of the four Fe-S clusters and of the Mo-cofactor, leading to inactive enzymes. In contrast, the C26A and C263A mutants retained significant nitrate reductase activities. The EPR analysis showed that the highest redox potential [4Fe-4S] cluster (center 1) was selectively removed by the C263A mutation and that the C26A replacement likely eliminated the lowest potential [4Fe-4S] cluster (center 4). In both mutants, the three remaining Fe-S clusters kept the same spectral and redox properties as in the wild type enzyme. These results enabled the determination of the Cys ligands of center 1 to be completed and led to a proposed model for the coordination of the four Fe-S centers by the four Cys groups of the beta subunit. In this model, the four clusters are organized in two pairs, (center 1, center 4) and (center 2, center 3), which is in good agreement with the magnitude of intercenter magnetic interactions observed by EPR and with the stability of the different mutants. The possible implications on the intramolecular electron transfer pathway are discussed.
Guigliarelli
B
B
Unité de Bioénergétique et Ingénierie des Protéines, UPR 9036, Marseille, France.
Magalon
A
A
Asso
M
M
Bertrand
P
P
Frixon
C
C
Giordano
G
G
Blasco
F
F
D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007501:Iron / Q000737:chemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D013455:Sulfur / Q000737:chemistry
D007527:Isoenzymes; D013455:Sulfur; D007501:Iron; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D003545:Cysteine