PMID:9260944
| Citation |
Magalon, A, Rothery, RA, Giordano, G, Blasco, F and Weiner, JH (1997) Characterization by electron paramagnetic resonance of the role of the Escherichia coli nitrate reductase (NarGHI) iron-sulfur clusters in electron transfer to nitrate and identification of a semiquinone radical intermediate. J. Bacteriol. 179:5037-45 |
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| Abstract |
We have used Escherichia coli cytoplasmic membrane preparations enriched in wild-type and mutant (NarH-C16A and NarH-C263A) nitrate reductase (NarGHI) to study the role of the [Fe-S] clusters of this enzyme in electron transfer from quinol to nitrate. The spectrum of dithionite-reduced membrane bound NarGHI has major features comprising peaks at g = 2.04 and g = 1.98, a peak-trough at g = 1.95, and a trough at g = 1.87. The oxidized spectrum of NarGHI in membranes comprises an axial [3Fe-4S] cluster spectrum with a peak at g = 2.02 (g(z)) and a peak-trough at g = 1.99 (g(xy)). We have shown that in two site-directed mutants of NarGHI which lack the highest potential [4Fe-4S] cluster (B. Guigliarelli, A. Magalon, P. Asso, P. Bertrand, C. Frixon, G. Giordano, and F. Blasco, Biochemistry 35:4828-4836, 1996), NarH-C16A and NarH-C263A, oxidation of the NarH [Fe-S] clusters is inhibited compared to the wild type. During enzyme turnover in the mutant enzymes, a distinct 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive semiquinone radical species which may be located between the hemes of NarI and the [Fe-S] clusters of NarH is observed. Overall, these studies indicate (i) the importance of the highest-potential [4Fe-4S] cluster in electron transfer from NarH to the molybdenum cofactor of NarG and (ii) that a semiquinone radical species is an important intermediate in electron transfer from quinol to nitrate. |
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| Keywords |
Benzoquinones/metabolism; Cell Membrane/enzymology; Cell Membrane/metabolism; Coenzymes; Electron Spin Resonance Spectroscopy; Electron Transport; Escherichia coli/enzymology; Escherichia coli/metabolism; Hydroquinones/metabolism; Hydroxyquinolines/pharmacology; Iron/metabolism; Metalloproteins/metabolism; Mutagenesis, Site-Directed; Nitrate Reductase; Nitrate Reductases/metabolism; Nitrates/metabolism; Oxidation-Reduction; Pteridines/metabolism; Sulfur/metabolism |
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