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.
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.
Magalon
A
A
Laboratoire de Chimie Bactérienne, IBSM-CNRS, Marseille, France.
Rothery
R A
RA
Giordano
G
G
Blasco
F
F
Weiner
J H
JH
D016227:Benzoquinones / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003067:Coenzymes; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D006873:Hydroquinones / Q000378:metabolism; D006912:Hydroxyquinolines / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D008667:Metalloproteins / Q000378:metabolism; D000090006:Molybdenum Cofactors; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D010084:Oxidation-Reduction; D011621:Pteridines / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism
D016227:Benzoquinones; D003067:Coenzymes; D006873:Hydroquinones; D006912:Hydroxyquinolines; D007501:Iron; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D009566:Nitrates; D011621:Pteridines; D013455:Sulfur; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; C025232:semiquinone radicals; C022261:molybdopterin