Kinetic studies of a soluble alpha beta complex of nitrate reductase A from Escherichia coli. Use of various alpha beta mutants with altered beta subunits.
A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits. The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre. To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits. Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished. The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.
Buc
J
J
Laboratoire de Chimie Bactérienne, Institut Fédératif Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, Marseille, France.
Santini
C L
CL
Blasco
F
F
Giordani
R
R
Cárdenas
M L
ML
Chippaux
M
M
Cornish-Bowden
A
A
Giordano
G
G
D001594:Benzyl Viologen / Q000378:metabolism; D001665:Binding Sites; D003067:Coenzymes; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D008956:Models, Chemical; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D011621:Pteridines / Q000378:metabolism; D012995:Solubility
D001594:Benzyl Viologen; D003067:Coenzymes; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D009566:Nitrates; D011621:Pteridines; C022261:molybdopterin