Kinetic studies of a soluble up complex of nitrate reductase A from Escherichia coli Use of various ap mutants with altered p subunits

A soluble up complex of nitrate reductase can be obtained from a strain of Escheri(:h:hia r:oli that lacks the narl gene and expresses only the n and p subunits. The subunit contains four Fe-S centres and the a subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Dcspite the lack of the y subunit of the complete enzyme, this complex can still catalyse the reduction of niirate 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 molybdenuin cofactor, or both, we have studied the stcady-state kinetics and the binding of benzyl viologen to the ap complex and mutants @* with altered 11 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 supplics its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on ihe square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the (ID complex of nitrate reductase with artificial electron donors.