Nitrate reductase electron transport systems in mutant and in wild-type strains of Neurospora.

Abstract

Nitrate-inducible NADPH cytochrome c reductase (CR) activity (there is also a constitutive NADPH cytochrome c reductase) in Neurospora is stimulated by FAD, is absent from extracts of nit-2, nit-3, and nit-5, but is present in preparations of nit-1. Two components of NADPH cytochrome c reductase activity can be detected by the use of sucrose density gradient centrifugation in extracts of wild-type induced mycelia. One is a heavy component (CR I), is not associated in gradients with NADPH nitrate reductase activity, is the predominant component in extracts of uninduced wild-type, of nit-2, nit-3, and of nit-5 mycelia, is a major component of the NADPH cytochrome c reductase activity of induced wild-type preparations, and a relatively minor component of induced nit-1 preparations. It is probably the “constitutive” NADPH cytochrome c reductase. The other is a lighter NADPH cytochrome c reductase component (CR II6.8), is associated in gradients with NADPH nitrate reductase activity, is a major component of freshly prepared extracts of induced wild-type mycelia, and a minor component in preparations of uninduced wild-type, nit-2, nit-3, nit nit-5. Preparations of induced nit-1 are similar to steapsin-treated extracts of induced wild-type in that they have in addition to the omnipresent CR I component, a CR II3.8 component. The CR II3.8 component has a lower s20,w value than the CR II6.8 component of wild-type preparations. Benzyl viologen nitrate reductase activity is stimulated much less than NADPH nitrate reductase by FAD, is absent from preparations of nit-1, nit-2, and nit-5, is inducible by nitrate in the wild-type strain, and is present in extracts of nit-3. All nit mutants by definition lack NADPH nitrate reductase activity. Taken together, the above observations suggest that NADPH nitrate reductase is an aggregate of two proteins: one, under the control of nit-1, that has benzyl viologen nitrate reductase activity, and another, under the control of nit-3, that has NADPH cytochrome c reductase activity.