Biochemical Characterizations of a Soybean (Glycine Max (L.) Merr. ) Nitrate Reductase Mutant and Crude and Purified Nitrate Reductases

77 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.Wild-type soybean Glycine max (L.) Merr. cv. Williams leaves contain three forms of nitrate reductase (NR), two functional in the absence of NO(,3)('-) in the growth medium (constitutive forms; c(,1)NR, c(,2)NR) and the third active after induction by NO(,3)('-) (inducible form; iNR). Experiments were conducted to determine the site of effect of a mutation which resulted in loss of cNR activity in the soybean mutant (gene symbol nr(,1)). In addition, crude and purified preparations of soybean leaf NR forms were biochemically characterized.The nr(,1) mutant, compared with the wild type, had decreased NADH-NR, FMNH(,2)-NR, and cytochrome c reductase (diaphorase) activities, all associated with the loss of cNR activity. However, nitrite reductase and xanthine oxidase activities were similar between nr(,1) and the wild type. Since the cNR forms are structurally similar to the assimilatory NADH:NR (EC 1.6.6.1) found in other higher plants (a valid statement based on results described in the following paragraph), absence of diaphorase activity and presence of xanthine oxidase activity indicated that the apoprotein and not the molybdenum cofactor (MoCo) of the cNR forms had been affected in nr(,1).In characterizing crude extracts, cNR and iNR activities were found to be similar in exhibiting diaphorase and FMNH(,2)-NR activities and susceptibility to inhibition by p-hydroxymercuribenzoate (pHMB). Purified c(,1)NR, c(,2)NR, and iNR showed further similarities in that for each form (1) pHMB inhibited initial electron transport e.g., from NAD(P)H to cytochrome c , (2) CN('-) inhibited the reduced state of the NR enzyme more than the oxidized state, and (3) complementation was achieved with a MoCo-defective NR (e.g., a cofactor with characteristics similar to a normal MoCo was present). However, in crude extracts, cNR activity had a more acidic pH optimum and a decreased ability to distinguish between NO(,3)('-) and HCO(,3)('-), compared with iNR activity. Among the purified NR forms, dissimilarities were found in sedimentation behaviors, absolute responses to CN('-) treatment, and absolute abilities to complement a MoCo-defective NR. It was concluded that iNR was identical, in all characteristics, to EC 1.6.6.1 and that c(,1)NR and c(,2)NR, although similar in most respects, had certain characteristics different from iNR. Specifically it was determined that the terminal cofactor-containing portions of the cNR forms were altered compared with the iNR form.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD