The role of tryptophan in the reaction catalyzed by spinach ferredoxin-dependent nitrite reductase

The role of tryptophan in the ferredoxin-dependent nitrite reductase of spinach

“…Earlier results of chemical modification studies carried out in our laboratory suggested that at least one conserved tryptophan residue in spinach nitrite reductase, Trp92, played an important role in the reduction of nitrite to ammonia catalyzed by this enzyme (Hirasawa et al 1994b). However, the observations, reported above, that the inhibition caused by replacing each of the eight nitrite reductase tryptophans, one-at-a-time, by either non-aromatic or non-aromatic amino acids appears to be completely nonspecific suggests that tryptophan residues do not play any specific role in the electron transfer reaction catalyzed by this enzyme.…”

“…Assays of nitrite reductase activity with either reduced ferredoxin or with reduced methyl viologen serving as the electron donor were carried out as described previously (Hirasawa et al 1993). Chemical modification of nitrite reductase tryptophan residues by treatment with N-bromosuccinimide (NBS) was carried out as described previously (Hirasawa et al 1994b). Dissociation constants (K d ) for the binding of nitrite (Hirasawa et al 1987) and the binding of ferredoxin (Hirasawa and Knaff 1985) to nitrite reductase were calculated from spectral perturbation measurements, carried out as described previously.…”

“…These observations were interpreted as indicating that at least one of the enzyme's eight tryptophan residues played an important role in the reduction of nitrite catalyzed by the enzyme (Hirasawa et al 1994b). The observation that formation of the 1:1 complex between ferredoxin and the enzyme completely protected the enzyme against inhibition during subsequent treatment with NBS (Hirasawa et al 1994b) was exploited in a differential peptide-mapping study that identified the conserved Trp92 as the most likely site for NBS inhibition of the enzyme (Hirasawa et al 1998).…”

“…Chemical modification studies of spinach nitrite reductase, using the tryptophan-specific modifier N-bromosuccinimide (NBS), indicated that treatment of the enzyme with this reagent produced substantial inhibition of nitrite reduction regardless of, whether, reduced ferredoxin or reduced methyl viologen was used as the electron donor (Hirasawa et al 1994b). These observations were interpreted as indicating that at least one of the enzyme's eight tryptophan residues played an important role in the reduction of nitrite catalyzed by the enzyme (Hirasawa et al 1994b).…”