Two-domain structure of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase.

NADH-cytochrome b₅ reductase is an amphiphilic protein consisting of a hydrophilic (FAD-containing) moiety and a hydrophobic (membrane-binding) segment and exists in aqueous media as an oligomeric aggregate. Circular dichroism studies have shown that denaturation of the reductase by guanidine hydrochloride in the presence of Emulgen 109P, a nonionic detergent, is a two-stage process as a function of the denaturant concentration. The first transition occurs at about 1 m guanidine hydrochloride and the second one at much higher concentrations. The guanidine hydrochloride concentration causing the second-stage unfolding depends on the concentration of Emulgen 109P. A hydrophilic fragment of the reductase lacking the hydrophobic segment undergoes one-stage denaturation at about 1 m guandine hydrochloride regardless of the presence and absence of Emulgen 109P. Both the reductase as well as the hydrophilic fragment lose their NADH-ferricyanide reductase activity and FAD also at about 1 m guanidine hydrochloride in the presence of the detergent. These findings suggest that the first-stage denaturation of the reductase represents the unfolding of the hydrophilic moiety and the second one that of the hydrophobic segment. Gel chromatography experiments have suggested that in the presence of Emulgen 109P the reductase exists as a mixed micelle with the detergent and this aggregation state persists even after the first-stage denaturation (unfolding of the hydrophilic moiety). The dissociation of the mixed micelle seems to take place concomitant with the second-stage denaturation. It is concluded that the two moieties of the reductase molecule, though linked to each other covalently, exist as independent domains undergoing unfolding separately at least in the presence of Emulgen 109P. This structural feature of the reductase is similar to that of cytochrome b₅ reported by us. The reductase is, therefore, another example of amphiphilic membrane proteins having two structurally independent domains in the molecule.