Binding of Cibacron Blue F3GA to the Flavin and NADH Sites in Cytochrome b5 Reductase

Open Access
Publisher Website
Google Scholar
Abstract
The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran–Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 μM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 μM NADH + 10 μM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitudes at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran-Sepharose: the NADH binding site and the flavin binding site. For the latter it is clear that the isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, an FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur, J. Biochem. 90, 563–569].