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

Abstract

The behaviour of cytochrome hs reductase holoenzyme and apoenzyme toward blue-dextranSepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 pM NADH or NAD'. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 pM NADH + 10 pM 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 hs 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 hs reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome h2, an FMN-dependent enzyme [D. Pompon and F. Lederer (1 978) Eur, J . Biochem. 90, 563-5691,

On the Transhydrogenase Activity of Baker's Yeast Flavocytochrome b2

“…Furthermore, the measured k,,, for the reverse reaction found in this work is 3.6 s -I ; this low rate cannot be due to slow bromolactate dissociation, for the following reason. The kinetic parameters K , and k,,, determined for bromolactate are not very different from those of lactate (see below and Table2); we can then reasonably assume that the two hydroxy acids will bind and dissociate at rates of a similar order of magnitude; in particular, the value of k,,, for lactate was calculated to lie within 50-100 s-' [23]. This reasoning indicated that under our experi- mental conditions oxidized flavin is reduced much more rapidly than it is produced.…”

“…It has been shown that in the tetramer there are privileged heme-flavin pairs, between which electron transfer is rapid [22, Electron transfer between any member of one pair to any member of another pair is supposed to be slow, with rates lower than 10 s-' [23]. The rate constant for lactate to flavin transfer, which is the slow step in the forward reaction, was estimated by simulation studies to be 120 s-' at 24 "C [20,22].…”

“…This piece of evidence indicates that, in the steady state, heme plays no significant role and supports our contention that oxidized flavin is reduced more rapidly than it is produced. Secondly, simulation studies were carried out according to the procedure described by Pompon, using his kinetic scheme for the normal reaction as a basis [23]. These studies are described in [24].…”

Reconstitution of Liver NADH: Cytochrome b5 Oxidoreductase and of Desulfovibrio vulgaris Flavodoxin with 1‐Carba‐1‐deazaflavin

“…In order to remove contaminating hemoglobin, an additional step was added to the purification, namely chromatography on blue-dextran-Sepharose [15]. For flavin removal, 15 pM reductase in 10 mM Tris/HCl buffer, 1 mM EDTA, 0.1 dithiothreitol, pH 8.5, was brought to pH 2.3 by rapid mixing with 0.8 M HCl at 0°C. The solution was stirred for a few seconds with HCI-washed charcoal and finally centrifuged for a few minutes.…”

“…Before use it was equilibrated against the required buffer by dialysis or filtration on Sephadex G-25. NADH cytochrome b5 reductase was solubilized by proteolysis from beef liver microsomes according to Takesue and Omura [14]. In order to remove contaminating hemoglobin, an additional step was added to the purification, namely chromatography on blue-dextran-Sepharose [15]. For flavin removal, 15 pM reductase in 10 mM Tris/HCl buffer, 1 mM EDTA, 0.1 dithiothreitol, pH 8.5, was brought to pH 2.3 by rapid mixing with 0.8 M HCl at 0°C.…”