Ubiquinone is a hysteretic modulator of the NADH:cytochrome b 5 reductase activity of human C b 5 R

ABSTRACT Background Cytochrome b 5 reductase is a flavoprotein that transfers electrons from NADH to multiple electron acceptors, such as cytochrome b 5 or ubiquinone. Hysteresis is a phenomenon characterized by a slow transition between active and inactive catalytic states, leading to a lag phase in enzymatic activity. In this study, the effect of the soluble analogue of ubiquinone named 2,3 dimethoxy-5-methyl-1,4 benzoquinone (CoQ 0 ) on the NADH:C b 5 reductase activity of recombinant human soluble C b 5 R, using recombinant human soluble C b 5 as a substrate was evaluated. The aim of this study was to determine whether ubiquinone exerts a hysteretic modulation of this activity based on previous studies supporting that microsomal reduction of cytochrome b 5 is controlled by redox hysteresis. Results The NADH:cytochrome b 5 reductase activity of C b 5 R was characterized at different concentrations of C b 5 R, cytochrome b 5 , and CoQ 0 by monitoring the reduction of cytochrome b 5 . The addition of CoQ 0 induced the appearance of a lag phase, whose duration increased with the concentration of CoQ 0 and decreased with higher concentrations of cytochrome b 5 or C b 5 R. Additionally, a concentration-dependent decrease in the maximum rate of reduction and the appearance of positive cooperativity was observed in the presence of CoQ 0 which resulted in leading to lower K M values for cytochrome b 5 . This suggests the formation of a CoQ 0 :C b 5 R complex altering the interaction between the reductase and cytochrome b 5 which increases the affinity for cytochrome b 5 . Cyclic voltammetry data support the formation of CoQ 0 /protein complex that could be responsible for the hysteretic behavior. Conclusions These results support the hypothesis that CoQ 0 is a hysteretic modulator and inhibitor of the NADH: cytochrome b 5 reductase activity of human C b 5 R.