Cytochrome b5 reductase (Cb5R) is a pleiotropic flavoprotein that catalyzes multiple one-electron reduction reactions with various redox partners in cells. In earlier work from our laboratory, we have shown its implication in the generation of reactive oxygen species (ROS), primarily a superoxide anion overshoot peak, which plays a major role as a triggering event for the acceleration of apoptosis in cerebellar granule neurons in culture. However, the results obtained in that work did not allow us to exclude the possibility that this superoxide anion production could be derived from Cb5R acting in concert with other cellular components. In this work, we have purified Cb5R from pig liver and we have experimentally shown that this enzyme catalyzed NADH-dependent production of superoxide anion, assayed with cytochrome c and nitroblue tetrazolium as detection reagents for this particular ROS. The basic kinetic parameters for this novel NADH-dependent activity of Cb5R at 37 1C are Vmax 1⁄4 3.070.5 μmol/min/mg of purified Cb5R and KM(NADH) 1⁄4 2.870.3 μM NADH. In addition, we report that apocynin, a widely used inhibitor of nonmitochondrial ROS production in mammalian cell cultures and tissues, is a potent inhibitor of purified Cb5R activity at the concentrations used in the experiments done with cell cultures. In the presence of apocynin the KM(NADH) value of Cb5R increases, and docking simulations indicate that apocynin can bind to a site near to or partially overlapping the NADH binding site of Cb5R. Other ROS, such as nitric oxide and peroxynitrite, have inhibitory effects on purified Cb5R, providing the basis for a feedback cellular protection mechanism through modulation of excessive extramitochondrial superoxide anion production by Cb5R. Both kinetic assays and docking simulations suggest that nitric oxide-induced nitrosylation (including covalent adduction of nitroso functional groups) of Cb5R cysteines and peroxynitrite-induced tyrosine nitration and cysteine oxidation modified the conformation of the NADH binding domain leading to a decreased affinity of Cb5R for NADH.
Purified NADH-cytochrome b5 reductase is a novel superoxide anion source inhibited by apocynin: sensitivity to nitric oxide and peroxynitrite
Alejandro K. Samhan-Arias
on 12 09 2019
12 05 2022
< 1 minute
Coupling stabilizers open KV1-type potassium channels
ABSTRACT: The opening and closing of voltage-gated ion channels are regulated by voltage sensors...
12 11 2021
< 1 minute
Responding to the Challenge Posed by the Generic Control of Substances
Drug monitoring organizations report that new psychoactive substances continue to emerge, posing...
12 10 2021
< 1 minute
Boost analytical experiments with phys-chem properties
Physicochemical properties have a fundamental impact on analytical experiment conditions,...
12 09 2021
< 1 minute
Efficient acetaminophen removal from water and hospital effluents treatment by activated carbons derived from Brazil nutshells
Abstract Activated carbons from Brazil nutshells were produced by ZnCl2-activation at different...