One-electron reduction of hepatic NADH-cytochrome b5 reductase as studied by pulse radiolysis.
The reduction of flavin in hepatic NADH-cytochrome b5 reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced the flavin of NADH-cytochrome b5 reductase to form the red semiquinone between pH 5 and 9. The spectrum of the red semiquinone differs from that of enzyme reduced by dithionite in the presence of NAD+. After the first phase of the reduction, conversion of the red to blue semiquinone was observed at acidic pH. Resulting products are the blue (neutral) or red (anionic) semiquinone or a mixture of the two forms. The pK value for this flavin radical was approximately 6.3. Subsequently, the semiquinone form reacted by dismutation to form the oxidized and the fully reduced forms of the enzyme with a rate constant of 1 x 10(3) M-1 s-1 at pH 7.1. In the presence of NAD+, eaq- reacted with NAD+ to yield NAD(.). Subsequently, NAD. transferred an electron to NAD+-bound oxidized enzyme to form the blue and red semiquinone or mixture of the two forms of the enzyme, where pK value of this flavin radical was approximately 6.3. The blue semiquinone obtained at acidic pH was found to convert to the red semiquinone with a first order rate constant of 90 s-1, where the rates were not affected by pH or the concentration of NAD+. The final product is NAD+-bound red semiquinone of the enzyme.
Kobayashi
K
K
Institute of Scientific and Industrial Research, Osaka University, Japan.
Iyanagi
T
T
Ohara
H
H
Hayashi
K
K
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011675:Pulse Radiolysis; D013552:Swine
D009243:NAD; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase