One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase.
The one-electron oxidation-reduction properties of flavin in hepatic NADH-cytochrome b5 reductase were investigated by optical absorption spectroscopy, electron paramagnetic resonance (EPR), and potentiometric titration. An intermediate with a peak at 375 nm previously described by Iyanagi (1977) [ Iyanagi , T. (1977) Biochemistry 16, 2725-2730] was confirmed to be a red anionic semiquinone. The NAD+-bound reduced enzyme was oxidized by cytochrome b5 via the semiquinone intermediate. This indicates that electron transfer from flavin to cytochrome b5 proceeds in two successive one-electron steps. Autoxidation of the NAD+-bound reduced enzyme was slower than that of the NAD+-free reduced enzyme and was accompanied by the appearance of an EPR signal. Midpoint redox potentials of the consecutive one-electron-transfer steps in the presence of excess NAD+ were Em,1 = -88 mV and Em,2 = 147 mV at pH 7.0. This corresponds to a semiquinone formation constant of 8. The values of Em,1 and Em,2 were also studied as a function of pH. A mechanism for electron transfer from NADH to cytochrome b5 is discussed on the basis of the one-electron redox potentials of the enzyme and is compared with the electron-transfer mechanism of NADPH-cytochrome P-450 reductase.
D000818:Animals; D016227:Benzoquinones; D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D004227:Dithionite; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011809:Quinones; D013552:Swine
D016227:Benzoquinones; D011809:Quinones; D004227:Dithionite; C025232:semiquinone radicals; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D010100:Oxygen