One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase

Biochemistry. 1984 Mar 27;23(7):1418-25. doi: 10.1021/bi00302a013.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzoquinones*
  • Cytochrome Reductases / metabolism*
  • Cytochrome-B(5) Reductase
  • Dithionite
  • Electron Spin Resonance Spectroscopy
  • Hydrogen-Ion Concentration
  • Microsomes, Liver / enzymology*
  • Oxidation-Reduction
  • Oxygen / metabolism
  • Quinones
  • Swine

Substances

  • Benzoquinones
  • Quinones
  • Dithionite
  • semiquinone radicals
  • Cytochrome Reductases
  • Cytochrome-B(5) Reductase
  • Oxygen