Although the oxidation and reduction of the FAD or FMN prosthetic groups of the flavin dehydrogenases are usually written formally as reactions involving simultaneous transfers of 2 hydrogen atoms or 2 electrons, there is evidence that these reactions occur in two separate one-electron steps. Transfer of one electron to FAD (or FMN) leads to formation of its semiquinone, or free radical, which contains an unpaired electron. The semiquinone may then accept a second electron to go into the fully reduced form. The semiquinone forms of some flavin dehydrogenases can be detected either by their characteristic absorption spectrum or by means of electron spin resonance spectroscopy, which can detect the occurrence of molecules with unpaired electron spins by their characteristic behavior in a magnetic field. FAD and FMN can also be reduced nonenzymatically with reducing agents such as dithionite (Lehninger, 1970).

Reduction of NAD(P)⁺ also requires transfer of 2 electrons.

Higher plant nitrate reductases can be either NADH or NAD(P)H specific. In vitro mutation of NAD(P)H --> NADH nitrate reductase can be accomplished by a single amino acid substitution in the FAD domain (Schondorf and Hachtel, 1995).

Lehninger AL 1970 Biochemistry. Worth Publishers, New York.

Schondorf T, Hachtel W 1995 The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch. Plant Physiol. 108: 203-210.