Nicotinamide adenine dinucleotide consists of two nucleosides joined by pyrophosphate. The nucleosides each contain a ribose ring, one with adenine attached to the first carbon atom (the 1' position) (adenosine diphosphate ribose) and the other with nicotinamide at this position.
The compound accepts or donates the equivalent of H. Such reactions (summarized in formula below) involve the removal of two hydrogen atoms from the reactant (R), in the form of a hydride ion (H), and a proton (H). The proton is released into solution, while the reductant RH is oxidized and NAD reduced to NADH by transfer of the hydride to the nicotinamide ring.
:RH + NAD → NADH + H + R;
From the hydride electron pair, one electron is attracted to the slightly more electronegative atom of the nicotinamide ring of NAD, becoming part of the nicotinamide moiety. The second electron and proton atom are transferred to the carbon atom adjacent to the N atom. The midpoint potential of the NAD/NADH redox pair is −0.32 volts, which makes NADH a moderately strong reducing agent. The reaction is easily reversible, when NADH reduces another molecule and is re-oxidized to NAD. This means the coenzyme can continuously cycle between the NAD and NADH forms without being consumed.
In appearance, all forms of this coenzyme are white amorphous powders that are hygroscopic and highly water-soluble. The solids are stable if stored dry and in the dark. Solutions of NAD are colorless and stable for about a week at 4 °C and neutral pH, but decompose rapidly in acidic or alkaline solutions. Upon decomposition, they form products that are enzyme inhibitors.
Both NAD and NADH strongly absorb ultraviolet light because of the adenine. For example, peak absorption of NAD is at a wavelength of 259 nanometers (nm), with an extinction coefficient of 16,900 Mcm. NADH also absorbs at higher wavelengths, with a second peak in UV absorption at 339 nm with an extinction coefficient of 6,220 Mcm. This difference in the ultraviolet absorption spectra between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in enzyme assays – by measuring the amount of UV absorption at 340 nm using a spectrophotometer.
NAD and NADH also differ in their fluorescence. Freely diffusing NADH in aqueous solution, when excited at the nicotinamide absorbance of ~335 nm (near-UV), fluoresces at 445–460 nm (violet to blue) with a fluorescence lifetime of 0.4 nanoseconds, while NAD does not fluoresce. The properties of the fluorescence signal changes when NADH binds to proteins, so these changes can be used to measure dissociation constants, which are useful in the study of enzyme kinetics. These changes in fluorescence are also used to measure changes in the redox state of living cells, through fluorescence microscopy.
NADH can be converted to NAD+ in a reaction catalysed by copper, which requires hydrogen peroxide. Thus, the supply of NAD+ in cells requires mitochondrial copper(II).