Nicotinamide adenine dinucleotide

Nicotinamide adenine dinucleotide, or NAD+, is a coenzyme that plays a key role in metabolism and is present in every living cell. Its name comes from its structure: it is a dinucleotide, meaning it is made of two nucleotides linked by their phosphate groups. One of these nucleotides contains adenine, and the other contains nicotinamide. The molecule exists in two states—an oxidized form (NAD+) and a reduced form (NADH), where the "H" stands for hydrogen.

NAD's main job is to participate in redox reactions, shuttling electrons between molecules. In its oxidized form, NAD+ acts as an electron acceptor, pulling electrons from other molecules and becoming reduced to NADH (with the addition of a proton). NADH can then act as a reducing agent, donating those electrons elsewhere. These electron transfers are reversible, allowing NAD to cycle between forms without being used up. Beyond redox work, NAD also serves as a substrate for enzymes that add or remove chemical groups from proteins, a process known as posttranslational modification. Because these functions are so vital, the enzymes that handle NAD metabolism are often targeted in drug development.

Organisms can build NAD from scratch using simple building blocks like the amino acids tryptophan or aspartic acid. Alternatively, they can take up more complex components from nutrients, such as vitamin B3 (niacin, or nicotinic acid, which gives nicotinamide its name). There are also salvage pathways that recycle breakdown products of NAD back into active forms.

The superscript plus sign in NAD+ indicates a positive formal charge on one of its nitrogen atoms. Some NAD is converted into a related coenzyme, nicotinamide adenine dinucleotide phosphate (NADP). NADP works much like NAD, but it has an extra phosphate group at the C-2′ position of the adenosyl part. NADP exists in oxidized (NADP+) and reduced (NADPH) forms, and its main role is as a reducing agent in anabolic reactions, such as the Calvin cycle and the synthesis of lipids and nucleic acids.

Physically, NAD is made of two nucleosides joined by a pyrophosphate group. Each nucleoside has a ribose ring: one carries adenine (adenosine diphosphate ribose), and the other carries nicotinamide. In redox reactions, NAD accepts or donates a hydride ion (H−). For example, when a molecule RH₂ is oxidized, it loses two hydrogen atoms: one hydride ion transfers to the nicotinamide ring of NAD+, and a proton is released into solution. This reduces NAD+ to NADH. The midpoint potential of the NAD+/NADH pair is −0.32 volts, making NADH a moderately strong reducing agent.

In pure form, NAD and NADH are white, hygroscopic, amorphous powders that dissolve easily in water. They 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 they break down quickly in acidic or alkaline conditions, forming products that inhibit enzymes.

Both NAD+ and NADH absorb ultraviolet light strongly due to their adenine component. NAD+ has a peak absorption at 259 nm, while NADH has an additional peak at 339 nm. This difference in UV absorption at higher wavelengths allows scientists to easily track the conversion between forms in enzyme assays by measuring absorption at 340 nm with a spectrophotometer. NADH also fluoresces when excited at around 335 nm, emitting light at 445–460 nm with a fluorescence lifetime of 0.4 nanoseconds; NAD+ does not fluoresce. When NADH binds to proteins, its fluorescence properties change, which can be used to measure dissociation constants and study enzyme kinetics. These fluorescence changes are also used to monitor the redox state of living cells via fluorescence microscopy. Additionally, NADH can be converted back to NAD+ in a reaction catalyzed by copper that requires hydrogen peroxide, meaning the supply of NAD+ in cells depends on mitochondrial copper(II).

In cells, the total amount of NAD+ and NADH is about 1 micromole per gram of wet weight in rat liver—roughly ten times the concentration of NADP+ and NADPH. The actual concentration of NAD+ in the cytosol of animal cells is estimated at around 0.3 mM, and in yeast it ranges from 1.0 to 2.0 mM. However, more than 80% of NADH fluorescence in mitochondria comes from bound forms, so the free concentration is much lower. NAD+ levels are highest in the mitochondria, where they make up 40% to 70% of the cell's total NAD+. Cytosolic NAD+ is transported into the mitochondrion by a specific membrane carrier.