Cofactors and coenzymes

Cofactors supply reactivity that amino acid side chains lack: good electron sinks, reversible redox at accessible potentials, and carriers for groups such as acyl, amino or one-carbon units. Coenzymes are typically regenerated and reused, acting as shuttles between enzymes; tightly or covalently bound ones are called prosthetic groups.

Redox coenzymes

• NAD^+/NADH and NADP^+/NADPH. The nicotinamide ring accepts a hydride at C4 to form the 1,4-dihydropyridine. Dehydrogenases transfer hydride stereospecifically to one face (pro-R or pro-S hydrogen). NADH is a biological equivalent of a mild hydride reducing agent; NAD^+ mostly serves catabolic oxidation, NADPH biosynthetic reduction.
• FAD and FMN. The isoalloxazine ring can accept two electrons at once or one at a time via a semiquinone radical, so flavins link two-electron substrates (such as succinate) to one-electron carriers like iron–sulfur clusters.

Group-transfer coenzymes

• Coenzyme A. Its terminal thiol forms thioesters, R–CO–SCoA. Thioesters are more reactive acyl donors than oxygen esters (poorer resonance overlap between S and C=O) and their \alpha-protons are more acidic, which favours enolate-type C–C bond formation, as in the citric acid cycle and fatty acid synthesis.
• Pyridoxal 5′-phosphate (PLP). The aldehyde forms an imine with an active-site lysine; an amino acid substrate swaps in by transimination. The protonated pyridinium ring then acts as an electron sink, stabilising a carbanion at C\alpha. Which bond the enzyme breaks (the one aligned perpendicular to the \pi-system) determines the outcome: loss of H^+ leads to transamination or racemisation, loss of CO_2 to decarboxylation. In transamination, the amino group is passed to PLP (giving pyridoxamine phosphate) and then to a keto acid.
• Thiamine diphosphate (ThDP). Deprotonation at C2 of the thiazolium ring gives an ylide/carbene nucleophile that adds to a keto acid carbonyl. Decarboxylation then leaves a carbanion stabilised by the thiazolium, an umpolung (reversal of carbonyl polarity) analogous to cyanide in the benzoin condensation. This drives pyruvate decarboxylation and transketolase reactions.
• Others include biotin (CO_2 carrier for carboxylases), tetrahydrofolate (one-carbon units) and cobalamin (radical rearrangements and methyl transfer).

Metal cofactors

Mg^{2+} binds ATP phosphates in kinases; Zn^{2+} is a Lewis acid in hydrolases; Fe and Cu cycle oxidation states in electron transfer and O_2 activation (haem in cytochrome P450, Fe–S clusters); Mo, Ni and Co enable more specialised chemistry.

Cofactor chemistry is a rich area of current research. Radical SAM enzymes use a [4Fe–4S] cluster to cleave S-adenosylmethionine reductively, generating a 5′-deoxyadenosyl radical that abstracts unactivated hydrogen atoms; this superfamily carries out tens of thousands of transformations in natural product biosynthesis and cofactor assembly. Flavin enzymes have revealed unexpected modes, including covalent flavin–substrate adducts, flavin-N5-oxide intermediates and photochemistry, as in the light-dependent fatty acid photodecarboxylase.

In biocatalysis, cofactor dependence brings both opportunity and cost. Transaminases (PLP) make chiral amines at manufacturing scale, ketoreductases (NAD(P)H) make chiral alcohols, and ThDP-dependent lyases form C–C bonds asymmetrically. Because nicotinamide cofactors are expensive, processes regenerate them in situ with a sacrificial substrate and a second enzyme (e.g. glucose dehydrogenase or formate dehydrogenase), or use cheaper synthetic nicotinamide biomimetics. "Photoenzymatic" catalysis exploits the excited states of flavins and of NAD(P)H in ene-reductases and ketoreductases to drive non-natural radical reactions.

Metallocofactors pose deep mechanistic questions. The FeMo-cofactor of nitrogenase, with its interstitial carbide, still lacks an agreed N_2-binding mode; the oxygen-evolving Mn_4CaO_5 cluster of photosystem II is studied by time-resolved XFEL crystallography and EPR. Artificial metalloenzymes, made by anchoring abiological metal complexes inside protein scaffolds or substituting the native metal (such as iridium in place of iron in haem proteins), combine organometallic reactivity with protein-controlled selectivity and can be optimised by directed evolution.