Lipoamide

E3 Reaction

E3 promotes the transfer of two hydrogen atoms from the reduced lipoamide group of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyl-lysine group of E2 and ultimately leading to the production of NADH and a free proton. Each E3 monomer has two active sites and four distinct domains: a FAD-binding domain, an NAD-binding domain, as well as central and interface domains. Each active site is formed at the junction of the two subunits with binding pockets for NAD and dihydrolipamide located on opposite sides of a centrally located flavin, which binds to each chain in an extended conformation to intersect the channel. The redox center of the enzyme consists of a reactive intramolecular disulfide bridge that couples with the flavin rings. Upon dihydrolipoyl binding, the first reductive half reaction occurs whereby electrons are transferred from dihydrolipoamide to open the reactive disulfide bridge, permitting one of the cysteine residues to form a charge transfer complex with the flavin. This half reaction is initiated by abstracting a proton from the substrate by a conserved histidine residue that interacts directly with the bound flavin. The FADH2 form of E3 is required for binding of NAD+, which orients so that its nicotinamide rings are closely juxtaposed with the isoalloxazine rings of the flavin. The second oxidative half reaction then occurs, in which the reduced FADH2 group transfers a hydride ion to NAD+ giving rise to a free proton and NADH, which enters into the respiratory chain to generate ATP. The reactive disulfide bridge of E3 is also regenerated during this half reaction.