(product). MurB is a monomer and contains one FAD molecule. Before the substrate can be converted to product, NADPH must first reduce FAD. Once NADP+ dissociates, the substrate can bind and the reduced flavin can reduce the product. Thiol/disulfide chemistry Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH). GR requires FAD and NADPH to facilitate this reaction; first a hydride must be transferred from NADPH to FAD. The reduced flavin can then act as a nucleophile to attack the disulfide, this forms the C4a-cysteine adduct. Elimination of this adduct results in a flavin-thiolate charge-transfer complex. Electron transfer reactions Cytochrome P450 type enzymes that catalyze monooxygenase (hydroxylation) reactions are dependent on the transfer of two electrons from FAD to the P450. Two types of P450 systems are found in eukaryotes. The P450 systems that are located in the endoplasmic reticulum are dependent on a cytochrome P-450 reductase (CPR) that contains both an FAD and an FMN. The two electrons on reduced FAD (FADH2) are transferred one at a time to FMN and then a single electron is passed from FMN to the heme of the P450. The P450 systems that are located in the mitochondria are dependent on two electron transfer proteins: An FAD containing adrenodoxin reductase (AR) and a small iron-sulfur group containing protein named adrenodoxin. FAD is embedded in the FAD-binding domain of AR. The FAD of AR is reduced to FADH2 by transfer of two electrons from NADPH that binds in the NADP-binding domain of AR. The structure of this enzyme is highly conserved to maintain precisely the alignment of electron donor NADPH and acceptor FAD for efficient electron transfer. The two electrons in reduced FAD are transferred one a time to adrenodoxin which in turn donates the single electron to the heme group of the mitochondrial P450. The structures of the reductase of the microsomal versus reductase of the mitochondrial P450 systems are completely different and show no homology. Redox p-Hydroxybenzoate hydroxylase (PHBH) catalyzes the oxygenation of p-hydroxybenzoate (pOHB) to 3,4-dihyroxybenzoate (3,4-diOHB); FAD, NADPH and molecular oxygen are all required for this reaction. NADPH first transfers a hydride equivalent to FAD, creating FADH−, and then NADP+ dissociates from the enzyme. Reduced PHBH then reacts with molecular oxygen to form the flavin-C(4a)-hydroperoxide. The flavin hydroperoxide quickly hydroxylates pOHB, and then eliminates water to regenerate oxidized flavin. An alternative flavin-mediated oxygenation mechanism involves the