other cytochrome P450 enzymes, 21-hydroxylase participates in the cytochrome P450 catalytic cycle and engages in one-electron transfer with NADPH-P450 reductase. 21-Hydroxylase is highly specific for hydroxylation of progesterone and 17-hydroxyprogesterone. This is in marked contrast to the evolutionarily and functionally related P450 enzyme 17-hydroxylase, which has a broad range of substrates. The chemical reaction in which 21-hydroxylase catalyzes the addition of hydroxyl (-OH) to the C21 position of progesterone, 17α-hydroxyprogesterone and 21-desoxycortisone was first described in 1952. Studies of the human enzyme expressed in yeast initially classified 17-hydroxyprogesterone as the preferred substrate for 21-hydroxylase, however, later analysis of the purified human enzyme found a lower KM and greater catalytic efficiency for progesterone over 17-hydroxyprogesterone. The catalytic efficiency of 21-hydroxylase for conversion of progesterone in humans is approximately 1.3 x 107 M−1s−1 at 37 °C. This makes it the most catalytically efficient P450 enzyme of those reported to date, and catalytically more efficient than the closely related bovine 21-hydroxylase enzyme. C-H bond breaking to create a primary carbon radical is thought to be the rate-limiting step in the hydroxylation. Genetics 21-hydroxylase is a protein encoded by the CYP21A2 gene in humans. A related pseudogene, CYP21A1P, is located nearby and retains 98% exonic sequence identity with the functional gene CYP21A2. Both genes are located on chromosome 6, in the major histocompatibility complex III close to the Complement component 4 genes C4A and C4B, the Tenascin X gene TNXB and STK19. In the mouse genome, the CYP21A2 is a pseudogene and the CYP21A1 is a functional gene. In the chicken and quail, there is only a single CYP21 gene, which locus is located between complement component C4 and TNX gene, along with CENPA. The CYP21A2 gene travels in tandem with a pseudogene, CYP21P1, and the high degree of sequence similarity between them indicates that these two genes are evolving in tandem through intergenic exchange of DNA. The CYP21A2 gene is located within the RCCX cluster (an abbreviation composed of the names of the genes RP (a former name for STK19 serine/threonine kinase 19), C4, CYP21 and TNX), which is the most complex gene cluster in the human genome. It is part of the major histocompatibility complex class III (MHC class III), which is the most gene-dense region of the human genome, containing many genes that yet have unknown function or structure. Due to the high degree of homology between the CYP21A2 gene and