Electron paramagnetic resonance investigation of cytochrome P-450c21 from bovine adrenocortical microsomes: a new enzymatic activity.
Cytochrome P-450c21 (CYP21A1) purified from bovine adrenocortical microsomes was investigated by electron paramagnetic resonance (EPR) spectroscopy to clarify the interactions among heme active center, protein surroundings, water molecules and bound substrates or analogues. The low-spin EPR signals of the oxidized enzyme (as purified) consisted of two species; one at gz = 2.39, gy = 2.23, and gx = 1.925 (component A) and the other at gz = 2.42, gy = 2.23, and gx = 1.916 (component B). The component A is probably representing a product-bound form, whereas the component B indicates either occupation of the substrate-binding site with a substrate analogue or absence of steroid at the site. Upon addition of progesterone, the component A signal completely disappeared and the intensity of high-spin signal (g = 8.06, 3.54) increased. Addition of 17 alpha-hydroxyprogesterone caused a development of a new low-spin signal at gz = 2.42, gy = 2.21, and gx = 1.966 (component C) and a further increase in intensity of the high-spin signal (g = 8.06, 3.54). Addition of 20 beta-hydroxyprogesterone caused an increase in intensity of the component C signal (and the g = 8 high-spin signal) even stronger than did 17 alpha-hydroxyprogesterone. These observations suggest that 20 beta-hydroxyprogesterone binds to the cytochrome P-450c21 active center in a very similar manner as 17 alpha-hydroxyprogesterone does and, therefore, may be a metabolizable substrate. A new enzymatic pathway catalyzed by cytochrome P-450c21 was confirmed with a reconstituted enzymatic system consisting of cytochrome P-450c21, NADPH-cytochrome P-450 reductase and an NADPH-generating system. 20 beta-Hydroxyprogesterone was converted to progesterone via a putative 20 beta-oxidase reaction in a comparable turnover number to that of the 21-hydroxylation of 17 alpha-hydroxyprogesterone.
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D013058:Mass Spectrometry; D008861:Microsomes / Q000201:enzymology*; D011374:Progesterone / Q000378:metabolism; D013255:Steroid 21-Hydroxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D011374:Progesterone; D013255:Steroid 21-Hydroxylase