ELECTRON-PARAMAGNETIC-RESONANCE INVESTIGATION OF CYTOCHROME P-450(C21) FROM BOVINE ADRENOCORTICAL MICROSOMES - A NEW ENZYMATIC-ACTIVITY

Cytochrome P-450(c21) (CYP21A1) purified from bovine adrenocortical mi crosomes was investigated by electron paramagnetic resonance (EPR) spe ctroscopy to clarify the interactions among heme active center, protei n surroundings, water molecules and bound substrates or analogues, The low-spin EPR signals of the oxidized enzyme (as purified) consisted o f two species; one at g(z) = 2.39, g(y) = 2.23, and g(x) = 1.925 (comp onent A) and the other at g(z) = 2.42, g(y) = 2.23, and g(x) = 1.916 ( component B), The component A is probably representing a product-bound form, whereas the component B indicates either occupation of the subs trate-binding site with a substrate analogue or absence of steroid at the site. Upon addition of progesterone, the component A signal comple tely disappeared and the intensity of high-spin signal (g = 8.06, 3.54 ) increased, Addition of 17 alpha-hydroxyprogesterone caused a develop ment of a new low-spin signal at g(z) = 2.42, g(y) = 2.21, and g(x) = 1.966 (component C) and a further increase in intensity of the high-sp in signal (g = 8.06, 3.54), Addition of 20 beta-hydroxyprogesterone ca used 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-450(c21) active center in a very similar manner as 1 7 alpha-hydroxyprogesterone does and, therefore, may be a metabolizabl e substrate, A new enzymatic pathway catalyzed by cytochrome P-450(c21 ) was confirmed with a reconstituted enzymatic system consisting of cy tochrome P-450(c21), NADPH-cytochrome P-450 reductase and an NADPH-gen erating system. 20 beta-Hydroxyprogesterone was converted to progester one via a putative 20 beta-oxidase reaction in a comparable turnover n umber to that of the 21-hydroxylation of 17 alpha-hydroxyprogesterone.