Active Site Structures of CYP11A1 in the Presence of Its Physiological Substrates and Alterations upon Binding of Adrenodoxin

The rate limiting step in the steroid synthesis pathway is catalyzed by CYP11A1 through three sequential reactions. The first two steps involve hydroxylations at the 22- and 20- positions, generating 20R,22R-dihydroxycholesterol (20R,22R-DiOHCH), with the third stage leading to a C20-C22 bond cleavage, forming pregnenolone. The present work provides detailed information on active site structure of CYP11A1 in the resting state and substrate-bound ferric forms as well as the CO-ligated adducts. In addition, high quality resonance Raman spectra are reported for the dioxygen complexes, providing new insight into the status of Fe-O-O fragments encountered in the enzymatic cycle. Results show that the three natural substrates of CYP11A1 have quite different effects on the active site structure, including variations of spin state populations, reorientations of heme peripheral groups and, most importantly, substrate-mediated distortions of Fe-CO and Fe-O2 fragments, as revealed by telltale shifts of the observed ...