Functional and dysfunctional enzymatic pathways of cytochrome P450s after formation of the reduced ferrous dioxygen species have been investigated using nonlocal density functional quantum chemical methods, employing a methyl mercapto iron porphine model of the cytochrome P450 heme complex. The goal of this study was to assess the validity of proposed pathways to both compound I and peroxide involving protonation of the distal and proximal oxygen atoms of the reduced ferrous dioxygen species. Optimized geometries, energies, and electrostatic and electronic properties of each putative heme intermediate in these pathways were calculated and these properties examined for consistency with the proposed role of the intermediate in compound I or peroxide formation. Single protonation of the distal oxygen resulted in significant weakening of the O-O bond. Addition of a second proton to the distal oxygen and energy optimization led directly to compound I and water products, without any apparent activation barrier or formation of a diprotonated intermediate. These results provide direct robust support for the proton-assisted mechanism of dioxygen bond cleavage to form compound I. The dysfunctional pathway to the formation of peroxide was explored by examining the properties of the distal and proximal singly protonated species. The proximal tautomer is thermodynamically less favorable than the distal species by 18.4 kcal/mol. Electrostatic features of both singly protonated species suggest preferred proton delivery to the remaining unprotonated oxygen in each case, favoring peroxide formation. Moreover, addition of a second proton to either of these singly protonated species results in formation of a stable hydrogen peroxide heme complex. These results, taken together, suggest that the simultaneous availability of two protons on the distal oxygen is a requirement for P450 enzymatic efficacy, while asynchronous delivery of protons to the dioxygen site favors decoupling.

I. Introduction and Background 407 II. The Heme Site of the Resting Form of CYP450s 410 III. Heme Site of the Substrate Bound Ferric CYP450s 412 IV. The Heme Site of the Ferrous Dioxygen Species and the Putative Transient Reduced Ferrous Dioxygen Species in CYP450s 413 V. Investigation of a Proposed Pathway to Formation of Compound I from the Twice Reduced Dioxygen Species of CYP450s 414 VI. Electronic Structure of a P450 Compound I Heme Species 415 VII. Role of the Protein in Compound I Formation 416 VIII. Conclusions 417 IX. Glossary of Abbreviations 417 X. Acknowledgment 417 XI. References 417 Gilda H. Loew received her B.A. degree in Chemistry and Physics from New York University, Her M.A. degree in Chemistry from Columbia University, and her Ph.D. degree in Chemical Physics from the University of California Berkeley. She has authored 304 technical publications and is co-inventor on three patents.

The retinoid 6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) and its active analogues induce cell-cycle arrest and programmed cell death (apoptosis) in cancer cells independently of retinoic acid receptor (RAR) interaction. Its analogue, (E)-4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-(3'-acetamidopropyloxy)cinnamic acid (3-A-AHPC) selectively antagonized cell apoptotic events (TR3/nur77/NGFI-B expression and nuclear-to-mitochondrial translocation) but not the proliferative events (cell-cycle arrest and p21(WAF1/CIP1) expression) induced by proapoptotic AHPN and its analogues. The syntheses of 3-A-AHPC and proapoptotic (E)-6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-5-chloronaphthalenecarboxylic acid (5-Cl-AHPN) are described. Computational studies on AHPN, AHPC, and three substituted analogues (5-Cl-AHPN, 3-Cl-AHPC, and 3-A-AHPC) suggested reasons for their diametric effects on RAR activation. Density functional theory studies indicated that the 1-adamantyl (1-Ad) groups of the AHPN and AHPC configurations assumed positions that were nearly planar with the aromatic rings of their polar termini. In contrast, in the configurations of the substituted analogues having chloro and 3-acetamidopropyloxy groups, rather than a hydrogen, ortho to the diaryl bonds, the diaryl bond torsion angles increased so that the 1-Ad groups were oriented out of this plane. Docking and molecular dynamics of AHPN, AHPC, and these substituted analogues in the RARgamma ligand-binding domain illustrated how specific substituents on the AHPN and AHPC scaffolds modulated the positions and dynamics of the 1-Ad groups. As a result, the position of RARgamma helix H12 in forming the coactivator-binding site was impacted in a manner consistent with the experimental effect of each analogue on RARgamma transcriptional activation.