Edited by Ruma Banerjee Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in the oxidation of steroids as well as many other compounds. Their versatility has been explained in part by flexibility of the proteins and complexity of the binding mechanisms. However, whether these proteins bind their substrates via induced fit or conformational selection is not understood. P450 17A1 has a major role in steroidogenesis, catalyzing the two-step oxidations of progesterone and pregnenolone to androstenedione and dehydroepiandrosterone, respectively, via 17␣-hydroxy (OH) intermediates. We examined the interaction of P450 17A1 with its steroid substrates by analyzing progress curves (UV-visible spectroscopy), revealing that the rates of binding of any of these substrates decreased with increasing substrate concentration, a hallmark of conformational selection. Further, when the concentration of 17␣-OH pregnenolone was held constant and the P450 concentration increased, the binding rate increased, and such opposite patterns are also diagnostic of conformational selection. Kinetic simulation modeling was also more consistent with conformational selection than with an induced-fit mechanism. Cytochrome b 5 partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17␣-OH pregnenolone or 17␣-OH progesterone, as judged by the apparent K d and binding kinetics. The P450 17A1 inhibitor abiraterone also bound to P450 17A1 in a multistep manner, and modeling indicated that the selective inhibition of the two P450 17A1 steps by the drug orteronel can be rationalized only by a multiple-conformation model. In conclusion, P450 17A1 binds its steroid substrates via conformational selection. Cytochrome P450 (P450) 2 enzymes are the main catalysts involved in the oxidation of steroids, drugs, fat-soluble vitamins, chemical carcinogens, and many other chemicals (1, 2). Collectively, they account for Ͼ95% of the oxidations and This work was supported by National Institutes of Health Grants R01 GM118122 (to F. P. G.) and T32 ES007028 (to F. P. G., support of S. M. G. and M. J. R.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article was selected as one of our Editors' Picks. This article contains Scheme S1 and Figs. S1-S8.

Cytochrome P450 (P450) enzymes are major catalysts involved in the oxidations of most drugs, steroids, carcinogens, fat-soluble vitamins, and natural products. The binding of substrates to some of the 57 human P450s and other mammalian P450s is more complex than a two-state system and has been proposed to involve mechanisms such as multiple ligand occupancy, induced-fit, and conformational-selection. Here, we used kinetic analysis of binding with multiple concentrations of substrates and computational modeling of these data to discern possible binding modes of several human P450s. We observed that P450 2D6 binds its ligand rolapitant in a mechanism involving conformational-selection. P450 4A11 bound the substrate lauric acid via conformational-selection, as did P450 2C8 with palmitic acid. Binding of the steroid progesterone to P450 21A2 was also best described by a conformational-selection model. Hexyl isonicotinate binding to P450 2E1 could be described by either a conformational-selection or an induced-fit model. Simulation of the binding of the ligands midazolam, bromocriptine, testosterone, and ketoconazole to P450 3A4 was consistent with an induced-fit or a conformational-selection model, but the concentration dependence of binding rates for varying both P450 3A4 and midazolam concentrations revealed discordance in the parameters, indicative of conformational-selection. Binding of the P450s 2C8, 2D6, 3A4, 4A11, and 21A2 was best described by conformational-selection, and P450 2E1 appeared to fit either mode. These findings highlight the complexity of human P450-substrate interactions and that conformational-selection is a dominant feature of many of these interactions.

Human cytochrome P450 (P450) Family 4 enzymes are involved in the metabolism of fatty acids and the bioactivation of carcinogenic arylamines and toxic natural products, e.g. 4-ipomeanol. These and other drug metabolizing P450s are redox sensitive, showing a loss of activity resulting from preincubation with H 2 O 2 and recovery with mild reducing agents (Albertolle, M.W. et al. (2017) J. Biol. Chem. 292, 11230-11242). The inhibition is due to sulfenylation of the hemethiolate ligand, as determined by chemopreoteomics and spectroscopy. This phenomenon may have implications in chemical toxicity and in observed disease-drug interactions, in which decreased metabolism of P450 substrates occurs in patients with inflammatory diseases (e.g., influenza, autoimmunity). Human P450 1A2 was determined to be redox insensitive. To determine the mechanism underlying the differential redox sensitivity, molecular dynamics (MD) simulations were employed using the crystal structure of rabbit P450 4B1 (PDB ID: 5T6Q). In simulating either the thiolate (Cys-S −) or the sulfenic acid (Cys-SOH) at the heme-ligation site, MD revealed Gln-451 in either an "open" or "closed" conformation, respectively, between the cytosol and heme-thiolate cysteine. Mutation to either an isosteric leucine (Q451L) or glutamate (Q451E) abrogated the redox sensitivity, suggesting that this "open" conformation allows for reduction of the sulfenic acid and thiolate religation to the heme iron. In summary, MD simulations suggest that Gln-451 in P450 4B1 adopts conformations that may stabilize and protect the heme-thiolate sulfenic acid; mutating this residue destabilizes the interaction, producing a redox insensitive enzyme.

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