The 14α‐demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three‐step reaction terminates in a C−C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO₂⁻) or Compound I (perferryl oxygen, FeO³⁺) reacting with either the aldehyde or its hydrate, respectively. Analysis of ¹⁸O incorporation into formic acid from ¹⁸O₂ provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % ¹⁸O (one atom) into formic acid, consistent with a major but not exclusive FeO₂⁻ mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X‐ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Å away from the heme iron atom. Experiments with human P450 51A1 and H₂¹⁸O yielded primarily one ¹⁸O atom but 14 % of the formic acid product with two ¹⁸O atoms, indicative of a minor contribution of a Compound I mechanism. LC–MS evidence for a Compound 0‐derived Baeyer–Villiger reaction product (a 14α‐formyl ester) was also found.

It has been recognized for >50 years that cytochrome b₅ (b₅) stimulates some cytochrome P450 (P450)-catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b₅ is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b₅ function. One of the issues in studying b₅-P450 interactions has been the limited solution assay methods. We constructed a fluorescently-labeled variant of human b₅ that can be used in titrations. The labeled b₅ bound to wild-type P450 17A1 with a K_d of 2.5 nM and rapid kinetics, on the order of 1 s^-1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b₅ binding. K_d values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b₅ fluorescence at higher concentrations. The addition of NADPH-P450 reductase (POR) to an Alexa 488-T70C-b₅:P450 17A1 complex resulted in a concentration-dependent, partial restoration of b₅ fluorescence, indicative of a ternary P450:b₅:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b₅ complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, with k_off rates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3β-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14α C-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450_scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)₂ cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)₂ cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and k_off rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)₂ cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d₃-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.

Cytochrome P450 (P450) enzymes dominate steroid metabolism. In general, the simple C-hydroxylation reactions are mechanistically straightforward and are generally agreed to involve a perferryl oxygen species (formally FeO³⁺). Several of the steroid transformations are more complex and involve C-C bond scission. We initiated mechanistic studies with several of these (i.e., 11A1, 17A1, 19A1, and 51A1) and have now established that the dominant modes of catalysis for P450s 19A1 and 51A1 involve a ferric peroxide anion (i.e., Fe³⁺O₂¯) instead of a perferryl ion complex (FeO³⁺), as demonstrated with ¹⁸O incorporation studies. P450 17A1 is less clear. The indicated P450 reactions all involve sequential oxidations, and we have explored the processivity of these multi-step reactions. P450 19A1 is distributive, i.e., intermediate products dissociate and reassociate, but P450s 11A1 and 51A1 are highly processive. P450 17A1 shows intermediate processivity, as expected from the release of 17-hydroxysteroids for the biosynthesis of key molecules, and P450 19A1 is very distributive. P450 11B2 catalyzes a processive multi-step oxidation process with the complexity of a chemical closure of an intermediate to a locked lactol form.

Cytochrome P450 (P450, CYP) 19A1 is the steroid aromatase, the enzyme responsible for the 3‐step conversion of androgens (androstenedione or testosterone) to estrogens. The final step is C−C bond scission (removing the 19‐oxo group as formic acid) that proceeds via a historically controversial reaction mechanism. The two competing mechanistic possibilities involve a ferric peroxide anion (Fe³⁺O₂⁻, Compound 0) and a perferryl oxy species (FeO³⁺, Compound I). One approach to discern the role of each species in the reaction is with the use of oxygen‐18 labeling, i.e., from ¹⁸O₂ and H₂¹⁸O of the reaction product formic acid. We applied this approach, using several technical improvements, to study the deformylation of 19‐oxo‐androstenedione by human P450 19A1 and of a model secosteroid, 3‐oxodecaline‐4‐ene‐10‐carboxaldehyde (ODEC), by rabbit P450 2B4. Both aldehyde substrates were sensitive to non‐enzymatic acid‐catalyzed deformylation, yielding 19‐norsteroids, and conditions were established to avoid issues with artifactual generation of formic acid. The Compound 0 reaction pathway predominated (i.e., Fe³⁺O₂⁻) in both P450 19A1 oxidation of 19‐oxo‐androstenedione and P450 2B4 oxidation of ODEC. The P450 19A1 results contrast with our prior conclusions (J. Am. Chem. Soc. 2014, 136, 15016–16025), attributed to several technical modifications.