Understanding the mechanism and regulation of androgen synthesis, catalyzed by Cytochrome P450 17A1 (CYP17), is imperative to guide drug development efforts for prostate cancer. CYP17 is a bifunctional P450 acting on multiple substrates in a spatio‐temporally regulated fashion. In the zona fasciculata of the adrenal cortex, it catalyzes the hydroxylation of progesterone and pregnenolone to produce precursors of glucocorticoids. In the adrenal zona reticularis and gonads, CYP17 also catalyzes a subsequent carbon‐carbon lyase reaction on these hydroxylated products, producing androgens. This chemically complex lyase step is not well understood, and is of key importance to help design androgen synthesis specific drugs which do not impact production of the vital corticoids.P450 mediated hydroxylation has been intensely studied and it is generally agreed that the active intermediate is the high valent oxy‐ferryl species‐Compound I (Cpd I). On the other hand, CYP17 lyase chemistry has also been hypothesized to occur through a nucleophilic attack by the peroxo anion on the carbonyl of the substrate, forming a peroxo‐hemiacetal intermediate which then decays to release product. In order to provide mechanistic insight, we made use of the proton‐dependence of the Cpd I scheme and determined the kinetic solvent isotope effects (KSIE). We observed an inverse KSIE, which is explained by the decrease in uncoupling through peroxide shunt in the D2O reactions. We cryotrapped oxy‐ferrous CYP17, employed cryoradiolysis to reduce the trapped oxy‐intermediate, followed by thermal annealing to follow the mechanism using optical spectroscopy and resonance Raman spectroscopy1. We were successful in observing a hemiacetal intermediate, which makes a strong case for peroxo anion mediated mechanism for lyase reaction.Cytochrome b5 (b5) is a small heme protein, which has been shown to enhance the lyase reaction rates 5–10 fold, but has no effect on hydroxylation. The mechanism of this selective modulation of lyase reaction is not clear. b5 has been shown to enhance product formation rates in some P450s by acting as an alternate redox donor, but a possible redox role in CYP17 lyase chemistry remains elusive. To this end, we prepared a redox inactive manganese substituted form of b5 (Mn b5) and compared the rate enhancement with Mn b5 vs. b52. To determine if b5 binding perturbs the CYP17 heme thereby affecting catalysis, we performed resonance Raman spectroscopy on substrate bound ferrous‐CO and oxy‐ferrous CYP17 as well as resting ferric CYP17 in the presence of Mn b5. We used the Nanodisc system in all our experiments to reconstitute these enzymes in known stoichiometries. We observed a 5 fold rate increase with b5 but no rate enhancement with Mn b5, suggesting a redox donor role for this protein. Although our results do not disprove the presence of allosteric interactions between b5 and CYP17, it can be argued that any allosteric interactions do not play a significant role in CYP17 mediated catalysis of lyase reaction.Support or Funding InformationThis work was supported by National Institutes of Health[GM110428]