TY - JOUR
T1 - Solvent isotope effects in the catalytic cycle of P450 CYP17A1
T2 - Computational modeling of the hydroxylation and lyase reactions
AU - Denisov, Ilia G.
AU - Sligar, Stephen G.
N1 - Funding Information:
This work is supported by a MIRA grant from NIGMS R35 GM118145 (S.G.S).
Publisher Copyright:
© 2023 Elsevier Inc.
PY - 2023/6
Y1 - 2023/6
N2 - The catalytic cycle of the cytochromes P450 (CYP) requires two electrons from a protein redox partner and two protons from water to generate the main catalytic intermediate, a ferryl-oxo complex with π-cation on the heme porphyrin ring, termed Compound 1. The protonation steps are at least partially rate-limiting, therefore the steady-state rates of P450 catalysis are usually slower in deuterated solvent (D2O) by a factor of 1.5–3. However, in several P450 systems a pronounced inverse kinetic solvent isotope effect (KSIE ∼0.4–0.7) is observed, where the reaction is faster in D2O. This raises an important mechanistic question: Is this inverse solvent isotope effect compatible with Compound 1 catalyzed reactions, or is it indicative of another catalytic intermediate being involved? In this communication we use exhaustive numerical modeling of the P450 steady-state kinetics to demonstrate that a significant inverse KSIE cannot be obtained for a pure Compound 1 driven catalytic cycle of P450. Rather, an alternative, protonation independent, catalytic intermediate needs to be introduced. This result is applicable to the broad spectrum of P450s in nature, but as an example we use the extensively documented inverse isotope effect in the human steroid biosynthetic P450 CYP17A1 where the involvement of a heme peroxo anion intermediate has been characterized. Based on this analysis, we show that the observation of an inverse KSIE can be used as a general mechanistic probe for reaction cycle intermediates in the cytochromes P450.
AB - The catalytic cycle of the cytochromes P450 (CYP) requires two electrons from a protein redox partner and two protons from water to generate the main catalytic intermediate, a ferryl-oxo complex with π-cation on the heme porphyrin ring, termed Compound 1. The protonation steps are at least partially rate-limiting, therefore the steady-state rates of P450 catalysis are usually slower in deuterated solvent (D2O) by a factor of 1.5–3. However, in several P450 systems a pronounced inverse kinetic solvent isotope effect (KSIE ∼0.4–0.7) is observed, where the reaction is faster in D2O. This raises an important mechanistic question: Is this inverse solvent isotope effect compatible with Compound 1 catalyzed reactions, or is it indicative of another catalytic intermediate being involved? In this communication we use exhaustive numerical modeling of the P450 steady-state kinetics to demonstrate that a significant inverse KSIE cannot be obtained for a pure Compound 1 driven catalytic cycle of P450. Rather, an alternative, protonation independent, catalytic intermediate needs to be introduced. This result is applicable to the broad spectrum of P450s in nature, but as an example we use the extensively documented inverse isotope effect in the human steroid biosynthetic P450 CYP17A1 where the involvement of a heme peroxo anion intermediate has been characterized. Based on this analysis, we show that the observation of an inverse KSIE can be used as a general mechanistic probe for reaction cycle intermediates in the cytochromes P450.
KW - C-C lyase reaction
KW - CYP17A1
KW - Cytochrome P450
KW - Kinetic solvent isotope effect
KW - Steady-state kinetics
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U2 - 10.1016/j.jinorgbio.2023.112202
DO - 10.1016/j.jinorgbio.2023.112202
M3 - Article
C2 - 37004494
AN - SCOPUS:85151268159
SN - 0162-0134
VL - 243
JO - Journal of Inorganic Biochemistry
JF - Journal of Inorganic Biochemistry
M1 - 112202
ER -