Oxidative Inhibition of the ω‐Hydroxylation Activity of Cytochrome P450 4A11 by Sulfenylation of the Heme‐Thiolate Cysteine

Abstract

Cytochrome P450 (CYP) 4A11 is an ω‐hydroxylase that catalyzes the oxidation of arachidonic acid to 20‐hydroxyeicosatetraenoic acid (20‐HETE). 20‐HETE can be pro‐hypertensive by constricting microvasculature or anti‐hypertensive by inhibiting salt reabsorption in the kidney. Thus, 20‐HETE and CYP4A11 have important roles in blood pressure regulation. Using isotope‐coded affinity tag (ICAT) technology, cysteine‐sulfenic acids were alkylated by d6‐dimedone followed by counter‐alkylation of free thiolates by d0‐iododimedone. Peptides were analyzed by nLC‐MS/MS and using the software, Skyline, peak integrals of heavy/light peptides were then compared to obtain a ratio of thiol sulfenylation. Loss/recovery of the heme‐thiolate ligand was monitored spectrophotometrically in anaerobic conditions using first P450 oxidoreductase and then dithionite as iron and thiol reducing agents, respectively. The ratio of sulfenic acid/thiolate of the heme ligand cysteine was correlated with the inhibition of enzymatic activity by H2O2 in a time‐ and concentration‐dependent manner. This loss and subsequent recovery of the heme‐thiolate ligand were observed spectrophotometrically. 20‐HETE production has been implicated in the development of hypertension in mice and correlates with vascular diseases in humans. Perturbations of the vascular system are known to induce oxidative signaling and oxidative stress. This redox switch in CYP4A11 may be part of a control mechanism to limit 20‐HETE production in vivo.Support or Funding InformationNIH T32 ES007028R01 GM118122