Glutamine-451 Confers Sensitivity to Oxidative Inhibition and Heme-Thiolate Sulfenylation of Cytochrome P450 4B1

Abstract

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.

Tight binding of cytochrome b5 to cytochrome P450 17A1 is a critical feature of stimulation of C21 steroid lyase activity and androgen synthesis

“…Protein concentrations were estimated from A 280 measurements using a Nanodrop spectrophotometer (Thermo Scientific) and the amino acid compositions of human P450 17A1 and b 5 . The estimated specific content of WT P450 17A1 was 7.3 nmol P450 (mg protein) −1 , which is somewhat low but within the historical range of 5.6 to 16.8 nmol P450 (mg protein) −1 for purified P450s expressed in E. coli and purified in this laboratory ( 69 , 70 , 71 , 72 , 73 ). The estimated specific contents of the purified E305G, R437H, and R358Q variants were 5.5, 2.3, and 4.2 nmol P450 (mg protein) −1 , respectively.…”

Identification of mtROS-sensitive processes in activated CD4+T cells

“…Therefore, the selectivity of mtROS is dependent on proteins which, by their structure, possess mtROS-sensitive thiolates, ensuring that mtROS only acts on certain redox active protein families. For example, a cysteine which is surrounded with positively charged glutamate residues will stabilize the conjugate thiolate anion, which is then capable of being oxidized (16).…”

Characterization of high-H2O2-tolerant bacterial cytochrome P450 CYP105D18: insights into papaverine N-oxidation

“…These conformational changes alter the solvent accessibility of heme-thiolate Cys488. Once the Cys488 was oxidized to sulfenic acid, the Gln451 forms an NH-bond with Phe441 for closed conformation, limiting the accessibility of oxidizing agents to the protein (Albertolle et al, 2019). Consistent with this, H 2 O 2 -stable CYP105D18 shows high sequential and structural similarity with P450 4B1.…”

“…Previous studies have reported that human P450 1A2 is thiol-insensitive to H 2 O 2 . The molecular dynamics simulation using rabbit P450 4B1 as a model suggested that the Gln451 of P450 4B1 is an essential residue for the thiol sensitivity of P450 (Albertolle et al, 2019). Gln451 in P450 4B1 showed heme thiolate and sulfenic acid-dependent open and closed conformations, respectively.…”