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Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration.

ABSTRACT: The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. et al. (2017) J. Biol. Chem. 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).

SUBMITTER: Albertolle ME

PROVIDER: S-EPMC5930400 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration.

Albertolle Matthew E ME Phan Thanh T N TTN Pozzi Ambra A Guengerich F Peter FP

Molecular & cellular proteomics : MCP 20180126 5

The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes vi ...[more]

PMID: 29374135

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Project description:This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O2 O-O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe2S2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam. P450cam is specific for its own Fe2S2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam-Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surrounding residues play in catalysis. Here we summarize these recent results which provide a much more dynamic picture of P450 catalysis.

Dataset Information

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration.

Publications

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration.

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