Growing evidence indicates that endogenously produced hydrogen peroxide acts as a cellular signaling molecule that (among other things) can regulate the activity of some protein phosphatases. Recent X-ray crystallographic studies revealed an unexpected chemical transformation underlying the redox regulation of protein tyrosine phosphatase 1B, in which oxidative inactivation of the enzyme yields an intrastrand protein cross-link between the catalytic cysteine residue and its neighboring amide nitrogen. This work describes a small organic molecule that serves as an effective model for the redox-sensing assembly of functional groups at the active site of PTP1B. Findings obtained using this model system suggest that the oxidative transformation of PTP1B to its "crosslinked" inactive form can proceed directly via oxidation of the active-site cysteine to a sulfenic acid (RSOH). The remarkably facile nature of this protein cross-link-forming reaction, along with the widespread cellular occurrence of protein sulfenic acids generated via oxidation of cysteine residues, suggests that the type of oxidative protein cross-link formation first seen in the context of PTP1B represents a potentially general mechanism for redox "switching" of protein function. Thus, the chemistry characterized here could have broad relevance to both redox-regulated signal transduction and the toxic effects of oxidative stress.
CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidation of the cholesterol side-chain terminal methyl group to the alcohol, aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products. The acid arises by protonation of the ferric–peroxo anion species and formation of the ferryl–oxene species, also known as Compound I, followed by hydrogen abstraction and oxygen transfer. The deformylation products arise by addition of the same ferric–peroxo anion to the aldehyde intermediate to give a peroxyhemiacetal that leads to C–C bond cleavage. This bifurcation of the catalytic sequence has allowed us to examine the effect of electron donation by the proximal ligand on the properties of the ferric–peroxo anion. Replacement of the cysteine thiolate iron ligand by a selenocysteine results in UV–vis, EPR, and resonance Raman spectral changes indicative of an increased electron donation from the proximal selenolate ligand to the iron. Analysis of the product distribution in the reaction of the selenocysteine substituted enzyme reveals a gain in the formation of the acid (Compound I pathway) at the expense of deformylation products. These observations are consistent with an increase in the pK a of the ferric–peroxo anion, which favors its protonation and, therefore, Compound I formation.
DosS/DosR is a two-component regulatory system in which DosS, a heme-containing sensor also known as DevS, under certain conditions undergoes autophosphorylation and then transfers the phosphate to DosR, a DNA-binding protein that controls the entry of Mycobacterium tuberculosis and other mycobacteria into a latent, dormant state. DosT, a second sensor closely related to DosS, is present in M. tuberculosis and participates in the control of the dormancy response mediated by DosR. The binding of phosphorylated DosR to DNA initiates the expression of approximately fifty dormancy-linked genes. DosT is accepted to be a gas sensor that is activated in the ferrous state by the absence of an oxygen ligand or by the binding of NO or CO. DosS functions in a similar fashion as a gas sensor, but contradictory evidence has led to the suggestion that it also functions as a redox state sensor. This review focuses on the structure, biophysical properties, and function of the DosS/DosT heme sensors.
Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [ 13 C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.Recently, the dynamic nature of enzymes has drawn much attention (1-3). Protein dynamics are not only important for ligand recognition and binding, but also for bringing catalytic residues in close proximity to the bound substrate so that a reaction can occur (4, 5). It has long been known that conformational flexibility is critical for the recognition of a wide variety of substrates and inhibitors by the human liver drug-metabolizing cytochrome P450 enzymes (6 -9). These enzymes are members of a superfamily of hemoproteins that catalyze oxidative transformations of xenobiotic compounds (10). These "promiscuous" enzymes utilize a conserved mechanism of oxygen activation to oxidize a host of structurally diverse molecules (10). The crystal structures of several human P450 isoforms have recently been obtained, in many cases co-crystallized with known ligands (9,(11)(12)(13). In some cases, ligands have been found bound at some distance from the heme iron or even outside the active site (11,14). These particular structures imply that concerted conformational changes have to take place in the enzyme to position the ligand favorably for oxidation. However, it is not clear how this type of conformational change manifests itself in this important enzyme family. Two competing, albeit not mutually exclusive, theories have emerged to explain how P450s are able to adapt themselves to accommodate such a large number of chemically diverse compounds. The first, a derivative of Koshland's classic induced fit model, relies on substrate binding to induce conformational changes in the enzyme in a stepwise fashion that ultimately advance the ligand into the active site and place it in a productive orientation for oxidation (9, 15-17). The second model, derived from Monod-WymanChangeux allostery theory, ...
Model reactions offer a chemical mechanism by which formation of a sulfenyl amide residue at the active site of the redox-regulated protein tyrosine phosphatase PTP1B protects the cysteine redox switch in this enzyme against irreversible oxidative destruction. The results suggest that "overoxidation" of the sulfenyl amide redox switch to the sulfinyl amide in proteins is a chemically reversible event, because the sulfinyl amide can be easily returned to the native cysteine thiol residue via reactions with cellular thiols.Intracellular concentrations of hydrogen peroxide (H 2 O 2 ) increase under conditions of oxidative stress and during some normal signal transduction processes. [1][2][3] An important mechanism by which cells issue temporary responses to such transitory increases in H 2 O 2 levels involves reversible oxidation of cysteine residues on critical "sensor" proteins.4 ,5 The ability of cysteine residues to serve as reversible redox switches relies upon the unique ability of the γ-sulfur atom in this amino acid to cycle easily between (at least) two oxidation states under physiological conditions. Specifically, oxidation of a cysteine thiol by H 2 O 2 yields a sulfenic acid residue (reaction i, Scheme 1A) that can, over time, be returned to the native thiol by reactions with biological thiols (reaction ii, Scheme 1A). [4][5][6][7][8] Protein sulfenic acid residues also have the potential to undergo further reaction with hydrogen peroxide to generate the corresponding sulfinic acid (reaction iii, Scheme 1A). [4][5][6][7][8][9] This reaction is irreversible, except in the case of some peroxiredoxins 8 and, therefore, yields an overoxidized, "broken" redox switch. Alternatively, in some proteins, the initially-formed sulfenic acid intermediate undergoes reaction with a neighboring "back door" cysteine thiol to generate a disulfide linkage (reaction ii, Scheme 1B). [10][11][12][13] Rudolph and Sohn provided evidence that, at least in the context of the phosphatase Cdc25B, disulfide formation protects the enzyme against irreversible overoxidation. 10,11 There are at least two possible mechanisms underlying this protection. First, the disulfide may be relatively resistant to further oxidation (reaction iii, Scheme 1B). 10,11 Second, if "overoxidation" does occur, the resulting thiosulfinate likely could be converted cleanly back to the native cysteine residues by reactions with biological thiols (reaction v, Scheme 1B). 14 © 2009 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +1-573-882-6763; fax: +1-573-882-2754 gatesk@missouri.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaim...
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