Catalytic Center Of Enzyme Research Articles

Resveratrol Inhibits Nucleosome Binding and Catalytic Activity of PARP1

The natural polyphenol resveratrol is a biologically active compound that interacts with DNA and affects the activity of some nuclear enzymes. Its effect on the interaction between nucleosomes and poly(ADP-ribose) polymerase-1 (PARP1) and on the catalytic activity of PARP1 was studied using Western blotting, spectrophotometry, electrophoretic mobility shift assay, and single particle Förster resonance energy transfer microscopy. Resveratrol inhibited PARP1 activity at micro- and sub-micromolar concentrations, but the inhibitory effect decreased at higher concentrations due to the aggregation of the polyphenol. The inhibition of PARP1 by resveratrol was accompanied by its binding to the enzyme catalytic center and a subsequent decrease in PARP1 affinity to nucleosomal DNA. Concurrent binding of talazoparib to the substrate binding pocket of PARP1, which occurs in the presence of resveratrol, restores the interaction of PARP1 with nucleosomes, suggesting that the binding sites of resveratrol and talazoparib overlap. The data suggest that resveratrol can be classified as a natural inhibitor of PARP1.

Genetically encoded Nδ-vinyl histidine for the evolution of enzyme catalytic center

Genetic code expansion has emerged as a powerful tool for precisely introducing unnatural chemical structures into proteins to improve their catalytic functions. Given the high catalytic propensity of histidine in the enzyme pocket, increasing the chemical diversity of catalytic histidine could result in new characteristics of biocatalysts. Herein, we report the genetically encoded Nδ-Vinyl Histidine (δVin-H) and achieve the wild-type-like incorporation efficiency by the evolution of pyrrolysyl tRNA synthetase. As histidine usually acts as the nucleophile or the metal ligand in the catalytic center, we replace these two types of catalytic histidine to δVin-H to improve the performance of the histidine-involved catalytic center. Additionally, we further demonstrate the improvements of the hydrolysis activity of a previously reported organocatalytic esterase (the OE1.3 variant) in the acidic condition and myoglobin (Mb) catalyzed carbene transfer reactions under the aerobic condition. As histidine is one of the most frequently used residues in the enzyme catalytic center, the derivatization of the catalytic histidine by δVin-H holds a great potential to promote the performance of biocatalysts.

Mechanistic insights into the inhibition of human placental glutathione S-transferase P1-1 by abscisic and gibberellic acids: An integrated experimental and computational study.

The interactions of the classic phytohormones gibberellic acid (gibberellin A3 , GA3 ) and abscisic acid (dormin, ABA), which antagonistically regulate several developmental processes and stress responses in higher plants, with human placental glutathione S-transferase P1-1 (hpGSTP1-1), an enzyme that plays a role in endo- or xenobiotic detoxification and regulation of cell survival and apoptosis, were investigated. The inhibitory potencies of ABA and GA3 against hpGSTP1, as well as the types of inhibition and the kinetic parameters, were determined by making use of both enzyme kinetic graphs and SPSS nonlinear regression models. The structural basis for the interaction between hpGSTP1-1 and phytohormones was predicted with the aid of molecular docking simulations. The IC50 values of ABA and GA3 were 5.3 and 5.0 mM, respectively. Both phytohormones inhibited hpGSTP1-1 in competitive manner with respect to the cosubstrates GSH and CDNB. When ABA was the inhibitor at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v , Vm , Km , and Ki values were statistically estimated to be 205 ± 16 μmol/min-mg protein, 1.32 ± 0.18 mM, 1.95 ± 0.25 mM and 175 ± 6 μmol/min-mg protein, 0.85 ± 0.06 mM, 1.85 ± 0.16 mM, respectively. On the other hand, the kinetic parameters Vm , Km , and Ki obtained with GA3 at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v were found to be 303 ± 14 μmol/min-mg protein, 1.77 ± 0.13 mM, 3.38 ± 0.26 mM and 249 ± 7 μmol/min-mg protein, 1.43 ± 0.07 mM, 2.89 ± 0.19 mM, respectively. Both phytohormones had the potential to engage in hydrogen-bonding and electrostatic interactions with the key residues that line the G- and H-sites of the enzyme's catalytic center. Inhibitory actions of ABA/GA3 on hpGSTP1-1 may guide medicinal chemists through the structure-based design of novel antineoplastic agents. It should be noted, however, that the same interactions may also render fetuses vulnerable to the potentially toxic effects of xenobiotics and noxious endobiotics.

Apocarotenoids are allosteric effectors of a dimeric plant glycosyltransferase involved in defense and lignin formation.

Glycosyltransferases are nature's versatile tools to tailor the functionalities of proteins, carbohydrates, lipids, and small molecules by transferring sugars. Prominent substrates are hydroxycoumarins such as scopoletin, which serve as natural plant protection agents. Similarly, C13-apocarotenoids, which are oxidative degradation products of carotenoids/xanthophylls, protect plants by repelling pests and attracting pest predators. We show that C13-apocarotenoids interact with the plant glycosyltransferase NbUGT72AY1 and induce conformational changes in the enzyme catalytic center ultimately reducing its inherent UDP-α-d-glucose glucohydrolase activity and increasing its catalytic activity for productive hydroxycoumarin substrates. By contrast, C13-apocarotenoids show no effect on the catalytic activity toward monolignol lignin precursors, which are competitive substrates. In vivo studies in tobacco plants (Nicotiana benthamiana) confirmed increased glycosylation activity upon apocarotenoid supplementation. Thus, hydroxycoumarins and apocarotenoids represent specialized damage-associated molecular patterns, as they each provide precise information about the plant compartments damaged by pathogen attack. The molecular basis for the C13-apocarotenoid-mediated interplay of two plant protective mechanisms and their function as allosteric enhancers opens up potential applications of the natural products in agriculture and pharmaceutical industry.

Multi-scale QM/QM/MM molecular dynamics through quantum embedding.

Physical and chemical processes in bio-molecular systems typically span a wide range of time and space scales. These coupled processes at various scales must be taken into account concurrently in simulations depending on the problems of interest, yet they usually do not need to be depicted with the same accuracy. Large-scale protein conformational changes, for example, can be represented by carefully calibrated force fields, whereas chemical reactions at an enzyme's catalytic center necessitate a quantum mechanical approach. However, the number of atoms involved in the reaction center may still be quite large, limiting the applicability of some high-accuracy quantum chemistry methods, but the challenging part of the electronic structure may be even more local. A transition metal in enzymes, for example, may require some high-level multi-reference approaches, but a low-level mean-field description, such as the density functional theory, may be sufficient for metal ligands, serving as its environments. In this study, we used quantum embedding theory to rigorously partition a quantum system into multiple fragments, each of which could be solved using high-level quantum chemistry methods, allowing us to develop a multi-scale QM(high-level)/QM(low-level)/MM approach to achieve varying levels of accuracy in different scaled phenomenon.

Revealing Escherichia coli type II l-asparaginase active site flexible loop in its open, ligand-free conformation

Since 1993, when the structure of Escherichia coli type II l-asparaginase (EcAII) in complex with l-aspartate was firstly reported, many structures of the wild type and mutated enzyme have been deposited in the Protein Data Bank. None of them report the full structure of the monomer in its ligand-free, open conformation, mainly because of the high dynamic and flexibility of the active site flexible loop. Here we report for the first time the structure of EcAII wild type in its open conformation comprising, for at least one protomer, clear electron density for the active site flexible loop (PDB ID: 6YZI). The structural element is highly mobile and it is transposed onto the rigid part of the active site upon substrate binding to allow completion of the enzyme catalytic center, thanks to key residues that serve as hinges and anchoring points. In the substrate binding pocket, several highly conserved water molecules are coordinated by residues involved in substrate binding, comprising two water molecules very likely involved in the enzyme catalytic process. We also describe, by molecular dynamics simulations, how the transposition of the loop, besides providing the proximity of residues needed for catalysis, causes a general stabilization of the protein.

Structural Evidence of Active Site Adaptability towards Different Sized Substrates of Aromatic Amino Acid Aminotransferase from Psychrobacter Sp. B6.

Aromatic amino acid aminotransferases present a special potential in the production of drugs and synthons, thanks to their ability to accommodate a wider range of substrates in their active site, in contrast to aliphatic amino acid aminotransferases. The mechanism of active site adjustment toward substrates of psychrophilic aromatic amino acid aminotransferase (PsyArAT) from Psychrobacter sp. B6 is discussed based on crystal structures of complexes with four hydroxy-analogs of substrates: phenylalanine, tyrosine, tryptophan and aspartic acid. These competitive inhibitors are bound in the active center of PsyArAT but do not undergo transamination reaction, which makes them an outstanding tool for examination of the enzyme catalytic center. The use of hydroxy-acids enabled insight into substrate binding by native PsyArAT, without mutating the catalytic lysine and modifying cofactor interactions. Thus, the binding mode of substrates and the resulting analysis of the volume of the catalytic site is close to a native condition. Observation of these inhibitors’ binding allows for explanation of the enzyme’s adaptability to process various sizes of substrates and to gain knowledge about its potential biotechnological application. Depending on the character and size of the used inhibitors, the enzyme crystallized in different space groups and showed conformational changes of the active site upon ligand binding.

Processing of the abasic sites clustered with the benzo[a]pyrene adducts by the base excision repair enzymes

The major enzyme in eukaryotic cells that catalyzes the cleavage of apurinic/apyrimidinic (AP or abasic) sites is AP endonuclease 1 (APE1) that cleaves the phosphodiester bond on the 5′-side of AP sites. We found that the efficiency of AP site cleavage by APE1 was affected by the benzo[a]pyrenyl-DNA adduct (BPDE-dG) in the opposite strand. AP sites directly opposite of the modified dG or shifted toward the 5′ direction were hydrolyzed by APE1 with an efficiency moderately lower than the AP site in the control DNA duplex, whereas AP sites shifted toward the 3′ direction were hydrolyzed significantly less efficiently. For all DNA structures except DNA with the AP site shifted by 3 nucleotides in the 3′ direction (AP+3-BP-DNA), hydrolysis was more efficient in the case of (+)-trans-BPDE-dG. Using molecular dynamic simulation, we have shown that in the complex of APE1 with the AP+3-BP-DNA, the BP residue is located within the DNA bend induced by APE1 and contacts the amino acids in the enzyme catalytic center and the catalytic metal ion. The geometry of the APE1 active site is perturbed more significantly by the trans-isomer of BPDE-dG that intercalates into the APE1-DNA complex near the cleaved phosphodiester bond. The ability of DNA polymerases β (Polβ), λ and ι to catalyze gap-filling synthesis in cooperation with APE1 was also analyzed. Polβ was shown to inhibit the 3′→5′ exonuclease activity of APE1 when both enzymes were added simultaneously and to insert the correct nucleotide into the gap arising after AP site hydrolysis. Therefore, further evidence for the functional cooperation of APE1 and Polβ in base excision repair was obtained.

Transformation of triclosan by laccase catalyzed oxidation: The influence of humic acid-metal binding process

Laccase is a widely present extracellular phenoloxidase excreted by fungi, bacteria, and high plants. It is able to catalyze one-electron oxidation of phenolic compounds into radical intermediates that can subsequently couple to each other via covalent bonds. These reactions are believed to play an important role in humification process and the transformation of contaminants containing phenolic functionalities in the environment. In this study, we investigated the kinetics of triclosan transformation catalyzed by laccase. It was found that the rate of triclosan oxidation was first order to the concentrations of both substrate and enzyme. Humic acid (HA) could inhibit the reaction by quenching the radical intermediate of triclosan generated by laccase oxidation. Such inhibition was more significant in the presence of divalent metal cations. This is because that binding to metal ions neutralized the negative charge of HA molecules, thus making them more accessible to laccase molecule that is also negatively charged. Therefore, it has greater chance to quench the radical intermediate that is very unstable and can only diffuse a limited distance after being released from the enzyme catalytic center. Based on these understandings, a reaction model was developed by integration of metal-HA binding equilibriums and kinetic equations. This model precisely predicted the transformation rate of triclosan in the presence of HA and divalent metal ions including Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ba2+, and Zn2+. Overall, this work reveals important insights into laccase catalyzed oxidative coupling process.

2-(4-Fluorophenyl)-quinazolin-4(3H)-one as a novel tyrosinase inhibitor: Synthesis, inhibitory activity, and mechanism

2-(4-Fluorophenyl)-quinazolin-4(3H)-one (FQ) was synthesized, and its structure was identified with 1H nuclear magnetic resonance (1H NMR), 13C nuclear magnetic resonance (13C NMR), fourier transform infrared spectroscopy (FTIR), and high resolution mass spectrometry (HRMS). From the enzyme analysis, the results showed that it could inhibit the diphenolase activity of tyrosinase (IC50=120±2μM). Furthermore, the results of kinetic studies showed that the compound was a reversible mixed-type inhibitor, and that the inhibition constants were determined to be 703.2 (KI) and 222.1μM (KIS). The results of fluorescence quenching experiment showed that the compound could interact with tyrosinase and the substrates (tyrosine and l-DOPA). Molecular docking analysis revealed that the mass transfer rate was affected by FQ blocking the enzyme catalytic center. In brief, current study identified a novel tyrosinase inhibitor which deserved further study for hyperpigmentation drugs.

Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket

Phospholipase D (PLD) has been implicated in many physiological functions, such as chemotaxis and phagocytosis, as well as pathological functions, such as cancer cell invasion and metastasis. New inhibitors have been described that hamper the role of PLD in those pathologies but their site of action is not known. We have characterized the biochemical and biological behavior of the PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), and the specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,­8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT), and found that both FIPI and NFOT are mixed-kinetics inhibitors. Mutagenesis studies indicate that FIPI binds at S757 of PLD2, which is within the HKD2 catalytic site of the enzyme, whereas NFOT binds to PLD2 at two different sites, one being at S757/S648 and another to an allosteric site that is a natural site occupied by PIP2 (R210/R212). This latter site, along with F244/L245/L246, forms a hydrophobic pocket in the PH domain. The mechanism of action of FIPI is a direct effect on the catalytic site (and as such inhibits both PLD1 and PLD2 isoforms), whereas PLD2 affects both the catalytic site (orthosteric) and blocks PIP2 binding to PLD2 (allosteric), which negates the natural enhancing role of PIP2. Moreover, NFOT prevents cell invasion of cancer cells, which does not occur in cells overexpressing PLD2-F244A/L245A/L246A, or PLD2-R210A/R212A, or PLD2-S757/S648 mutants. This study provides new specific knowledge of enzyme regulation and mechanisms of activation and inhibition of PLD2 that are necessary to understand its role in cell signaling and to develop new inhibitors for cancer cell invasion and metastasis.

Electropolymerized carbonic anhydrase immobilization for carbon dioxide capture.

Biomimetic carbonation carried out with carbonic anhydrase (CA) in CO2-absorbing solutions, such as methyldiethanolamine (MDEA), is one approach that has been developed to accelerate the capture of CO2. However, there are several practical issues, such as high cost and limited enzyme stability, that need to be overcome. In this study, the capacity of CA immobilization on a porous solid support was studied to improve the instability in the tertiary amine solvent. We have shown that a 63% porosity macroporous carbon foam support makes separation and reuse facile and allows for an efficient supply and presentation of CO2 to an aqueous solvent and the enzyme catalytic center. These enzymatic supports conserved 40% of their initial activity after 42 days at 70 °C in an amine solvent, whereas the free enzyme shows no activity after 1 h in the same conditions. In this work, we have overcome the technical barrier associated with the recovery of the biocatalyst after operation, and most of all, these electropolymerized enzymatic supports have shown a remarkable increase of thermal stability in an amine-based CO2 sequestration solvent.

Hierarchical Stable Enzyme Microenvironments for High‐Temperature Stability in Amine Solvents

Several methods have been proposed for capturing the CO2 emitted into the atmosphere by human activity. To date, mainly amine‐based absorption processes are currently among the more promising systems for post‐combustion CO2 capture. Tertiary amine solvents obviate the need for a high solvent regeneration temperature and fast absorption can be achieved with the use of carbonic anhydrase (CA), as an activator. In this study, the capacity of CA immobilization on nanoporous microparticles hierarchically structured to enhance their stability in tertiary amines is investigated. These microstructures allow for an efficient supply and presentation of substrate in the non‐aqueous solvent to the enzyme catalytic center and the particles' large size is attractive to make separation and reuse facile. These hierarchically structured particles conserve 70% of their initial activity after 30 d at 50 °C in amine solvent, whereas the free enzyme shows no activity after 1 h in the same conditions. In this work, we have overcome the technical hurdle linked to the recovery of the biocatalyst after operation thereby reducing costs of the system and importantly these micro‐bioparticles have shown a remarkable increase of the thermal stability of CA in an amine‐based CO2 sequestration solvent as determined by a para‐nitrophenyl acetate assay.

The Mechanism of Citryl-Coenzyme A Formation Catalyzed by Citrate Synthase

The enzyme citrate synthase is used by all living cells to catalyze the first step of the citric acid cycle. In this work, we have investigated the enolization and condensation steps catalyzed by citrate synthase, using ab initio (B3LYP/def2-TZVP and MP2/aug-cc-pVDZ) quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. The results of the latter indicate that the catalytic His238 residue is in its neutral form, and also argue strongly for the presence of a water molecule in the enzyme's catalytic center. Such a water is observed in some, but not all, of the experimentally resolved structures of the protein. The mechanism itself starts with an enolization that proceeds via an enolate intermediate rather than the enol form, which is much more unstable. This is in agreement with the results of other workers. For the condensation step, we investigated two mechanisms in which there is a direct nucleophilic attack of the enolate intermediate on the oxaloacetate carbonyl carbon, and found the one in which there is no proton transfer from the neighboring arginine to be preferred. Although this residue, Arg329, is not implicated directly in the reaction, it helps to stabilize the negative citryl-CoA formed during the condensation step.

Mechanisms of copper homeostasis in bacteria

Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu+ transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of these components, their irregular presence in different organisms, functional interactions, and resulting system architectures.

Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1

ERAP1 trims antigen precursors to fit into MHC class I proteins. To perform this function, ERAP1 has unique substrate preferences, trimming long peptides while sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analysis suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long but not short substrates induces a conformational change with reorientation of a key catalytic residue towards the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.

Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1

ERAP1 trims antigen precursors to fit into MHC class I proteins. To perform this function, ERAP1 has unique substrate preferences, trimming long peptides while sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X‐ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analysis suggest a mechanism for ERAP1's length‐dependent trimming activity, whereby binding of long but not short substrates induces a conformational change with reorientation of a key catalytic residue towards the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.

Designing small-molecule catalysts for CO2 capture

One method for CO2 capture is to dissolve CO2 in water to form carbonic acid. This reaction (CO2+H20→H2CO3(aq)) is remarkably slow but is catalyzed in biological systems by an enzyme called carbonic anhydrase (CA). The catalyzed reaction is diffusion limited and occurs at near neutral pH. The enzyme catalytic center is composed of a Zn(II) ion that is coordinated by 3 histidine residues and an axial water/hydroxyl group. A nucleophilic attack by the hydroxyl group on the CO2 molecule is the first step in the reaction mechanism. Cu(II), Hg(II), Cd(II), Ni(II), Co(II) and Mn(II) can bind the CA binding site and substitute the zinc ion; however, only Co(II) yields rates comparable to Zn(II). Unfortunately, an enzyme, such as carbonic anhydrase, is not amenable for industrial applications where a wide range of physico-chemical conditions exist. Enzymes are vulnerable to large pressures, high temperature, and high ionic strength. Efforts to isolate the key structural features responsible for catalysis led to the development of small-molecule mimetics of the CA catalytic site. These mimetics can, in turn, be used as catalyst for CO2 sequestration. Two of the fastest catalyst are the cyclic molecules: 1, 4, 7, 10-tetraazacyclododedacane and 1, 5, 9-triazacyclododedacane (both complexed with a Zn(II) ion). Nitrogen atoms in these cyclic molecules mimic the imidazole nitrogens of the CA active site. It is possible to examine the energetics of these compounds using transition state theory for the purposes of designing more efficient catalysts. Transition state theory predicts that the reaction rate constant is proportional to exp(−Ea/kT), where Ea denotes the activation energy, k the Boltzmann constant, and T the temperature of the reaction. The activation energy is the energetic cost of forming the reaction transition state from the reactants. Ab initio calculations can yield the activation energy, which can, in principle, be used as a design metric for more efficient catalysts. Using this approach, the difference in kinetic rate constant between the tetra- and tri-aza dodecane catalysts can be determined. Furthermore, the rates of the corresponding Co(II) catalysts were explored. Our data suggests this is a viable method for the design of inorganic small-molecule catalysts.

Requirement of glutathione for Sod1 activation during lifespan extension

It has been shown that the activation of cytosolic superoxide dismutase (Sod1) in Saccharomyces cerevisiae is only dependent on Ccs1, which is responsible for insertion of copper into the enzyme catalytic center, and that glutathione (GSH) is not necessary for this process. In this work, we addressed an important role of GSH in Sod1 activation by a Ccs1-dependent mechanism during oxidative stress and its role in yeast lifespan. Exponential cells of Saccharomyces cerevisiae, treated or not with 0.5 mM menadione for 1 h, were used for evaluation of the effect of a mild oxidative stress pre-treatment on chronological lifespan. The results showed that menadione induced a lifespan extension in the wild-type (WT) strain but this adaptive response was repressed in gsh1 and in sod1 strains. Interestingly, menadione treatment increased SOD1 and CCS1 gene expression in both WT and gsh1 strains. However, while these strains showed the same Sod1 activity before treatment, only the WT presented an increase of Sod1 activity after menadione exposure. Glutathionylation seems to be essential for Sod1 activation since no increase in activity was observed after menadione treatment in grx1 and grx2 null mutants. Our results suggest that GSH and glutathionylation are fundamental to protect Sod1 sulfhydryl residues under mild oxidative stress, enabling Sod1 activation and lifespan extension.

New p22-Phox Monoclonal Antibodies: Identification of a Conformational Probe for Cytochrome b558

The phagocyte NADPH oxidase, belonging to the NADPH oxidase family (Nox), is dedicated to the production of bactericidal reactive oxygen species. The enzyme catalytic center is the cytochrome b₅₅₈, formed by 2 subunits, Nox2 (gp91-phox) and p22-phox. Cytochrome b₅₅₈ activation results from a conformational change induced by cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac). The catalytic subunit is Nox2, while p22-phox is essential for both Nox2 maturation and the membrane anchorage of regulatory proteins. Moreover, it has been shown to be necessary for novel Nox activity. In order to characterize both p22-phox topology and cytochrome b₅₅₈ conformational change, 6 monoclonal antibodies were produced against purified cytochrome b₅₅₈. Phage display epitope mapping combined with a truncation analysis of recombinant p22-phox allowed the identification of epitope regions. Some of these antibodies almost completely inhibited in vitro reconstituted NADPH oxidase activity. Data analysis identified antibodies that recognized epitopes involved in either Nox2 maturation or Nox2 activation. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils showed that the monoclonal antibody 12E6 bound preferentially active cytochrome b₅₅₈. These monoclonal antibodies provided novel and unique probes to investigate maturation, activation and activity, not only of Nox2 but also of novel Nox.