LC-AMP-F1 Derived from the Venom of the Wolf Spider Lycosa coelestis, Exhibits Antimicrobial and Antibiofilm Activities.

In recent years, there has been a growing interest in antimicrobial peptides as innovative antimicrobial agents for combating drug-resistant bacterial infections, particularly in the fields of biofilm control and eradication. In the present study, a novel cationic antimicrobial peptide, named LC-AMP-F1, was derived from the cDNA library of the Lycosa coelestis venom gland. The sequence, physicochemical properties and secondary structure of LC-AMP-F1 were predicted and studied. LC-AMP-F1 was tested for stability, cytotoxicity, drug resistance, antibacterial activity, and antibiofilm activity in vitro compared with melittin, a well-studied antimicrobial peptide. The findings indicated that LC-AMP-F1 exhibited inhibitory effects on the growth of various bacteria, including five strains of multidrug-resistant bacteria commonly found in clinical settings. Additionally, LC-AMP-F1 demonstrated effective inhibition of biofilm formation and disruption of mature biofilms. Furthermore, LC-AMP-F1 exhibited favorable stability, minimal hemolytic activity, and low toxicity towards different types of eukaryotic cells. Also, it was found that the combination of LC-AMP-F1 with conventional antibiotics exhibited either synergistic or additive therapeutic benefits. Concerning the antibacterial mechanism, scanning electron microscopy and SYTOX Green staining results showed that LC-AMP-F1 increased cell membrane permeability and swiftly disrupted bacterial cell membranes to exert its antibacterial effects. In summary, the findings and studies facilitated the development and clinical application of novel antimicrobial agents.

Facile synthesis of L10-PtFe/C intermetallic catalysts with superior catalytic durability for the oxygen reduction reaction.

An o-PtFe/C intermetallic catalyst was prepared by a facile thermal reduction method with the average particle size of only 6.6 nm in the presence of urea. The loss of mass activity is only 25.9% after 50 000 cycles. This work provides guidance on the suppression of grain coarsening for high-temperature synthesis of Pt-based intermetallic catalysts.

Enhanced Oxygen Reduction Reaction Performance by Adsorbed Water on Edge Sites.

Pt-based alloy nanoparticles have broad application prospects as cathode catalyst materials for proton-exchange membrane fuel cells (PEMFCs). Optimization of the oxygen adsorption energy is crucial to boost the performance of oxygen reduction catalysis. We successfully synthesized well-dispersed Pt1.2Ni tetrahedra and obtained the Pt1.2Ni/C catalyst adopting the one-pot synthetic protocol, which exhibits superb activity and good long-term stability for oxygen reduction reaction (ORR), achieving a mass activity of 1.53 A/mgPt at 0.90 VRHE, which is 12 times higher than that of commercial Pt/C. On combining X-ray photoelectron spectroscopy and density functional theory calculations, abundant water is adsorbed stably on the Pt1.2Ni alloy surface. We find that the intense interaction between the adsorbed O atom and adsorbed water can weaken the adsorption of oxygen, contributing to the ORR performance.

Microfibril-Associated Glycoprotein-2 Promoted Fracture Healing via Integrin αvß3/PTK2/AKT Signaling.

Fracture healing is a complex physiological process in which angiogenesis plays an essential role. Microfibril-associated glycoprotein-2 (MAGP2) has been reported to possess a proangiogenic activity via integrin αvß3, yet its role in bone repair is unexplored. In this study, a critical-sized femoral defect (2 mm) was created in mice, followed by the delivery of an adenovirus-based MAGP2 overexpression vector or its negative control at the fracture site. At days 7, 14, 21, and 28 postfracture, bone fracture healing was evaluated by radiography, micro-computed tomography, and histopathologic analysis. Adenovirus-based MAGP2 overexpression vector-treated mice exhibited increased bone mineral density and bone volume fraction. MAGP2 overexpression contributed to an advanced stage of endochondral ossification and induced cartilage callus into the bony callus. Further analysis indicated that MAGP2 was associated with enhanced angiogenesis, as evidenced by marked MAGP2 and integrin αvß3 costaining and increased endothelial cell markers such as endomucin and CD31 levls, as well as elevated phosphorylation of protein tyrosine kinase 2 (PTK2) and AKT serine/threonine kinase 1 (AKT) in the callus. In vitro, recombinant human MAGP2 treatment enhanced the viability, migration, and tube formation ability of human microvascular endothelial cells, which was partially reversed by integrin αvß3 inhibition or MK-2206, a specific AKT inhibitor. Inhibition of integrin αvß3 abolished MAGP2-induced PTK2 and AKT activation. Taken together, our data provide the first evidence that MAGP2 promotes angiogenesis and bone formation by activating the integrin αvß3/PTK2/AKT signaling pathway.

Gas Diffusion Layer with a Regular Hydrophilic Structure Boosts the Power Density of Proton Exchange Membrane Fuel Cells via the Construction of Water Highways.

The gas diffusion layer (GDL) is an essential carrier for the mass transmission of proton exchange membrane fuel cells (PEMFCs), which decides the peak power density of PEMFCs. Herein, a gas diffusion layer with a regularly arranged hydrophilic and hydrophobic pattern structure was prepared by a template method combined with the ultrasonic spray process. The peak power density was enhanced by 30% (from 520 to 678 mW/cm2) compared to an unpatterned structure, and the breakthrough pressure of the GDL was reduced from 13.61 to 2.96 kPa. In addition, the finite element analysis (FEA) results indicate that the polarization curve of calculation was highly consistent with the experimental results. Importantly, the capillary pressure of the hydrophilic area was about 0.3 kPa, much lower than that of the hydrophobic area (2 kPa), demonstrating that the hydrophilic and hydrophobic synergistic structure reduced the water transmission resistance in separating water and oxygen and builds a high-speed channel for water transmission.

Left ventricular dysfunction and reversible pulmonary hypertension secondary to severe pneumonia in a background of sepsis: a case report and review of the literature.

Pneumonia is a well-recognized respiratory infection associated with substantial morbidity and mortality. Despite its effects on the respiratory system, pneumonia can cause or exacerbate cardiovascular complications through various mechanisms. The two main mechanisms that are described in this case report are hypoxia-induced pulmonary hypertension and the effect of sepsis on the cardiovascular system. Pulmonary hypertension (PH) is a disease characterized by raised pulmonary arterial pressure due to a progressive increase in pulmonary vascular resistance, inevitably leading to right ventricular (RV) afterload. For our case, the situation was complicated by sepsis, which further worsened the myocardial function causing left ventricular hypertrophy and left ventricular dysfunction. The main goal of this case report is to highlight the fact that cardiovascular events due to pneumonia are a potential complication even in young patients who are without any comorbidities. We present a case of a 14-year-old patient who presented with symptoms of cough, hemoptysis, fever, chest pain, and dyspnea. After the necessary investigations, he was diagnosed with severe pneumonia, sepsis, moderate PH, and left ventricular dysfunction. The treatment course was focused on stabilizing the patient by oxygen supplementation, treating the underlying cause with the use of antibiotics, and decreasing the already raised arterial pressures through vasodilator therapy. After the patient went through the proper course of treatment, there was a marked improvement in his general condition.Cardiac complications due to pneumonia are potential complications even in relatively young patients who have no noted comorbidities. Clinicians should be aware of these potentially fatal complications of pneumonia and appreciate the significance of this association for timely recognition, diagnosis, and management of these complications.

Network pharmacology-based analysis of the role of traditional Chinese herbal medicines in the treatment of COVID-19.

BACKGROUND: The novel coronavirus named COVID-19, which originated in Wuhan, China, has spread to many countries around the world. Currently, no effective medical treatment exists to combat this disease. Traditional Chinese herbal medicines (CHM) have unique roles in the treatment of viral infections. In this article we analyzed the effectiveness and possible molecular mechanisms of CHM formulas for the prevention of COVID-19. METHODS: The active ingredients and action targets of CHM formulas were obtained from the TCMSP database. Genes related to severe acute respiratory syndromes (SARS) and Middle East respiratory syndrome (MERS) were queried on the GeneCards database. The action mechanisms of these genes were predicted using a Gene Ontology (GO)-based functional enrichment and annotation tool and the Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: CHM formulas played a positive role in preventing COVID-19 and warrant further application. CONCLUSIONS: Our research provides new evidence to support the possible value of CHM formulas for the prevention of COVID-19. However, further clinical studies with large sample sizes are required to verify their effectiveness.

A general carbon monoxide-assisted strategy for synthesizing one-nanometer-thick Pt-based nanowires as effective electrocatalysts.

To balance the Pt utilization and the durability is the key issue for developing Pt-based oxygen reduction reaction (ORR) catalysts, and constructing ultrathin one-dimensional (1D) structure provides a practical solution. Here, a facile CO-assisted strategy has been proposed for synthesizing PtFe nanowires (NWs) with an ultrathin diameter of one-nanometer and high aspect ratio for the first time, which demonstrates great universality and can be extended to a ternary system. The NWs are found to grow following an oriented attachment mechanism facilitated by the preferential adsorption and reducibility of CO. Based on composition regulation, PtFe NWs and PtFeCo NWs exhibit superior catalytic performance, of which the electrochemical active surface areas are extremely high, achieving 1.5 folds of that of Pt/C catalyst. Benefiting from the synergistic effect endowed by alloying and the ultrathin anisotropic structure, PtFe NWs and PtFeCo NWs show remarkable mass activity of 0.57 and 0.58 A mg-1Pt, respectively, and the durability also meet the 2020 standard of DOE, holding great application potential.

Locally-ordered PtNiPb ternary nano-pompons as efficient bifunctional oxygen reduction and methanol oxidation catalysts.

To accurately control the composition and structure of Pt-based alloys is essential for engineering highly active and stable catalysts, yet challenging. Here, ternary PtNiPb nano pompons (NPs) combining the features of a highly open structure, local-ordering and the introduction of Pb have been synthesized via a seed-mediated growth method. Taking advantage of the reduction potential differences, gradient distribution of Pb and Ni throughout the NPs is realized, and both the elemental composition and distribution can be facilely regulated by reaction time. The PtNiPb NPs exhibit much enhanced catalytic performance for both the oxygen reduction reaction (ORR) and methanol oxidation reaction (MOR), of which the ORR specific activity is 7.4- and 2.3-fold larger than those of the commercial Pt/C catalyst and binary PtNi NPs. Density functional theory (DFT) calculations reveal that the weak coupling between Pb-p and Pt-d orbitals together with the regulation of the over-compressed Pt surface by the local-ordering structure and embedded Pb atoms optimize the surface oxygen adsorption character and eventually boost the ORR.

A radical on the Met-Tyr-Trp modification required for catalase activity in catalase-peroxidase is established by isotopic labeling and site-directed mutagenesis.

A transient tyrosyl-like radical with a narrow doublet X-band EPR signal is present during catalase turnover by Mycobacterium tuberculosis catalase-peroxidase (KatG). Labeling of KatG with beta-methylene-deuterated tyrosine causes a collapse of the doublet to a singlet, while for 3,5-ring-deuterated tyrosine-labeled enzyme, no changes occur in the EPR signal. Except for the replacement Tyr229Phe, all other single-tyrosine mutants of KatG exhibit the same narrow doublet EPR signal and catalase activity similar to that of the wild-type enzyme. These findings confirm that this catalytically competent radical is associated with Tyr229, whose 3' and 5' protons are replaced as a result of cross-links with neighboring Met255 and Trp107 side chains in the post-translationally modified enzyme containing a distal-side Met255-Tyr229-Trp107 adduct.

Role of the oxyferrous heme intermediate and distal side adduct radical in the catalase activity of Mycobacterium tuberculosis KatG revealed by the W107F mutant.

Catalase-peroxidase (KatG) is essential in Mycobacterium tuberculosis for oxidative stress management and activation of the antitubercular pro-drug isoniazid. The role of a unique distal side adduct found in KatG enzymes, involving linked side chains of residues Met255, Tyr229, and Trp107 (MYW), in the unusual catalase activity of KatG is addressed here and in our companion paper (Suarez, J., Ranguelova, K., Jarzecki, A. A., Manzerova, J., Krymov, V., Zhao, X., Yu, S., Metlitsky, L., Gerfen, G. J., and Magliozzo, R. S. (2009) J. Biol. Chem. 284, in press). The KatG[W107F] mutant exhibited severely reduced catalase activity yet normal peroxidase activity, and as isolated contains more abundant 6-coordinate heme in high spin and low spin forms compared with the wild-type enzyme. Most interestingly, oxyferrous heme is also found in the purified enzyme. Oxyferrous KatG[W107F] was prepared by photolysis in air of the carbonyl enzyme or was generated using hydrogen peroxide decayed with a t1/2 of 2 days compared with 6 min for wild-type protein. The stability of oxyenyzme was modestly enhanced in KatG[Y229F] but was not affected in KatG[M255A]. Optical stopped-flow experiments showed rapid formation of Compound I in KatG[W107F] and facile formation of oxyferrous heme in the presence of micromolar hydrogen peroxide. An analysis of the relationships between catalase activity, stability of oxyferrous enzyme, and a proposed MYW adduct radical is presented. The loss of catalase function is assigned to the loss of the MYW adduct radical and structural changes that lead to greatly enhanced stability of oxyenzyme, an intermediate of the catalase cycle of native enzyme.

An oxyferrous heme/protein-based radical intermediate is catalytically competent in the catalase reaction of Mycobacterium tuberculosis catalase-peroxidase (KatG).

A mechanism accounting for the robust catalase activity in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. In Mycobacterium tuberculosis, KatG is the sole catalase and is also responsible for peroxidative activation of isoniazid, an anti-tuberculosis pro-drug. Here, optical stopped-flow spectrophotometry, rapid freeze-quench EPR spectroscopy both at the X-band and at the D-band, and mutagenesis are used to identify catalase reaction intermediates in M. tuberculosis KatG. In the presence of millimolar H2O2 at neutral pH, oxyferrous heme is formed within milliseconds from ferric (resting) KatG, whereas at pH 8.5, low spin ferric heme is formed. Using rapid freeze-quench EPR at X-band under both of these conditions, a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical and the unique heme intermediates persist in wild-type KatG only during the time course of turnover of excess H2O2 (1000-fold or more). Mutation of Met255, Tyr229, or Trp107, which have covalently linked side chains in a unique distal side adduct (MYW) in wild-type KatG, abolishes this radical and the catalase activity. The D-band EPR spectrum of the radical exhibits a rhombic g tensor with dual gx values (2.00550 and 2.00606) and unique gy (2.00344) and gz values (2.00186) similar to but not typical of native tyrosyl radicals. Density functional theory calculations based on a model of an MYW adduct radical built from x-ray coordinates predict experimentally observed hyperfine interactions and a shift in g values away from the native tyrosyl radical. A catalytic role for an MYW adduct radical in the catalase mechanism of KatG is proposed.

Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase-peroxidase (KatG).

The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.

Characterization of the binding of isoniazid and analogues to Mycobacterium tuberculosis catalase-peroxidase.

The first-line antituberculosis drug isonicotinic hydrazide (INH) is a prodrug whose bactericidal function requires activation by Mycobacterium tuberculosis catalase-peroxidase (KatG) to produce an acyl-NAD adduct. Peroxidation of INH is considered a required catalytic process for drug action. The binding of INH and a series of hydrazide analogues to resting KatG was examined using optical and calorimetric techniques to provide thermodynamic parameters, binding stoichiometries, and kinetic constants (on and off rates). This work revealed high-affinity binding of these substrates to a small fraction of ferric enzyme in a six-coordinate heme iron form, a species most likely containing a weakly bound water molecule, which accumulates during storage of the enzyme. The binding of hydrazides is associated with a large enthalpy loss (>100 kcal/mol); dissociation constants are in the range of 0.05-1.6 microM, and optical stopped-flow measurements demonstrated kon values in the range of 0.5-27 x 10(3) M-1 s-1 with very small koff rates. Binding parameters did not depend on pH in the range 5-8. High-affinity binding of INH is disrupted in two mutant enzymes bearing replacements of key distal side residues, KatG[W107F] and KatG[Y229F]. The rates of reduction of KatG Compound I by hydrazides parallel the on rates for association with the resting enzyme. In a KatG-mediated biomimetic activation assay, only isoniazid generated in good yield the acyl-NAD adduct which is considered a key molecule in INH action, providing a better understanding of the action mechanism of INH.

Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings.

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met-Tyr-Trp cross-linked adduct.

Mycobacterium tuberculosis catalase-peroxidase (Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous-CO complex of KatG(Y229F) suggest a non-linear Fe-CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met-Tyr-Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.

Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant.

Inhibition of the enzyme Mycobacterium tuberculosis InhA (enoyl-acyl carrier protein reductase) due to formation of an isonicotinoyl-NAD adduct (IN-NAD) from isoniazid (INH) and nicotinamide adenine dinucleotide cofactor is considered central to the mode of action of INH, a first-line treatment for tuberculosis infection. INH action against mycobacteria requires catalase-peroxidase (KatG) function, and IN-NAD adduct formation is catalyzed in vitro by M. tuberculosis KatG under a variety of conditions, yet a physiologically relevant approach to the process has not emerged that allows scrutiny of the mechanism and the origins of INH resistance in the most prevalent drug-resistant strain bearing KatG[S315T]. In this report, we describe how hydrogen peroxide, delivered at very low concentrations to ferric KatG, leads to efficient inhibition of InhA due to formation of the IN-NAD adduct. The rate of adduct formation mediated by wild-type KatG was about 20-fold greater than by the isoniazid-resistant KatG[S315T] mutant under optimal conditions (H2O2 supplied along with NAD+ and INH). Slow adduct formation also occurs starting with NADH and INH, in the presence of KatG even in the absence of added peroxide, due to endogenous peroxide. The poor efficiency of the KatG[S315T] mutant can be enhanced merely by increasing the concentration of INH, consistent with this enzyme's reduced affinity for INH binding to the resting enzyme and the catalytically competent enzyme intermediate (Compound I). Origins of drug resistance in the KatG[S315T] mutant enzyme are analyzed at the structural level through examination of the three-dimensional X-ray crystal structure of the mutant enzyme.

Resonance Raman spectroscopy of Compound II and its decay in Mycobacterium tuberculosis catalase-peroxidase KatG and its isoniazid resistant mutant S315T.

The reaction of Mycobacterium tuberculosis KatG and the mutant KatG(S315T) with two different organic peroxides is studied using resonance Raman spectroscopy. For the first time, an intermediate is observed in a catalase-peroxidase with vibrations that are characteristic of Compound II. The observation of this intermediate is consistent with photoreduction of Compound I and is in agreement with the formation of Compound I during the catalytic cycle of KatG. The same intermediate is detected in KatG(S315T), a mutant associated with resistance to isoniazid (INH), but with a lower yield, indicating that the organic peroxides cannot react with the heme iron in KatG(S315T) as efficiently as in wild-type KatG. Our results are consistent with catalytic competence of the S315T mutant and support the model that the S315T mutation confers antibiotic resistance by modifying the interaction between the enzyme and the drug.

Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function.

Mycobacterium tuberculosis (Mtb) KatG is a catalase-peroxidase that is thought to activate the antituberculosis drug isoniazid (INH). The local environment of Mtb KatG and its most prevalent INH-resistant mutant, KatG(S315T), is investigated with the exogenous ligands CO and NO in the absence and presence of INH by using resonance Raman, FTIR, and transient absorption spectroscopy. The Fe-His stretching vibration is detected at 244 cm(-)(1) in the ferrous forms of both the wild-type enzyme and KatG(S315T). The ferrous-CO complex of both enzymes exhibits nu(CO), nu(Fe-CO), and delta(Fe-C-O) vibrations at 1925, 525, and 586 cm(-)(1), respectively, indicating a positive electrostatic environment for the CO complex, which is probably weakly hydrogen-bonded to a distal residue. The CO geometry is nonlinear as indicated by the unusually high intensity of the Fe-C-O bending vibration. The nu(Fe(III)-NO) and delta(Fe(III)-N-O) vibrations are detected at 596 and 571 cm(-)(1), respectively, in the ferric forms of wild-type and mutant enzyme and are indicative of a nonlinear binding geometry in support of the CO data. Although the presence of INH does not affect the vibrational frequencies of the CO- and NO-bound forms of either enzyme, it seems to perturb slightly their Raman intensities. Our results suggest a minimal, if any, perturbation of the distal heme pocket in the S315T mutant. Instead, the S315T mutation seems to induce small changes in the KatG conformation/dynamics of the ligand access channel as indicated by CO rebinding kinetics in flash photolysis experiments. The implications of these findings for the catalytic mechanism and mechanism of INH resistance in KatG(S315T) are discussed.

Evidence for radical formation at Tyr-353 in Mycobacterium tuberculosis catalase-peroxidase (KatG).

Mycobacterium tuberculosis KatG is a heme-containing catalase-peroxidase responsible for activation, through its peroxidase cycle, of the front line antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide). Formation of Compound I (oxyferryl heme-porphyrin pi-cation radical), the classical peroxidase intermediate generated when the resting enzyme turns over with alkyl peroxides, is rapidly followed by production of a protein-centered tyrosyl radical in this enzyme. In our efforts to identify the residue at which this radical is formed, nitric oxide was used as a radical scavenging reagent. Quenching of the tyrosyl radical generated in the presence of NO was shown using electron paramagnetic resonance spectroscopy, and formation of nitrotyrosine was confirmed by proteolytic digestion followed by high performance liquid chromatography analysis of the NO-treated enzyme. These results are consistent with formation of nitrosyltyrosine by addition of NO to tyrosyl radical and oxidation of this intermediate to nitrotyrosine. Two predominant nitrotyrosine-containing peptides were identified that were purified and sequenced by Edman degradation. Both peptides were derived from the same M. tuberculosis KatG sequence spanning residues 346-356 with the amino acid sequence SPAGAWQYTAK, and both peptides contained nitrotyrosine at residue 353. Some modification of Trp-351 most probably into nitrosotryptophan was also found in one of the two peptides. Control experiments using denatured KatG or carried out in the absence of peroxide did not produce nitrotyrosine. In the mutant enzyme KatG(Y353F), which was constructed using site-directed mutagenesis, a tyrosyl radical was also formed upon turnover with peroxide but in poor yield compared with wild-type KatG. Residue Tyr-353 is unique to M. tuberculosis KatG and may play a special role in the function of this enzyme.