The multifunctional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates.

Mycobacterium tuberculosis has evolved a number of strategies to survive within the hostile environment of host phagocytes. Reactive nitrogen and oxygen intermediates (RNI and ROI) are among the most effective antimycobacterial molecules generated by the host during infection. Lsr2 is a M. tuberculosis protein with histone-like features, including the ability to regulate a variety of transcriptional responses in mycobacteria. Here we demonstrate that Lsr2 protects mycobacteria against ROI in vitro and during macrophage infection. Furthermore, using macrophages derived from NOS(-/-) and Phox(-/-) mice, we demonstrate that Lsr2 is important in protecting against ROI but not RNI. The protection provided by Lsr2 protein is not the result of its ability to either bind iron or scavenge hydroxyl radicals. Instead, electron microscopy and DNA-binding studies suggest that Lsr2 shields DNA from reactive intermediates by binding bacterial DNA and physically protecting it. Thus, Lsr2 appears to be a unique protein with both histone-like properties and protective features that may be central to M. tuberculosis pathogenesis. In addition, evidence indicates that lsr2 is an essential gene in M. tuberculosis. Because of its essentiality, Lsr2 may represent an excellent candidate as a drug target.

Inactivation of pyruvate formate-lyase by dioxygen: defining the mechanistic interplay of glycine 734 and cysteine 419 by rapid freeze-quench EPR.

Pyruvate formate-lyase from Escherichia coli (EC 2.3.1.54; PFL) catalyzes the reversible anaerobic conversion of pyruvate and CoA into acetyl-CoA and formate. Active PFL contains a novel alpha-carbon centered glycyl radical at G734 that is required for its catalytic activity. Two adjacent cysteine residues, C418 and C419, are essential for PFL activity according to site-directed mutagenesis studies. Upon exposure to air, active PFL loses its activity with the concomitant loss of the glycyl radical. Previous EPR studies of dioxygen inactivation of PFL revealed protein-based peroxyl and sulfinyl radicals during the manual mixing and quenching process [Reddy et al. (1998) Biochemistry 37, 558-563]. To probe the mechanism of this process, we carried out experiments using rapid freeze-quench EPR spectroscopy. Upon mixing of active wild type or C418A PFL with oxygenated solution, a short-lived radical intermediate appears at the earliest time point (10 ms), followed by the appearance of a long-lived sulfinyl radical. The axial EPR spectrum of this short-lived radical (g = 2.034, 2.007) is characteristic of a peroxyl radical. When C419A PFL or the double mutant [C418A/C419A] PFL was mixed with oxygenated solution, the peroxyl radical was also observed at 10 ms but in this case persisted over 12 s. These observations provide compelling evidence to support a proposed mechanism in which dioxygen quenches the glycyl radical in the active enzyme and the resulting peroxyl radical may react further with the sulfhydryl group of the C419 residue to form the sulfinyl radical.

Catalase-peroxidase (Mycobacterium tuberculosis KatG) catalysis and isoniazid activation.

Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates.

A commercial preparation of catalase inhibits nitric oxide production by activated murine macrophages: role of arginase.

Catalase is widely used as a pharmacological probe to evaluate the role of hydrogen peroxide in antimicrobial activities of phagocytic cells. This report demonstrates that the ability of a commercial preparation of catalase to inhibit concomitantly macrophage antimycobacterial activity and production of reactive nitrogen intermediates can be attributed, at least in part, to the depletion of L-arginine by contaminating arginase. In experimental systems that employ pharmacological probes, the existence of nonspecific effects should be considered in data interpretation.

Impact of either elevated or decreased levels of cytochrome bd expression on Shigella flexneri virulence.

Shigella spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that S. flexneri cytochrome bd expression is necessary for normal intracellular survival and virulence. Cytochrome bd is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain. S. flexneri mutants that contain a disruption in the cydC locus, which leads to defective cytochrome bd expression, or in the riboflavin (ribE) or ubiquinol-8 (ubiH) biosynthetic pathway, which leads to elevated cytochrome bd expression, were evaluated in intracellular survival and virulence assays. The cydC mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. The ribE and ubiH mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome bd is required for S. flexneri intracellular survival and virulence.

Toxicity of nitrogen oxides and related oxidants on mycobacteria: M. tuberculosis is resistant to peroxynitrite anion.

OBJECTIVE: To test the toxicity of reactive nitrogen intermediates (RNI), including authentic nitric oxide (NO), nitrogen dioxide (NO2), and peroxynitrite anion (ONOO-), a potent oxidant derived from NO and superoxide anion, on various mycobacterial strains including M. tuberculosis. DESIGN: Relatively avirulent mycobacteria including M. smegmatis and BCG, as well as the pathogenic M. Bovis Ravenel and M. tuberculosis Erdman and the clinical isolate M160 (also known as the C strain) were tested for their susceptibility to the toxic effects of NO, NO2, and ONOO-, Deaerated, NO-saturated solutions as well as an anaerobic in vitro system in which mycobacteria can be exposed to desired concentrations of authentic NO or NO2, were employed in these studies. An in vitro ONOO- killing assay was used to examine the adverse effects of this NO-derived oxidant on the various strains of mycobacteria. RESULTS: Both NO and NO2 exhibit antimycobacterial activity, with the former being more potent. Results obtained using ONOO- killing assay revealed that while avirulent mycobacteria including BCG and M. smegmatis are susceptible to this NO-derived oxidant, the virulent Erdman strain of M. tuberculosis and M. bovis, as well as the clinical tuberculous isolate M160, are remarkably resistant. CONCLUSION: These results suggest that the interactions between RNI and various species of mycobacteria could be highly specific. And since activated macrophages produce peroxynitrite, the significance of the ONOO- resistance of M. tuberculosis strains in relation to intracellular survival deserves further investigation.

Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the formation of protein-based sulfinyl and peroxyl radicals.

We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation. Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO*). The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation. Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the alpha-carbon peroxyl radical (ROO*) of the active-site glycine, G734. These findings suggest that C419 is the site of sulfinyl radical formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical. The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen-dependent inactivation and cleavage of the protein. Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium. A mechanism that accounts for the formation of the radical intermediates is proposed.

The role of Mn(II)-peroxidase activity of mycobacterial catalase-peroxidase in activation of the antibiotic isoniazid.

The catalase-peroxidase of Mycobacteria smegmatis exhibits Mn(II)-peroxidase activity characterized by a low Km for Mn(II) (5 microM) and a high Km for t-butyl hydroperoxide (100 mM). This activity, monitored by the formation of Mn(III)-malate or -malonate, is inhibited by Co(II) but not by superoxide dismutase. Optical evidence for binding of Mn(II) to the resting (ferric) enzyme is found in a change in intensity of the Soret peak upon titration with Mn(II). A potential role for Mn(III) in the antimycobacterial action of the antibiotic isoniazid is suggested by the rapid reduction of Mn(III)-malonate by this drug. The stoichiometry of the reaction is consistent with two single electron transfer steps per mole of isoniazid.

Purification and characterization of the Mycobacterium smegmatis catalase-peroxidase involved in isoniazid activation.

The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s-1. The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1/2 = approximately 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN3, isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.

Electron paramagnetic resonance evidence for a cysteine-based radical in pyruvate formate-lyase inactivated with mercaptopyruvate.

Pyruvate formate-lyase (PFL) is a glycyl radical-containing enzyme that catalyzes the reversible, nonoxidative conversion of pyruvate and CoA into acetyl-CoA and formate. The radical is located on the alpha-carbon of glycine 734 and is required for catalysis. Two cysteine residues, C418 and C419, are also essential for catalysis. Mercaptopyruvate, a biologically relevant pyruvate analog, is shown here to be a mechanism-based inactivator of PFL. Upon addition of mercaptopyruvate to active PFL, an EPR spectrum is generated which exhibits components from two sulfur-based radicals. For one of these radicals, a disulfide radical, the hyperfine coupling to a single beta-methylene hydrogen is resolved in features at g = 2.057 and 2.023. The effects of deuterium labeling of the enzyme on the EPR spectrum for this species are consistent with the new radical being on a cysteine residue, probably cysteine 418 or 419. This spectrum is not formed upon addition of the inactivator to mutant enzymes, C418S and C419S, indicating that both active site cysteines are required for formation of the new radicals. The identity of the second species is also ascribed to be a sulfur-based radical on the basis of the EPR feature found at g = 2.01. Our results constitute the first direct evidence of sulfur-based radical formation in an enzyme. A mechanism for formation of the cysteine-based disulfide radical is proposed which requires the participation of the two active site cysteines as well as the glycyl radical.

Hydrogen exchange of the glycyl radical of pyruvate formate-lyase is catalyzed by cysteine 419.

Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of CoA and pyruvate into acetyl-CoA and formate. Active enzyme contains a glycyl radical whose alpha-hydrogen undergoes rapid exchange with solvent (t1/2 approximately 5 min at 0 degree C). We have investigated this exchange using site-directed mutagenesis and mechanism-based inactivation. Mutation of the active-site cysteine 419 into a serine, which renders the enzyme catalytically inactive, abolishes alpha-hydrogen exchange in the radical. This suggests that the exchange process is not an intrinsic property of the glycyl radical but is a consequence of its interaction with cysteine 419. This residue is also demonstrated to be involved in the transfer of the radical to acetylphosphinate, a mechanism-based inactivator of the enzyme. In contrast, mutation of the other essential cysteine 418 to a serine has no effect on the hydrogen exchange or the transfer of the radical to acetylphosphinate. A mechanism for the hydrogen exchange catalyzed by cysteine 419 consistent with a redox role for this residue in the normal catalytic reaction is proposed.

Cu(II) coordination in arthropod and mollusk green half-methemocyanins analyzed by electron spin-echo envelope modulation spectroscopy.

Hemocyanin (Hc) is a dinuclear copper protein that binds oxygen reversibly. The structure of the Cu(II) site in a derivative of hemocyanin known as green half-met (GHM) has been analyzed using the pulsed EPR technique of electron spin-echo envelope modulation (ESEEM) spectroscopy. The derivative, prepared by treating the native protein with nitrite at low pH, contains a mixed-valent binuclear copper center. It was shown through chemical assays and the ligand exchange reaction products identified by EPR spectroscopy to contain a nitrite ligand bound to Cu(II). The ESEEM spectra of green half-methemocyanins from mollusks and arthropods indicated that three imidazole ligands are coordinated to Cu(II). Therefore, a tetragonal N3O ligand structure (O is an oxygen of nitrite) is proposed. For GHM Hc from the mollusks Octopus vulgaris and Rapana thomasiana, the isotropic nitrogen nuclear hyperfine coupling constant, aiso, for the N delta (or remote) nitrogen of two imidazoles was approximately 1.4 MHz, while for the third, aiso congruent to 2.2 MHz. The difference between the two weaker nitrogens and the single, more strongly coupled nitrogen was smaller by 0.2 MHz in the GHM Hcs from the arthropods Carcinus maenas, Homarus americanus and Panulirus interruptus. The nitrogen nuclear quadrupole coupling constants and asymmetry parameters, e2Qq and eta, for the N delta nitrogens in nearly all cases were near 1.4 MHz and 0.8, respectively, although Rapana thomasiana GHM Hc exhibited a reduction in eta that may indicate weaker hydrogen bonding in the active site of this protein. The g and ACu (copper nuclear hyperfine coupling) values for the derivatives, and the finding of three similar nuclear hyperfine coupling constants for the N delta sites of imidazole ligands, when considered with the orientation-specific information obtained using angle-selection methods for simulation of ESEEM spectra, suggest a distorted tetragonal Cu(II) structure in which three imidazoles and a nitrite ligand are bound near the equatorial plane. The finding that the two molluscan GHM Hcs exhibit differences associated with the remote nitrogen of imidazoles bound to Cu(II) may be related to a structural variability in the active sites of these proteins not found in the arthropodan GHM Hcs examined.

Structural characterization of mononuclear Cu(II) and its nitrite complex in the active site of Carcinus maenas hemocyanin.

The preparation of a mononuclear Cu(II) derivative of Carcinus maenas hemocyanin (Cu(II)-Hc) and a nitrite complex of the derivative (Cu(II)-Hc-NO2-) are described. Several techniques have been used in their characterization, including X-ray absorption, continuous wave (cw) EPR, and electron spin-echo envelope modulation (ESEEM) spectroscopies. EXAFS results for Cu(II)-Hc indicate the presence of three ligands at 1.99 +/- 0.01 A and a fourth one at 2.26 +/- 0.01 A from the copper. The same coordination number and very similar bond lengths were obtained for Cu(II)-Hc-NO2-. On the basis of simulations of three-pulse ESEEM spectra, three equivalent imidazole nitrogens coupled to Cu(II) were identified in Cu(II)-Hc. Upon the binding of nitrite, a decrease in the hyperfine interaction for two of the three imidazole nitrogens was observed by ESSEM. Further, the results of a two-pulse ESEEM experiment are consistent with the assignment of the protons of a water ligand to Cu(II), which is displaced when nitrite is added. An analysis of X-ray absorption K-edge spectra suggests a coordination geometry intermediate between square-planar and tetrahedral for the metal centers in Cu(II)-Hc and Cu(II)-Hc-NO2-, in agreement with the g and ACu values determined by cw-EPR. On the basis of these results, an equivalent structure is suggested for Cu(II)-Hc-NO2- and the Cu(II) site in green half-methemocyanin, a partially oxidized binuclear derivative formed in the reaction of the native protein with nitrite.

Evaluation of nitrogen nuclear hyperfine and quadrupole coupling parameters for the proximal imidazole in myoglobin-azide, -cyanide, and -mercaptoethanol complexes by electron spin echo envelope modulation spectroscopy.

Electron spin echo envelope modulation (ESEEM) spectroscopy and computer simulation of spectra has been used to evaluate the nitrogen nuclear hyperfine and quadrupole coupling parameters for the proximal imidazole nitrogen directly coordinated to iron in three low-spin heme complexes, myoglobin-azide, -cyanide, and -mercaptoethanol (MbN3, MbCN, and MbRS). The variability in the weak electron-nuclear coupling parameters reveals the electronic flexibility within the heme group that depends on properties of the exogenous ligands. For example, the isotropic component of the nitrogen nuclear hyperfine coupling ranges from 4.4 MHz for MbN3 to 2.2 MHz for both MbCN and MbRS. The weaker coupling in MbCN and MbRS is taken as evidence for delocalization of unpaired electron spin from iron into the exogenous anionic ligands. The value of e2Qq, the nuclear quadrupole coupling constant for the axial imidazole nitrogen in MbCN and MbRS, was 2.5 MHz but was significantly larger, 3.2 MHz, in MbN3. This large value is considered evidence for a weakened sigma bond between the proximal imidazole and ferric iron in this form, and for a feature contributing to the origin of the high spin-low spin equilibrium exhibited by MbN3 [Beetlestone, J., & George, P. (1964) Biochemistry 5, 707-714]. The ESEEM results have allowed a correlation to be made between the orientation of the g tensor axes, the orientation of the p-pi orbital of the proximal imidazole nitrogen, and sigma- and pi-bonding features of the axial ligands. Furthermore, the proximal imidazole is suggested to act as a pi-acceptor in low-spin heme complexes in order to support strong sigma electron donation from the lone pair orbital to iron. An evaluation of the nitrogen nuclear hyperfine coupling parameters for the porphyrin pyrrole sites in MbRS reveals a large inequivalence in isotropic components consistent with an orientation of rhombic axes (and g tensor axes) that eclipses the Fe-Npyrrole vector directions.

Preparation and spectroscopic characterization of a coupled binuclear center in cobalt(II)-substituted hemocyanin.

A binuclear cobalt derivative of arthropod hemocyanin (Hc) has been prepared by the reaction of apo-Hc with Co(II) in the presence of thiocyanate. The crude product of the reaction contains specifically and adventitiously bound metal, the latter being removable by EDTA treatment. The specifically bound Co(II) constitutes a binuclear metal center that exhibits optical and CD spectra typical in their absorption maxima and extinction coefficients of Co(II) complexes with near-tetrahedral geometry. The EPR spectrum of the binuclear Co(II) derivative contains a resonance at g approximately 13, which is characteristic of integer spin systems and indicates coupled metal ions; the excess Co(II) bound to crude products exhibits an EPR signal at g approximately 4. The time course of derivative formation was followed by EPR, optical and atomic absorption techniques, and by fluorimetry. The intensity of the optical absorption in the visible region due to Co(II) increases with increasing stoichiometry of specifically bound metal [up to 2 Co(II) per protein monomer], but the intensity of the Co(II) EPR signal increases only during the formation of a mononuclear derivative. As the reaction proceeds over approximately 100 h to the formation of the binuclear derivative, the EPR signal intensity decreases to 10% of the value expected for 2 mol of EPR-active Co(II)/mol of protein. The binuclear cobalt derivative cannot be reconstituted to native Hc with Cu(I), indicating the stable loading of Co(II) in the active site. EPR and optical spectroscopic evidence is presented showing that the binuclear derivative does not bind oxygen.

Hydrogen bonding to the bound dioxygen in oxy cobaltous myoglobin reduces the superhyperfine coupling to the proximal histidine.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to study the electron-nuclear coupling in two oxygenated cobalt-substituted hemoproteins, myoglobin (oxyCoMb) and a monomeric hemoglobin from Glycera dibranchiata (oxyCoHbgly). The modulation frequency components in ESEEM spectra of both proteins arose from the coupling to the N epsilon of the proximal histidyl imidazole. The hyperfine and quadrupole coupling parameters for these two nitrogens, calculated by computer spectral simulation, are Aiso = 2.46 MHz, e2qQ = 2.15 MHz, and eta = 0.4 for oxyCoMb and Aiso = 3.70 MHz, e2qQ = 2.70 MHz, and eta = 0.5 for oxyCoHbgly. A hyperfine coupling of 0.6 MHz, found for oxyCoMb in D2O but not for oxyCoHbgly in D2O, was assigned to the coupling to a deuteron that is hydrogen-bonded to the O2 ligand in oxyCoMb. This hydrogen bonding is believed to be responsible for the reduction in hyperfine and nuclear quadrupole coupling to the proximal histidyl imidazole N epsilon in oxyCoMb. A molecular orbital model for O2 adducts of cobaltous compounds [Tovrog et al. (1976) J. Am. Chem. Soc. 98, 5144] was used to understand the hydrogen bond-induced reduction in 14N superhyperfine coupling in oxyCoMb.

Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages.

Tuberculosis remains one of the major infectious causes of morbidity and mortality in the world, yet the mechanisms by which macrophages defend against Mycobacterium tuberculosis have remained obscure. Results from this study show that murine macrophages, activated by interferon gamma, and lipopolysaccharide or tumor necrosis factor alpha, both growth inhibit and kill M. tuberculosis. This antimycobacterial effect, demonstrable both in murine macrophage cell lines and in peritoneal macrophages of BALB/c mice, is independent of the macrophage capacity to generate reactive oxygen intermediates (ROI). Both the ROI-deficient murine macrophage cell line D9, and its ROI-generating, parental line J774.16, expressed comparable antimycobacterial activity upon activation. In addition, the oxygen radical scavengers superoxide dismutase (SOD), catalase, mannitol, and diazabicyclooctane had no effect on the antimycobacterial activity of macrophages. These findings, together with the results showing the relative resistance of M. tuberculosis to enzymatically generated H2O2, suggest that ROI are unlikely to be significantly involved in killing M. tuberculosis. In contrast, the antimycobacterial activity of these macrophages strongly correlates with the induction of the L-arginine-dependent generation of reactive nitrogen intermediates (RNI). The effector molecule(s) that could participate in mediating this antimycobacterial function are toxic RNI, including NO, NO2, and HNO2, as demonstrated by the mycobacteriocidal effect of acidified NO2. The oxygen radical scavenger SOD adventitiously perturbs RNI production, and cannot be used to discriminate between cytocidal mechanisms involving ROI and RNI. Overall, our results provide support for the view that the L-arginine-dependent production of RNI is the principal effector mechanism in activated murine macrophages responsible for killing and growth inhibiting virulent M. tuberculosis.

Electron spin echo envelope modulation spectroscopic study of iron-nitrogen interactions in myoglobin hydroxide and Fe(III) tetraphenylporphyrin models.

The electron-nuclear coupling in low-spin iron complexes including myoglobin hydroxide (MbOH) and two related model compounds, Fe(III) tetraphenylporphyrin(pyridine)(OR-) (R = H or CH3) and Fe(III) tetraphenylporphyrin(butylamine)(OR-) was investigated using electron spin echo envelope modulation (ESEEM) spectroscopy. The assignment of frequency components in ESEEM spectra was accomplished through the use of nitrogen isotopic substitution wherever necessary. For example, the proximal imidazole coupling in MbOH was investigated without interference from the contributions of porphyrin 14N nuclei after substitution of the heme in native Mb with 15N-labeled heme. Computer simulation of spectra using angle selected techniques enabled the assignment of parameters describing the hyperfine and quadrupole interactions for axially bound nitrogen of imidazole in MbOH, of axial pyridine and butylamine in the models, and for the porphyrin nitrogens of the heme in native MbOH. The isotropic component of axial nitrogen hyperfine interactions exhibits a trend from 5 to 4 MHz, with imidazole (MbOH) greater than pyridine greater than amine. The nuclear quadrupole interaction coupling constant e2Qq was near 2 MHz for all nitrogens in these complexes. The Qzz axis of the nuclear quadrupole interaction tensor for the proximal imidazole nitrogen in MbOH was found to be aligned near gz (gmax) in MbOH, suggesting that gz is near the heme normal. A crystal field analysis, that allows a calculation of rhombic and axial splittings for the d orbitals of the t2g set in a low-spin heme complex, based on the g tensor assignment gz greater than gy greater than gx, yielded results that are consistent with the poor pi-acceptor properties expected for the closed shell oxygen atom of the hydroxide ligand in MbOH. A discussion is presented of the unusual results reported in a linear electric field effect in EPR (LEFE) study of MbOH published previously [Mims, W. B., & Peisach, J. (1976) J. Chem. Phys. 64, 1074-1091].

Transfer RNA is cleaved by activated bleomycin.

Activated bleomycin is shown for the first time to cleave tRNA in a specific and dose-dependent manner. Adenine and uracil are released in the reaction. Bleomycin and Fe(III)-bleomycin bind to yeast tRNAPhe) in analogy with the known behavior of the drug with B-DNA.

A comparative study of the interactions of bleomycin with nuclei and purified DNA.

Fe(III)-bleomycin associates strongly with rat liver nuclei and binds to nuclear DNA. Metal-free and Cu(II)-bleomycin, however, do not bind to nuclei. The treatment of nuclei with activated iron-bleomycin results in nucleic base and base propenal release from the DNA, and also gives membrane peroxidation. Isolation and quantitation of the base propenals and free bases released subsequent to activated bleomycin treatment reveal an alteration in the stoichiometry of these products compared to those released from purified DNA. With nuclei, significantly less propenal is formed, although the yield of free base is equivalent to that from purified DNA. The membrane peroxidation products from nuclei are the same as those obtained from microsomal membranes treated with activated bleomycin. Superoxide dismutase inhibits the membrane peroxidation but has no effect on the DNA breakage reactions. The results implicate a role for iron in mediating the in vivo action of bleomycin and also reveal a potentially toxic effect, membrane peroxidation, separate from DNA damage.