Reprint of: Purification and Characterization of an Extracellular Mn(ll)-Dependent Peroxidase from the Lignin-Degrading Basidiomycete, Phanerochaete chrysosporium.

A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group.

Modeling low-pH hemoproteins.

A tetracoordinate ferrous heme (iron-porphyrin) has been proposed as an intermediate at low pH (less than 3.0) for respiratory hemoproteins, peroxidases, and model heme complexes. This intermediate is believed to arise via protonation of the N(epsilon) atom of the proximal histidine and consequent cleavage of the Fe-N(epsilon) bond.

General mechanism for the bacterial toxicity of hypochlorous acid: abolition of ATP production.

The adenylate energy charges (EC) of Escherichia coli 25922, Pseudomonas aeruginosa 27853, and Streptococcus lactis 7962 rapidly fell in nutrient-rich media from values in excess of 0.9 to below 0.1 when the organisms were exposed to lethal levels of HOCl.

Thiol-mediated oxidation of nonphenolic lignin model compounds by manganese peroxidase of Phanerochaete chrysosporium.

In the presence of MnII, H2O2, and glutathione (GSH), manganese peroxidase oxidized veratryl alcohol (I) to veratraldehyde (IV). Anisyl alcohol (II) and benzyl alcohol (III) were also oxidized by this system to their corresponding aldehydes (V and VI). In the presence of GSH, chemically prepared MnIII or gamma-irradiation also catalyzed the oxidation of I, II, and III to IV, V, and VI, respectively.

Comparative spectral analysis of mammalian, fungal, and bacterial catalases. Resonance Raman evidence for iron-tyrosinate coordination.

Resonance Raman spectra are reported for catalases from bovine liver, the ascomycete fungus Aspergillus niger, and the bacterium Micrococcus luteus. The vibrational frequencies of the oxidation-, spin-, and coordination number-sensitive spectral bands are indicative of high spin pentacoordinate hemes in the resting ferric enzymes of each of these organisms. This result is in accord with the crystal structure of bovine catalase (Fita, I.

Diffusion of the Interspecies Electron Carriers H(2) and Formate in Methanogenic Ecosystems and Its Implications in the Measurement of K(m) for H(2) or Formate Uptake.

We calculated the potential H(2) and formate diffusion between microbes and found that at H(2) concentrations commonly found in nature, H(2) could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H(2) concentration was 63 nM (10.

Spectroscopic analysis of chlorophyll model complexes: methyl ester ClFe(III)pheophorbides.

As models for chlorophyll a (Chl a), methyl ester ClFe(III)pheophorbides (1, pheophorbide a; 2, mesopheophorbide a; and 3, mesopyropheophorbide a) were examined by Fourier transform infrared (FTIR) absorption and resonance Raman (RR) spectroscopy. The infrared (IR) chlorin band above 1600 cm-1, assigned as a Ca-Cm mode (Andersson et al. (1987) J.

Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation.

Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N.

Description of an estuarine methylotrophic methanogen which grows on dimethyl sulfide.

Characteristics of an obligately methylotrophic coccoid methanogen (strain GS-16) previously isolated from estuarine sediment are described. Growth was demonstrated on dimethyl sulfide (DMS) or trimethylamine (TMA), but not on methane thiol, methane thiol plus hydrogen, dimethyl disulfide, or methionine. DMS-grown cells were able to metabolize DMS and TMA simultaneously when inoculated into media containing substrate levels of these compounds.

Characterization of a cDNA encoding a manganese peroxidase, from the lignin-degrading basidiomycete Phanerochaete chrysosporium.

A cDNA clone of a manganese peroxidase (MnP) from Phanerochaete chrysosporium was isolated and characterized. The cDNA contains 1314 nucleotides excluding the poly(A) tail and the coding region has 68% G + C content. The deduced mature MnP protein contains 357 amino acids and is preceded by a 21-amino acid leader sequence.

Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism.

Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.

Resonance Raman spectroscopy of ribonucleotide reductase. Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center.

Native ribonucleotide reductase from Escherichia coli exhibits a resonance-enhanced Raman mode at 1498 cm-1 that is characteristic of a tyrosyl radical. The Raman frequency as well as the absorption maximum at 410 nm identifies the radical as being in a deprotonated state. The B2 subunit of ribonucleotide reductase shows an additional resonance Raman mode at 493 cm-1 that has been assigned to the symmetric stretch of an Fe-O-Fe moiety.

Transformation by Complementation of an Adenine Auxotroph of the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium.

Swollen basidiospores of an adenine auxotroph of Phanerochaete chrysosporium were protoplasted with Novozyme 234 and transformed to prototrophy by using a plasmid containing the gene for an adenine biosynthetic enzyme from Schizophyllum commune. Transformation frequencies of 100 transformants per mug of DNA were obtained. Southern blot analysis of DNA extracted from transformants demonstrated that plasmid DNA was integrated into the chromosomal DNA in multiple tandem copies.

Leukocytic oxygen activation and microbicidal oxidative toxins.

Following a brief introduction of cellular response to stimulation comprising leukocyte activation, three major areas are discussed: (1) the neutrophil oxidase; (2) myeloperoxidase (MPO)-dependent oxidative microbicidal reactions; and (3) MPO-independent oxidative reactions. Topics included in section (A) are current views on the activation mechanism, redox composition, structural and topographic organization of the oxidase, and its respiratory products. In section (B), emphasis is placed on recent research on cidal mechanisms of HOCl, including the oxidative biochemistry of active chlorine compounds, identification of sites of lesions in bacteria, and attendant metabolic consequences.

Chemosensory responses in two species of elephants to constituents of temporal gland secretion and musth urine.

This report discusses three areas of investigation: (1) The chemical components in the temporal gland secretion (TGS) of Asian (Elephas maximus) and African (Loxodonta africana) elephants were characterized by radioimmunoassay (RIA) for testosterone (T) and dihydrotestosterone (DHT) levels and by on-column capillary column gas chromatographic analysis of volatiles. An inverse relationship between TGS testosterone levels and (E)-farnesol levels was observed. (2).

Control of the Life Cycle of Methanosarcina mazei S-6 by Manipulation of Growth Conditions.

The morphology of Methanosarcina mazei was controlled by magnesium, calcium, and substrate concentrations and by inoculum size; these factors allowed manipulation of the morphology and interconversions between pseudosarcinal aggregates and individual, coccoid cells. M. mazei grew as aggregates in medium with a low concentration of catabolic substrate (either 50 mM acetate, 50 mM methanol, or 10 mM trimethylamine) unless Ca and Mg concentrations were high.

Viability and metabolic capability are maintained by Escherichia coli, Pseudomonas aeruginosa, and Streptococcus lactis at very low adenylate energy charge.

Metabolic regulation by nucleotides has been examined in several bacteria within the context of the adenylate energy charge (EC) concept. The ECs of bacteria capable of only fermentative metabolism (Streptococcus lactis and the ATPase-less mutant Escherichia coli AN718) fell to less than 0.2 under carbon-limiting conditions, but the bacteria were able to step up the EC to greater than 0.

Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium: spectral characterization of the oxidized states and the catalytic cycle.

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the Mn(II)-dependent oxidation of a variety of phenols. Herein, we spectroscopically characterize the oxidized states of MnP compounds I, II, and III and clarify the role of Mn in the catalytic cycle of the enzyme. Addition of 1 equiv of H2O2 to the native ferric enzyme yields compound I, characterized by absorption maxima at 407, 558, 605, and 650 nm.

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium.

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties.

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans.

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region.

Involvement of oxo-bridged binuclear iron centers in oxygen transport, oxygen reduction, and oxygenation.

The occurrence of an oxo-bridged binuclear iron site is well-established for the oxygen transport protein, hemerythrin, and strongly implicated in ribonucleotide reductase, purple acid phosphatase, ferritin, and methane monooxygenase. Key identifying characteristics are an antiferromagnetic interaction between the two iron atoms, an Fe-O-Fe vibrational mode in the resonance Raman spectrum, and an S = 1/2 EPR signal upon one-electron reduction. In hemerythrin the oxo bridge serves as a hydrogen bond acceptor which stabilizes the bound hydroperoxide.

Hydrogen bonding of sulfur ligands in blue copper and iron-sulfur proteins: detection by resonance Raman spectroscopy.

The resonance Raman spectrum of the blue copper protein azurin from Alcaligenes denitrificans exhibits nine vibrational modes between 330 and 460 cm-1, seven of which shift 0.4-3.0 cm-1 to lower energy after incubation of the protein in D2O.

Hypochlorous acid-promoted loss of metabolic energy in Escherichia coli.

Oxidation of Escherichia coli by hypochlorous acid (HOCl) or chloramine (NH2Cl) gives rise to massive hydrolysis of cytosolic nucleotide phosphoanhydride bonds, although no immediate change occurs in either the nucleotide pool size or the concentrations of extracellular end products of AMP catabolism. Titrimetric curves of the extent of hydrolysis coincide with curves for loss of cell viability, e.g.

Improved optical local-oscillator isolation using multiple acousto-optic modulators and frequency diversity.

A technique is described and experimental evidence presented for obtaining ultrahigh isolation between the optical local oscillator and the transmitter in heterodyne systems. A degree of isolation that results in an equivalent fed-back optical signal that is more than 200 dB below the transmitted power level has been achieved.

Mating System and Basidiospore Formation in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium.

Prototrophic strains recovered from crosses between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The progeny of most of these self-crosses were prototrophic, indicating that the nuclei of the original prototroph were wild-type recombinants rather than complementary heterokaryons and that the binucleate basidiospores of this organism are homokaryotic. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium.