Rapid polyether cleavage via extracellular one-electron oxidation by a brown-rot basidiomycete

Fungi that cause brown rot of wood are essential biomass recyclers and also the principal agents of decay in wooden structures, but the extracellular mechanisms by which they degrade lignocellulose remain unknown. To test the hypothesis that brown-rot fungi use extracellular free radical oxidants as biodegradative tools, Gloeophyllum trabeum was examined for its ability to depolymerize an environmentally recalcitrant polyether, poly(ethylene oxide) (PEO), that cannot penetrate cell membranes. Analyses of degraded PEOs by gel permeation chromatography showed that the fungus cleaved PEO rapidly by an endo route. 13C NMR analyses of unlabeled and perdeuterated PEOs recovered from G. trabeum cultures showed that a major route for depolymerization was oxidative C—C bond cleavage, a reaction diagnostic for hydrogen abstraction from a PEO methylene group by a radical oxidant. Fenton reagent (Fe(II)/H2O2) oxidized PEO by the same route in vitro and therefore might account for PEO biodegradation if it is produced by the fungus, but the data do not rule out involvement of less reactive radicals. The reactivity and extrahyphal location of this PEO-degrading system suggest that its natural function is to participate in the brown rot of wood and that it may enable brown-rot fungi to degrade recalcitrant organopollutants.

Repair of hydantoins, one electron oxidation product of 8-oxoguanine, by DNA glycosylases of Escherichia coli

8-Oxoguanine (8-oxoG), induced by reactive oxygen species and arguably one of the most important mutagenic DNA lesions, is prone to further oxidation. Its one-electron oxidation products include potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) because of their mispairing with A or G. All three oxidized base-specific DNA glycosylases of Escherichia coli, namely endonuclease III (Nth), 8-oxoG-DNA glycosylase (MutM) and endonuclease VIII (Nei), excise Gh and Sp, when paired with C or G in DNA, although Nth is less active than the other two. MutM prefers Sp and Gh paired with C (kcat/Km of 0.24–0.26 min–1 nM–1), while Nei prefers G over C as the complementary base (kcat/Km – 0.15–0.17 min–1 nM–1). However, only Nei efficiently excises these paired with A. MutY, a 8-oxoG·A(G)-specific A(G)-DNA glycosylase, is inactive with Gh(Sp)·A/G-containing duplex oligonucleotide, in spite of specific affinity. It inhibits excision of lesions by MutM from the Gh·G or Sp·G pair, but not from Gh·C and Sp·C pairs. In contrast, MutY does not significantly inhibit Nei for any Gh(Sp) base pair. These results suggest a protective function for MutY in preventing mutation as a result of A (G) incorporation opposite Gh(Sp) during DNA replication.

The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Opposite effects of nitric oxide and nitroxyl on postischemic myocardial injury

Recent experimental evidence suggests that reactive nitrogen oxide species can contribute significantly to postischemic myocardial injury. The aim of the present study was to evaluate the role of two reactive nitrogen oxide species, nitroxyl (NO−) and nitric oxide (NO⋅), in myocardial ischemia and reperfusion injury. Rabbits were subjected to 45 min of regional myocardial ischemia followed by 180 min of reperfusion. Vehicle (0.9% NaCl), 1 μmol/kg S-nitrosoglutathione (GSNO) (an NO⋅ donor), or 3 μmol/kg Angeli’s salt (AS) (a source of NO−) were given i.v. 5 min before reperfusion. Treatment with GSNO markedly attenuated reperfusion injury, as evidenced by improved cardiac function, decreased plasma creatine kinase activity, reduced necrotic size, and decreased myocardial myeloperoxidase activity. In contrast, the administration of AS at a hemodynamically equieffective dose not only failed to attenuate but, rather, aggravated reperfusion injury, indicated by an increased left ventricular end diastolic pressure, myocardial creatine kinase release and necrotic size. Decomposed AS was without effect. Co-administration of AS with ferricyanide, a one-electron oxidant that converts NO− to NO⋅, completely blocked the injurious effects of AS and exerted significant cardioprotective effects similar to those of GSNO. These results demonstrate that, although NO⋅ is protective, NO− increases the tissue damage that occurs during ischemia/reperfusion and suggest that formation of nitroxyl may contribute to postischemic myocardial injury.

No ·NO from NO synthase

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of l-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form l-citrulline (Cit) and nitric oxide (·NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify ·NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable ·NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal ·NO standards were only partially recovered and the dismutation of superoxide (O2⨪), which otherwise scavenges ·NO to yield ONOO−, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO− resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8–1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to ·NO or that ·NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal ·NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO−) to ·NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from ·NO decomposition, was consistent with formation of an ·NO precursor molecule such as NO−. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that ·NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.

Stability of nonrelativistic quantum mechanical matter coupled to the (ultraviolet cutoff) radiation field

We announce a proof of H-stability for the quantized radiation field, with ultraviolet cutoff, coupled to arbitrarily many non-relativistic quantized electrons and static nuclei. Our result holds for arbitrary atomic numbers and fine structure constant. We also announce bounds for the energy of many electrons and nuclei in a classical vector potential and for the eigenvalue sum of a one-electron Pauli Hamiltonian with magnetic field.

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 μM. The LiP–DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI–DHP and LiPII–DHP interactions were calculated to be 350 and 250 μM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s−1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His–Asp⋅⋅⋅proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.

Identification of the proton pathway in bacterial reaction centers: Both protons associated with reduction of QB to QBH2 share a common entry point

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, QB. This process involves the transfer of two protons from the aqueous solution to the protein-bound QB molecule. The second proton, H+(2), is supplied to QB by Glu-L212, an internal residue protonated in response to formation of QA− and QB−. In this work, the pathway for H+(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of QA (DQA→D+QA−) and QB (DQB→D+QB−), studied by using pH-sensitive dyes. The other was the electron transfer kAB(1) (QA−QB→QAQB−). At pH 8.5, binding of Zn2+, Cd2+, or Ni2+ reduced the rates of proton uptake upon QA− and QB− formation as well as kAB(1) by ≈an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D+QA− is formed 105-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212→Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn2+ binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H+(2) was thus identified to be the same as for the first proton H+(1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Redox transitions between oxygen intermediates in cytochrome-c oxidase.

Some intermediates in the reduction of O2 to water by cytochrome-c oxidase have been characterized by optical, Raman, and magnetic circular dichroism spectroscopy. The so-called "peroxy" (P) and "ferryl" (F) forms of the enzyme, which have been considered to be intermediates of the oxygen reaction, can be generated when the oxidized enzyme reacts with H2O2, or when the two-electron reduced ("CO mixed-valence") enzyme reacts with O2. The structures as well as the overall redox states of P and F have recently been controversial. We show here, using tris(2,2'-bipyridyl)ruthenium(II) as a photoinducible reductant, that one-electron reduction of P yields F, and that one-electron reduction of F yields the oxidized enzyme. This confirms that the overall redox states of P and F differ from the oxidized enzyme by two and one electron equivalents, respectively. The structures of the P and F states are discussed.

Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins.

Association of glycolate oxidation with photosynthetic electron transport in plant and algal chloroplasts.

Photosynthetic carbon metabolism is initiated by ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which uses both CO2 and O2 as substrates. One 2-phosphoglycolate (P-glycolate) molecule is produced for each O2 molecule fixed. P-glycolate has been considered to be metabolized exclusively via the oxidative photosynthetic carbon cycle. This paper reports an additional pathway for P-glycolate and glycolate metabolism in the chloroplasts. Light-dependent glycolate or P-glycolate oxidation by osmotically shocked chloroplasts from the algae Dunaliella or spinach leaves was measured by three electron acceptors, methyl viologen (MV), potassium ferricyanide, or dichloroindophenol. Glycolate oxidation was assayed with 3-(3,4)-dichlorophenyl)-1,1-dimethylurea (DCMU) as oxygen uptake in the presence of MV at a rate of 9 mol per mg of chlorophyll per h. Washed thylakoids from spinach leaves oxidized glycolate at a rate of 22 mol per mg of chlorophyll per h. This light-dependent oxidation was inhibited completely by SHAM, an inhibitor of quinone oxidoreductase, and 75% by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which inhibits electron transfer from plastoquinone to the cytochrome b6f complex. SHAM stimulated severalfold glycolate excretion by algal cells, Dunaliella or Chlamydomonas, and by isolated Dunaliella chloroplasts. Glycolate and P-glycolate were oxidized about equally well to glyoxylate and phosphate. On the basis of results of inhibitor action, the possible site which accepts electrons from glycolate or P-glycolate is a quinone after the DCMU site but before the DBMIB site. This glycolate oxidation is a light-dependent, SHAM-sensitive, glycolate-quinone oxidoreductase system that is associated with photosynthetic electron transport in the chloroplasts.

Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from Rhodobacter sphaeroides

The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetry-related protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll–protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to ≈60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron–nuclear–nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from ≈2:1 to ≈5:1 in the mutants compared with ≈2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation–reduction midpoint potential.

Mössbauer and electron paramagnetic resonance studies of chloroperoxidase following mechanism-based inactivation with allylbenzene

We have used Mössbauer and electron paramagnetic resonance (EPR) spectroscopy to study a heme-N-alkylated derivative of chloroperoxidase (CPO) prepared by mechanism-based inactivation with allylbenzene and hydrogen peroxide. The freshly prepared inactivated enzyme (“green CPO”) displayed a nearly pure low-spin ferric EPR signal with g = 1.94, 2.15, 2.31. The Mössbauer spectrum of the same species recorded at 4.2 K showed magnetic hyperfine splittings, which could be simulated in terms of a spin Hamiltonian with a complete set of hyperfine parameters in the slow spin fluctuation limit. The EPR spectrum of green CPO was simulated using a three-term crystal field model including g-strain. The best-fit parameters implied a very strong octahedral field in which the three 2T2 levels of the (3d)5 configuration in green CPO were lowest in energy, followed by a quartet. In native CPO, the 6A1 states follow the 2T2 ground state doublet. The alkene-mediated inactivation of CPO is spontaneously reversible. Warming of a sample of green CPO to 22°C for increasing times before freezing revealed slow conversion of the novel EPR species to two further spin S = ½ ferric species. One of these species displayed g = 1.82, 2.25, 2.60 indistinguishable from native CPO. By subtracting spectral components due to native and green CPO, a third species with g = 1.86, 2.24, 2.50 could be generated. The EPR spectrum of this “quasi-native CPO,” which appears at intermediate times during the reactivation, was simulated using best-fit parameters similar to those used for native CPO.

Theoretical investigation of the [1,2]-sigmatropic hydrogen migration in the mechanism of oxidation of 2-aminobenzoyl-CoA by 2-aminobenzoyl-CoA monooxygenase/reductase

The flavin hydroperoxide at the active site of the mixed-function oxidase 2-aminobenzoyl-CoA monooxygenase/reductase (Azoarcus evansii) transfers an oxygen to the 5-position of the 2-aminobenzoyl-CoA substrate to provide the alkoxide intermediate II−. Hydrogen migration from C5 to C6 follows this monooxygenation. The nature of the monooxygenation intermediate and plausible competing reactions leading to hydrogen migration have been considered. Ab initio molecular orbital theory has been used to calculate structures and electron distributions in intermediate and transition state structures. Electrostatic potential surface calculations establish that the transition state and product, associated with the C5 to C6 hydrogen transfer, are stabilized by electron distribution to the benzoyl-CoA thioester carbonyl oxygen. This is not so for the transition state and product associated with hydrogen transfer from C5 to C4. The activation energy for the 5,6-shift is 2.5 kcal/mol lower than that for the 5,4-shift. In addition, the product of the hydrogen 5,6-shift is more stable than is the product of the hydrogen 5,4-shift, by ≈6 kcal/mol. These results explain why only the shift of hydrogen from C5 to C6 is observed experimentally. Oxygen transfer and hydrogen migration almost coincide in the gas phase (activation energy of ≈0.6 kcal/mol, equivalent to a single bond vibration). Enzymatic formation of alkoxide II− requires its stabilization; thus, the rate constant for its breakdown would be slower than in the gas phase.

Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid β-oxidation

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the β-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the β-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.