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Papers by Paul Wood

Research paper thumbnail of EPR Evidence for hydroxyl- and substrate-derived radicals in Fe(II)-oxalate/hydrogen peroxide reactions. The importance of the reduction of Fe(III)-oxalate by oxygen-conjugated radicals to regenerate Fe(II) in reactions of carbohydrates and model compounds

Journal of the Chemical Society, Perkin Transactions 2

ABSTRACT

Research paper thumbnail of SHORT COMMUNICATION Haemoprotein 6-90 (EscheicAio cofi); Redesignation of a Bacterial 'Cytochrome q

Research paper thumbnail of Continuous monitoring of cellulase action on microcrystalline cellulose

Appl Microbiol Biotechnol, 1992

Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrysta... more Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for cellobiose oxidase, in Ixmol" m i n-~. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a Km of 4.8-+ 1.0 (g cellulose). 1-i , which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375 + 25 gmol. (g cellulase)-~. m i n-~ in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6 + 1.3 Ixmol. (g cellulose)-1. min-1.

Research paper thumbnail of Characterization of a split respiratory pathway in the wheat ""take-all"" fungus Gaeumannomyces graminis var. i tritici

This article describes the first detailed analysis of mitochondrial electron transfer and oxidati... more This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a ⌬ across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.

Research paper thumbnail of The kinetics and specificity of electron transfer from cytochromes and copper proteins to P700

Bba Bioenergetics, 1975

The rates of electron transfer to P700 from plastocyanin and cytochrome f have been compared with... more The rates of electron transfer to P700 from plastocyanin and cytochrome f have been compared with those from three other c-type cytochromes and azurin, a copper protein resembling plastocyanin. Three different disruptive techniques were used to expose P700; digitonin, Triton X-100 and sonication. The following rate constants were measured at 25 degrees C, pH 7.0, with digitonin-treated chloroplasts: plastocyanin, 8 x 10(7)M(-1) x s(-1); red-algal cytochrome c-553, 1.9 x 10(7)M(-1) x s (-1); Pseudomonas cytochrome c-551, 8 x 10(6)M(-1) x s (-1); azurin, less than or = 3 x 10(5)M(-1) x s (-1); cytochrome f, less than or = 2 x 10(4)M(-1) x s (-1); mammalian cytochrome c, less than or = 2 x 10(4)M(-1) x s (-1). For electron transfer from plastocyanin, the effects of ionic strength, pH and temperature were also studied, and saturation effects found in earlier work were avoided by a full consideration of the various secondary reactions and inclusion of superoxide dismutase. The relative rates are discussed in relation to photosynthetic electron transport.

Research paper thumbnail of Iron uptake by fungi: Contrasted mechanisms with internal or external reduction

Advances in Microbial Physiology, 2000

Almost all iron uptake by fungi involves reduction from Fe(III) to Fe(II) in order to facilitate ... more Almost all iron uptake by fungi involves reduction from Fe(III) to Fe(II) in order to facilitate ligand exchange. This leads to two mechanisms: uptake before reduction, or reduction before uptake. Many fungi secrete specific hydroxamate siderophores when short of iron. The mechanism with uptake before reduction is described in the context of siderophore synthesis and usage, since it applies to many (but not all) siderophores. The hydroxamate functional group is synthesized from ornithine by N5 hydroxylation and acylation. In most fungal siderophores, two or three modified ornithines are joined together by a non-ribosomal peptide synthetase. The transcription of these genes is regulated by an iron activated repressor. There is evidence that the iron-free siderophore may be stored in intracellular vesicles until secretion is required. After loading with iron, re-entry is likely to be via a proton symport. In some fungi, siderophores are used for iron storage. The iron is liberated by an NADPH-linked reductase. The second mechanism starts with Fe(III) reduction. In yeast, this is catalysed by an NADPH-linked transmembrane reductase, which has homology with the NADPH oxidase of neutrophils. There are two closely similar reductases with overlapping roles in Fe(III) and Cu(II) reduction, while the substrates for reduction include Fe(III)-siderophores. External reductants, which may be important in certain fungi, include 3-hydroxyanthranilic acid, melanin, cellobiose dehydrogenase and 2,5-dimethylhydroquinone. In yeast, a high-affinity iron uptake pathway involves reoxidation of Fe(II) to Fe(III), probably to confer specificity for iron. This is catalysed by a copper protein which has homology with ceruloplasmin, and is closely coupled to Fe(III) transport. The transcription of these genes is regulated by an iron-inhibited activator. Because of its copper requirement, the high-affinity pathway is blocked by disruption of genes for copper metabolism. A low-affinity uptake transports Fe(II) directly and is important in anoxic growth. In many fungi, mechanisms with internal or external reduction are both important. The external reduction is applicable to almost any Fe(III) complex, while internal reduction is more efficient at low iron but requires a siderophore permease through which toxins might enter. Both mechanisms require close coupling of Fe(III) reduction and Fe(II) utilization in order to minimize production of active oxygen.

Research paper thumbnail of Production of Fenton's reagent by cellobiose oxidase from cellulolytic cultures of Phanerochaete chrysosporium

European Journal of Biochemistry, 1992

The reduction of dioxygen by cellobiose oxidase leads to accumulation of H202, with either cellob... more The reduction of dioxygen by cellobiose oxidase leads to accumulation of H202, with either cellobiose or microcrystalline cellulose as electron donor. Cellobiose oxidase will also reduce many Fe(II1) complexes, including Fe(II1) acetate. Many Fe(I1) complexes react with H 2 0 2 to produce hydroxyl radicals or a similarly reactive species in the Fenton reaction as shown: H 2 0 2 + Fe2+ + HO' + HO-+ Fe3+. The hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid and 2,5dihydroxybenzoic acid is a standard test for hydroxyl radicals. Hydroxylation was observed in acetate buffer (pH 4.0), both with Fe(I1) plus H 2 0 2 and with cellobiose oxidase plus cellobiose, O2 and Fe(II1). The hydroxylation was suppressed by addition of catalase or the absence of iron [Fe(II) or Fe(II1) as appropriate]. Another test for hydroxyl radicals is the conversion of deoxyribose to malondialdehyde; this gave positive results under similar conditions. Further experiments used an O 2 electrode. Addition of H 2 0 2 to Fe(I1) acetate (pH 4.0) or Fe(I1) phosphate (pH 2.8) in the absence of enzyme led to a pulse of O2 uptake, as expected from production of hydroxyl radicals as shown: RH + HO'-f R' + H 2 0 ; R' + O 2-+ RO, + products. With phosphate (pH 2.8) or 10 mM acetate (pH 4.0), the O2 uptake pulse was increased by Avicel, suggesting that the Avicel was being damaged.

Research paper thumbnail of Evidence that cellobiose oxidase from Phanerochaete chrysosporium is primarily an Fe(III) reductas. Kinetic comparison with neutrophil NADPH oxidase and yeast flavocytochrome b2

European Journal of Biochemistry, 1992

Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30°C... more Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30°C , with excess cellobiose as substrate and O2 or Fe(II1) as acceptor. With 0, at 230 pM as sole electron acceptor, the O2 uptake rate corresponded to a one-electron turnover number of 0.13 & 0.01 s-', Measurements at different O2 concentrations indicated Km(Oz) > 120 pM. In separate experiments, the reduction of Fe(II1) acetate was monitored at 340 nm in the absence of oxygen. The maximum velocity of Fe(II1)-acetate reduction (VmJ was 4.5 L-0.7 s-', while K,[Fe(III) acetate] was 34 12 pM. With ferricyanide in place of Fe(IJ1) acetate, the corresponding values were 6.9 f 0.7 s-l and 23-t 5 pM, Redox titrations established the potential of the haem prosthetic group of the oxidase at pH 4.0 as +165 mV. The midpoint potential for Fe(III)/Fe(II) acetate at pH 4.0 is much higher, a value of +535 mV being obtained with 200 pM Fe. Cellobiose oxidase resembles yeast flavocytochrome b2 and differs from the neutrophil NADPH oxidase in having the potential of its haem group far above the potential for one-electron reduction of O 2 to superoxide (Em,4 =-110 mV). A kinetic comparison led to the conclusion that the role of cellobiose oxidase is as an Fe(1II) reductase. Fe(I1) may have a biological importance as a component of Fenton's reagent [Fe(II)/ HzOz]. The concentration of cellobiose oxidase in the growth medium at harvest (0.3 pM) can provide a far higher flux of Fe(I1) than a non-enzymic proposal in the literature.

Research paper thumbnail of The Roles of c-Type Cytochromes in Algal Photosynthesis. Extraction from Algae of a Cytochrome Similar to Higher Plant Cytochrome f

European Journal of Biochemistry, 1977

A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extrac... more A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas, Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome & with a-band maxima near 554 nm, very asymmetrical fl-bands, and y-band maxima at 421 nm. The cytochrome from Anacystis had c1 and y-bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as + 350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31 000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c-552, and was unaffected by Euglena cytochrome c-552 antiserum.

Research paper thumbnail of Interchangeable Copper and Iron Proteins in Algal Photosynthesis. Studies on Plastocyanin and Cytochrome c-552 in Chlamydomonas

European Journal of Biochemistry, 1978

The interrelation of the copper protein plastocyanin, and a soluble c-type cytochrome, c-552, in ... more The interrelation of the copper protein plastocyanin, and a soluble c-type cytochrome, c-552, in photosynthetic electron transport has been studied in the genus Chlamydomonas. With C. reinhardtii the plastocyanin : cytochrome c-552 ratio could be changed from 300: 1 to < 1 : 16 simply by omitting copper from the medium, without any other detectable change. Plastocyanin was indetectable in a second species, C. mundana, for which the cytochrome c-552 level was always very high. The properties of Levine's C. reinhardtii mutant lacking plastocyanin, ac-208, were studied and it was found that the photosynthetic capabilities of a suppressed phenotype and suppressed genotype could be explained by reference to the cytochrome c-552 levels. Both proteins were successfully used in reconstitution experiments with chloroplast fragments. Both showed very fast kinetics for reduction by purified Chlamydomonas cytochromef, but the rate of electron transfer from one to the other was much slower. It is concluded that they constitute an interchangeable pair, and the rationale for this and possible analogies are both discussed.

Research paper thumbnail of Autotrophic and heterotrophic mechanisms for ammonia oxidation

Soil Use and Management, 1990

Autotrophic nitrifiers such as Nitmsomonm use ammonia mono-oxygenase for the initial stage of amm... more Autotrophic nitrifiers such as Nitmsomonm use ammonia mono-oxygenase for the initial stage of ammonia oxidation. Nitrification inhibitors have this enzyme as their site of action. Their mechanisms include alternative substrates, suicide substrates and cuprous copper chelators. In heterotrophs, organic nitrogen is normally in the fully reduced state, but a few cell metabolites contain N-0 bonds. The synthesis and breakdown of such compounds provides a mechanism for heterotrophic nitrification. A non-enzymic mechanism for nitrogen-oxidation involves hydroxyl radicals produced by the Fenton reaction. Heterotrophic nitrification is particularly important in woodland soils, where wood-rotting fungi use free radicals to break down lignin. Tests for a radical mechanism are described.

Research paper thumbnail of Alternative oxidase reduces the sensitivity ofMycosphaerella graminicola to QOI fungicides

Pest Management Science, 2004

Forty-six (1.5%) of nearly 3000 isolates of Mycosphaerella graminicola assayed in vitro were resi... more Forty-six (1.5%) of nearly 3000 isolates of Mycosphaerella graminicola assayed in vitro were resistant to the QOI fungicide azoxystrobin, but on sub-culturing only ten remained resistant. Cross-resistance extended to other QOIs, but varied between different isolates. In planta the resistant isolates were not well controlled, especially at lower azoxystrobin dose rates. Propyl gallate, an inhibitor of alternative oxidase, potentiated the activity of azoxystrobin in vitro so that resistance was no longer observed. The growth of resistant strains in the presence of azoxystrobin led to alternative oxidase activation. This increased flexibility in respiration allows resistant strains to survive in the presence of a QOI fungicide. Under these conditions, selection for target-site mutations can occur. Using QOIs preventatively reduces the risk of resistance since the alternative oxidase cannot by itself generate all the energy needed for germination and early infection.

Research paper thumbnail of A critical evaluation of the role of alternative oxidase in the performance of strobilurin and related fungicides acting at the Qo site of Complex III

Pest Management Science, 2003

Mitochondrial respiration conserves energy by linking NADH oxidation and electron-coupled proton ... more Mitochondrial respiration conserves energy by linking NADH oxidation and electron-coupled proton translocation with ATP synthesis, through a core pathway involving three large protein complexes. Strobilurin fungicides block electron flow through one of these complexes (III), and disrupt energy supply. Despite an essential need for ATP throughout fungal disease development, strobilurins are largely preventative; indeed some diseases are not controlled at all, and several pathogens have quickly developed resistance. Target-site variation is not the only cause of these performance difficulties. Alternative oxidase (AOX) is a strobilurin-insensitive terminal oxidase that allows electrons from ubiquinol to bypass Complex III. Its synthesis is constitutive in some fungi but in many others is induced by inhibition of the main pathway. AOX provides a strobilurin-insensitive pathway for oxidation of NADH. Protons are pumped as electrons flow through Complex I, but energy conservation is less efficient than for the full respiratory chain. Salicylhydroxamic acid (SHAM) is a characteristic inhibitor of AOX, and several studies have explored the potentiation of strobilurin activity by SHAM. We present a kinetic-based model which relates changes in the extent of potentiation during different phases of disease development to a changing importance of energy efficiency. The model provides a framework for understanding the varying efficacy of strobilurin fungicides. In many cases, AOX can limit strobilurin effectiveness once an infection is established, but is unable to interfere significantly with strobilurin action during germination. A less stringent demand for energy efficiency during early disease development could lead to insensitivity towards this class of fungicides. This is discussed in relation to Botrytis cinerea, which is often poorly controlled by strobilurins. Mutations with a similar effect may explain evidence implicating AOX in resistance development in normally well-controlled plant pathogens, such as Venturia inaequalis.

Research paper thumbnail of Involvement of the alternative oxidase in cellular energy production in the wheat ‘take all’ fungus, Gaeumannomyces graminis var tritici

Pesticide Science, 1999

Abstract This summary describes a functional role for the alternative respiratory pathway of the ... more Abstract This summary describes a functional role for the alternative respiratory pathway of the wheat &#x27;take-all&#x27;fungus, Gaeumannomyces graminis var tritici (Ggt), which utilises the alternative oxidase as its terminal oxidase. Unlike other plant and fungal alternative ...

Research paper thumbnail of The Soluble Cytochrome Oxidase of Nitrosomonas europaea

Microbiology, 1983

The soluble cytochrome oxidase of Nitrosomonas europaea has been highly purified and shown to be ... more The soluble cytochrome oxidase of Nitrosomonas europaea has been highly purified and shown to be a copper protein devoid of haem, not a cytochrome o as was previously assumed. The native molecular weight was 120000 and the subunit molecular weight 35000. Soluble cytochrome oxidase activity co-purified with nitrite reductase activity; both activities were almost certainly associated with the same protein. The possible physiological role of the nitrite reductase activity is discussed.

Research paper thumbnail of Characterization of a Split Respiratory Pathway in the Wheat "Take-all" Fungus, Gaeumannomyces graminis var. tritici

Journal of Biological Chemistry, 1998

This article describes the first detailed analysis of mitochondrial electron transfer and oxidati... more This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a ⌬ across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.

Research paper thumbnail of A Kinetic and ESR Investigation of Iron(II) Oxalate Oxidation by Hydrogen Peroxide and Dioxygen as a Source of Hydroxyl Radicals

Free Radical Research, 1997

The reaction of Fe(II) oxalate with hydrogen peroxide and dioxygen was studied for oxalate concen... more The reaction of Fe(II) oxalate with hydrogen peroxide and dioxygen was studied for oxalate concentrations up to 20 mM and pH 2-5, under which conditions mono- and bis-oxalate complexes (Fe[II](ox) and Fe[II](ox)2[2-]) and uncomplexed Fe2+ must be considered. The reaction of Fe(II) oxalate with hydrogen peroxide (Fe2+ + H2O2 --&amp;amp;gt; Fe3+ + .OH + OH-) was monitored in continuous flow by ESR with t-butanol as a radical trap. The reaction is much faster than for uncomplexed Fe2+ and a rate constant, k = 1 x 10(4) M(-1) s(-1) is deduced for Fe(II)(ox). The reaction of Fe(II) oxalate with dioxygen is strongly pH dependent in a manner which indicates that the reactive species is Fe(II)(ox)2(2-), for which an apparent second order rate constant, k = 3.6 M(-1) s(-1), is deduced. Taken together, these results provide a mechanism for hydroxyl radical production in aqueous systems containing Fe(II) complexed by oxalate. Further ESR studies with DMPO as spin trap reveal that reaction of Fe(II) oxalate with hydrogen peroxide can also lead to formation of the carboxylate radical anion (CO2-), an assignment confirmed by photolysis of Fe(II) oxalate in the presence of DMPO.

Research paper thumbnail of Pathways for production of Fenton's reagent by wood-rotting fungi

FEMS Microbiology Reviews, 1994

Many forms of Fe(II) react with H20 2 to generate hydroxyl radicals (Fenton reaction). There is e... more Many forms of Fe(II) react with H20 2 to generate hydroxyl radicals (Fenton reaction). There is evidence that hydroxyl radicals are important in brown-rot, while they can be formed by secondary reactions during lignin breakdown by white-rot fungi. Their involvement in cellulose breakdown creates a range of oxidized sugars. The two reactants of Fenton's reagent can be generated by Fe(II) autoxidation, or by superoxide in reaction with Fe(III). A rapid autoxidation is not possible for complexes with a high Fe(III)/Fe(II) redox potential. Turning to specific pathways for formation of Fenton's reagent, decomposition of Fe(III)-oxalate is probably solely a photochemical process. Lignin peroxidases can act indirectly as a source of superoxide, either by reactions that lead to a peroxyradical, or by 1-electron oxidation of an aliphatic compound creating a strong reductant. Cellobiose dehydrogenase can provide a direct enzymic source for Fenton's reagent (S.M. Kremer and P.M. Wood (1992) Eur. J. Biochem. 208. 807-814). In the experiments as published, hydroxyl radical production was limited by the slow interaction of cellobiose dehydrogenase with 0 2. This limitation can be removed by the presence of an iron complex with an autoxidizable Fe(lI) state. The successful use of Fenton's reagent by a living organism requires a spatial separation between initiating enzyme(s) and the site of production of hydroxyl radicals. The mobility of the extra electron on Fe(II) by intermolecular transfer may be important for achieving this separation.

Research paper thumbnail of Kinetic and antigenic similarities for cellobiose dehydrogenase from the brown rot fungus Coniophora puteana and the white rot fungus Phanerochaete chrysosporium

FEMS Microbiology Letters, 1996

Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana. Strong cross-... more Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana. Strong cross-reaction was observed with antibodies to cellobiose:quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium. Kinetic measurements were made with cellobiose as electron donor. Ferricyanide and DCPIP both showed a pH optimum close to pH 4, but activity with ferricyanide declined more rapidly when the pH was raised. Dioxygen reduction to hydrogen peroxide was observed, but at a much lower rate than for other acceptors. These properties are similar to those of cellobiose dehydrogenase from P. chrysosporium, despite differences between brown and white rot modes of decay.

Research paper thumbnail of Tri- and tetramethylhydroquinone as electron donors for ammonia monooxygenase in whole cells of Nitrosomonas europaea

FEMS Microbiology Letters, 1986

Ten redox reagents have been tested as electron donors to ammonia monooxygenase in whole ceils of... more Ten redox reagents have been tested as electron donors to ammonia monooxygenase in whole ceils of Nitrosomonas europaea. Positive results were ol~tained with tri-and tetramethylhydroquinone. An earlier study showed that phenol was converted into hydroquinone by the monooxygenase. Cells were therefore incubated with trimethyiphenol, to see if its hydroxylation to trimethylhydroquinone would lead to a self-sufficient conversion of trimethylphenol into trimethylquinone. No trimethylquinone could be detected. The maximal rates of propene epoxidation obtained with triand tetramethylhydroquinone were 1.8 and 4.6 #mol. h-1. mg protein-1, respectively.

Research paper thumbnail of EPR Evidence for hydroxyl- and substrate-derived radicals in Fe(II)-oxalate/hydrogen peroxide reactions. The importance of the reduction of Fe(III)-oxalate by oxygen-conjugated radicals to regenerate Fe(II) in reactions of carbohydrates and model compounds

Journal of the Chemical Society, Perkin Transactions 2

ABSTRACT

Research paper thumbnail of SHORT COMMUNICATION Haemoprotein 6-90 (EscheicAio cofi); Redesignation of a Bacterial 'Cytochrome q

Research paper thumbnail of Continuous monitoring of cellulase action on microcrystalline cellulose

Appl Microbiol Biotechnol, 1992

Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrysta... more Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for cellobiose oxidase, in Ixmol" m i n-~. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a Km of 4.8-+ 1.0 (g cellulose). 1-i , which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375 + 25 gmol. (g cellulase)-~. m i n-~ in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6 + 1.3 Ixmol. (g cellulose)-1. min-1.

Research paper thumbnail of Characterization of a split respiratory pathway in the wheat ""take-all"" fungus Gaeumannomyces graminis var. i tritici

This article describes the first detailed analysis of mitochondrial electron transfer and oxidati... more This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a ⌬ across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.

Research paper thumbnail of The kinetics and specificity of electron transfer from cytochromes and copper proteins to P700

Bba Bioenergetics, 1975

The rates of electron transfer to P700 from plastocyanin and cytochrome f have been compared with... more The rates of electron transfer to P700 from plastocyanin and cytochrome f have been compared with those from three other c-type cytochromes and azurin, a copper protein resembling plastocyanin. Three different disruptive techniques were used to expose P700; digitonin, Triton X-100 and sonication. The following rate constants were measured at 25 degrees C, pH 7.0, with digitonin-treated chloroplasts: plastocyanin, 8 x 10(7)M(-1) x s(-1); red-algal cytochrome c-553, 1.9 x 10(7)M(-1) x s (-1); Pseudomonas cytochrome c-551, 8 x 10(6)M(-1) x s (-1); azurin, less than or = 3 x 10(5)M(-1) x s (-1); cytochrome f, less than or = 2 x 10(4)M(-1) x s (-1); mammalian cytochrome c, less than or = 2 x 10(4)M(-1) x s (-1). For electron transfer from plastocyanin, the effects of ionic strength, pH and temperature were also studied, and saturation effects found in earlier work were avoided by a full consideration of the various secondary reactions and inclusion of superoxide dismutase. The relative rates are discussed in relation to photosynthetic electron transport.

Research paper thumbnail of Iron uptake by fungi: Contrasted mechanisms with internal or external reduction

Advances in Microbial Physiology, 2000

Almost all iron uptake by fungi involves reduction from Fe(III) to Fe(II) in order to facilitate ... more Almost all iron uptake by fungi involves reduction from Fe(III) to Fe(II) in order to facilitate ligand exchange. This leads to two mechanisms: uptake before reduction, or reduction before uptake. Many fungi secrete specific hydroxamate siderophores when short of iron. The mechanism with uptake before reduction is described in the context of siderophore synthesis and usage, since it applies to many (but not all) siderophores. The hydroxamate functional group is synthesized from ornithine by N5 hydroxylation and acylation. In most fungal siderophores, two or three modified ornithines are joined together by a non-ribosomal peptide synthetase. The transcription of these genes is regulated by an iron activated repressor. There is evidence that the iron-free siderophore may be stored in intracellular vesicles until secretion is required. After loading with iron, re-entry is likely to be via a proton symport. In some fungi, siderophores are used for iron storage. The iron is liberated by an NADPH-linked reductase. The second mechanism starts with Fe(III) reduction. In yeast, this is catalysed by an NADPH-linked transmembrane reductase, which has homology with the NADPH oxidase of neutrophils. There are two closely similar reductases with overlapping roles in Fe(III) and Cu(II) reduction, while the substrates for reduction include Fe(III)-siderophores. External reductants, which may be important in certain fungi, include 3-hydroxyanthranilic acid, melanin, cellobiose dehydrogenase and 2,5-dimethylhydroquinone. In yeast, a high-affinity iron uptake pathway involves reoxidation of Fe(II) to Fe(III), probably to confer specificity for iron. This is catalysed by a copper protein which has homology with ceruloplasmin, and is closely coupled to Fe(III) transport. The transcription of these genes is regulated by an iron-inhibited activator. Because of its copper requirement, the high-affinity pathway is blocked by disruption of genes for copper metabolism. A low-affinity uptake transports Fe(II) directly and is important in anoxic growth. In many fungi, mechanisms with internal or external reduction are both important. The external reduction is applicable to almost any Fe(III) complex, while internal reduction is more efficient at low iron but requires a siderophore permease through which toxins might enter. Both mechanisms require close coupling of Fe(III) reduction and Fe(II) utilization in order to minimize production of active oxygen.

Research paper thumbnail of Production of Fenton's reagent by cellobiose oxidase from cellulolytic cultures of Phanerochaete chrysosporium

European Journal of Biochemistry, 1992

The reduction of dioxygen by cellobiose oxidase leads to accumulation of H202, with either cellob... more The reduction of dioxygen by cellobiose oxidase leads to accumulation of H202, with either cellobiose or microcrystalline cellulose as electron donor. Cellobiose oxidase will also reduce many Fe(II1) complexes, including Fe(II1) acetate. Many Fe(I1) complexes react with H 2 0 2 to produce hydroxyl radicals or a similarly reactive species in the Fenton reaction as shown: H 2 0 2 + Fe2+ + HO' + HO-+ Fe3+. The hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid and 2,5dihydroxybenzoic acid is a standard test for hydroxyl radicals. Hydroxylation was observed in acetate buffer (pH 4.0), both with Fe(I1) plus H 2 0 2 and with cellobiose oxidase plus cellobiose, O2 and Fe(II1). The hydroxylation was suppressed by addition of catalase or the absence of iron [Fe(II) or Fe(II1) as appropriate]. Another test for hydroxyl radicals is the conversion of deoxyribose to malondialdehyde; this gave positive results under similar conditions. Further experiments used an O 2 electrode. Addition of H 2 0 2 to Fe(I1) acetate (pH 4.0) or Fe(I1) phosphate (pH 2.8) in the absence of enzyme led to a pulse of O2 uptake, as expected from production of hydroxyl radicals as shown: RH + HO'-f R' + H 2 0 ; R' + O 2-+ RO, + products. With phosphate (pH 2.8) or 10 mM acetate (pH 4.0), the O2 uptake pulse was increased by Avicel, suggesting that the Avicel was being damaged.

Research paper thumbnail of Evidence that cellobiose oxidase from Phanerochaete chrysosporium is primarily an Fe(III) reductas. Kinetic comparison with neutrophil NADPH oxidase and yeast flavocytochrome b2

European Journal of Biochemistry, 1992

Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30°C... more Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30°C , with excess cellobiose as substrate and O2 or Fe(II1) as acceptor. With 0, at 230 pM as sole electron acceptor, the O2 uptake rate corresponded to a one-electron turnover number of 0.13 & 0.01 s-', Measurements at different O2 concentrations indicated Km(Oz) > 120 pM. In separate experiments, the reduction of Fe(II1) acetate was monitored at 340 nm in the absence of oxygen. The maximum velocity of Fe(II1)-acetate reduction (VmJ was 4.5 L-0.7 s-', while K,[Fe(III) acetate] was 34 12 pM. With ferricyanide in place of Fe(IJ1) acetate, the corresponding values were 6.9 f 0.7 s-l and 23-t 5 pM, Redox titrations established the potential of the haem prosthetic group of the oxidase at pH 4.0 as +165 mV. The midpoint potential for Fe(III)/Fe(II) acetate at pH 4.0 is much higher, a value of +535 mV being obtained with 200 pM Fe. Cellobiose oxidase resembles yeast flavocytochrome b2 and differs from the neutrophil NADPH oxidase in having the potential of its haem group far above the potential for one-electron reduction of O 2 to superoxide (Em,4 =-110 mV). A kinetic comparison led to the conclusion that the role of cellobiose oxidase is as an Fe(1II) reductase. Fe(I1) may have a biological importance as a component of Fenton's reagent [Fe(II)/ HzOz]. The concentration of cellobiose oxidase in the growth medium at harvest (0.3 pM) can provide a far higher flux of Fe(I1) than a non-enzymic proposal in the literature.

Research paper thumbnail of The Roles of c-Type Cytochromes in Algal Photosynthesis. Extraction from Algae of a Cytochrome Similar to Higher Plant Cytochrome f

European Journal of Biochemistry, 1977

A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extrac... more A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas, Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome & with a-band maxima near 554 nm, very asymmetrical fl-bands, and y-band maxima at 421 nm. The cytochrome from Anacystis had c1 and y-bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as + 350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31 000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c-552, and was unaffected by Euglena cytochrome c-552 antiserum.

Research paper thumbnail of Interchangeable Copper and Iron Proteins in Algal Photosynthesis. Studies on Plastocyanin and Cytochrome c-552 in Chlamydomonas

European Journal of Biochemistry, 1978

The interrelation of the copper protein plastocyanin, and a soluble c-type cytochrome, c-552, in ... more The interrelation of the copper protein plastocyanin, and a soluble c-type cytochrome, c-552, in photosynthetic electron transport has been studied in the genus Chlamydomonas. With C. reinhardtii the plastocyanin : cytochrome c-552 ratio could be changed from 300: 1 to < 1 : 16 simply by omitting copper from the medium, without any other detectable change. Plastocyanin was indetectable in a second species, C. mundana, for which the cytochrome c-552 level was always very high. The properties of Levine's C. reinhardtii mutant lacking plastocyanin, ac-208, were studied and it was found that the photosynthetic capabilities of a suppressed phenotype and suppressed genotype could be explained by reference to the cytochrome c-552 levels. Both proteins were successfully used in reconstitution experiments with chloroplast fragments. Both showed very fast kinetics for reduction by purified Chlamydomonas cytochromef, but the rate of electron transfer from one to the other was much slower. It is concluded that they constitute an interchangeable pair, and the rationale for this and possible analogies are both discussed.

Research paper thumbnail of Autotrophic and heterotrophic mechanisms for ammonia oxidation

Soil Use and Management, 1990

Autotrophic nitrifiers such as Nitmsomonm use ammonia mono-oxygenase for the initial stage of amm... more Autotrophic nitrifiers such as Nitmsomonm use ammonia mono-oxygenase for the initial stage of ammonia oxidation. Nitrification inhibitors have this enzyme as their site of action. Their mechanisms include alternative substrates, suicide substrates and cuprous copper chelators. In heterotrophs, organic nitrogen is normally in the fully reduced state, but a few cell metabolites contain N-0 bonds. The synthesis and breakdown of such compounds provides a mechanism for heterotrophic nitrification. A non-enzymic mechanism for nitrogen-oxidation involves hydroxyl radicals produced by the Fenton reaction. Heterotrophic nitrification is particularly important in woodland soils, where wood-rotting fungi use free radicals to break down lignin. Tests for a radical mechanism are described.

Research paper thumbnail of Alternative oxidase reduces the sensitivity ofMycosphaerella graminicola to QOI fungicides

Pest Management Science, 2004

Forty-six (1.5%) of nearly 3000 isolates of Mycosphaerella graminicola assayed in vitro were resi... more Forty-six (1.5%) of nearly 3000 isolates of Mycosphaerella graminicola assayed in vitro were resistant to the QOI fungicide azoxystrobin, but on sub-culturing only ten remained resistant. Cross-resistance extended to other QOIs, but varied between different isolates. In planta the resistant isolates were not well controlled, especially at lower azoxystrobin dose rates. Propyl gallate, an inhibitor of alternative oxidase, potentiated the activity of azoxystrobin in vitro so that resistance was no longer observed. The growth of resistant strains in the presence of azoxystrobin led to alternative oxidase activation. This increased flexibility in respiration allows resistant strains to survive in the presence of a QOI fungicide. Under these conditions, selection for target-site mutations can occur. Using QOIs preventatively reduces the risk of resistance since the alternative oxidase cannot by itself generate all the energy needed for germination and early infection.

Research paper thumbnail of A critical evaluation of the role of alternative oxidase in the performance of strobilurin and related fungicides acting at the Qo site of Complex III

Pest Management Science, 2003

Mitochondrial respiration conserves energy by linking NADH oxidation and electron-coupled proton ... more Mitochondrial respiration conserves energy by linking NADH oxidation and electron-coupled proton translocation with ATP synthesis, through a core pathway involving three large protein complexes. Strobilurin fungicides block electron flow through one of these complexes (III), and disrupt energy supply. Despite an essential need for ATP throughout fungal disease development, strobilurins are largely preventative; indeed some diseases are not controlled at all, and several pathogens have quickly developed resistance. Target-site variation is not the only cause of these performance difficulties. Alternative oxidase (AOX) is a strobilurin-insensitive terminal oxidase that allows electrons from ubiquinol to bypass Complex III. Its synthesis is constitutive in some fungi but in many others is induced by inhibition of the main pathway. AOX provides a strobilurin-insensitive pathway for oxidation of NADH. Protons are pumped as electrons flow through Complex I, but energy conservation is less efficient than for the full respiratory chain. Salicylhydroxamic acid (SHAM) is a characteristic inhibitor of AOX, and several studies have explored the potentiation of strobilurin activity by SHAM. We present a kinetic-based model which relates changes in the extent of potentiation during different phases of disease development to a changing importance of energy efficiency. The model provides a framework for understanding the varying efficacy of strobilurin fungicides. In many cases, AOX can limit strobilurin effectiveness once an infection is established, but is unable to interfere significantly with strobilurin action during germination. A less stringent demand for energy efficiency during early disease development could lead to insensitivity towards this class of fungicides. This is discussed in relation to Botrytis cinerea, which is often poorly controlled by strobilurins. Mutations with a similar effect may explain evidence implicating AOX in resistance development in normally well-controlled plant pathogens, such as Venturia inaequalis.

Research paper thumbnail of Involvement of the alternative oxidase in cellular energy production in the wheat ‘take all’ fungus, Gaeumannomyces graminis var tritici

Pesticide Science, 1999

Abstract This summary describes a functional role for the alternative respiratory pathway of the ... more Abstract This summary describes a functional role for the alternative respiratory pathway of the wheat &#x27;take-all&#x27;fungus, Gaeumannomyces graminis var tritici (Ggt), which utilises the alternative oxidase as its terminal oxidase. Unlike other plant and fungal alternative ...

Research paper thumbnail of The Soluble Cytochrome Oxidase of Nitrosomonas europaea

Microbiology, 1983

The soluble cytochrome oxidase of Nitrosomonas europaea has been highly purified and shown to be ... more The soluble cytochrome oxidase of Nitrosomonas europaea has been highly purified and shown to be a copper protein devoid of haem, not a cytochrome o as was previously assumed. The native molecular weight was 120000 and the subunit molecular weight 35000. Soluble cytochrome oxidase activity co-purified with nitrite reductase activity; both activities were almost certainly associated with the same protein. The possible physiological role of the nitrite reductase activity is discussed.

Research paper thumbnail of Characterization of a Split Respiratory Pathway in the Wheat "Take-all" Fungus, Gaeumannomyces graminis var. tritici

Journal of Biological Chemistry, 1998

This article describes the first detailed analysis of mitochondrial electron transfer and oxidati... more This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a ⌬ across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.

Research paper thumbnail of A Kinetic and ESR Investigation of Iron(II) Oxalate Oxidation by Hydrogen Peroxide and Dioxygen as a Source of Hydroxyl Radicals

Free Radical Research, 1997

The reaction of Fe(II) oxalate with hydrogen peroxide and dioxygen was studied for oxalate concen... more The reaction of Fe(II) oxalate with hydrogen peroxide and dioxygen was studied for oxalate concentrations up to 20 mM and pH 2-5, under which conditions mono- and bis-oxalate complexes (Fe[II](ox) and Fe[II](ox)2[2-]) and uncomplexed Fe2+ must be considered. The reaction of Fe(II) oxalate with hydrogen peroxide (Fe2+ + H2O2 --&amp;amp;gt; Fe3+ + .OH + OH-) was monitored in continuous flow by ESR with t-butanol as a radical trap. The reaction is much faster than for uncomplexed Fe2+ and a rate constant, k = 1 x 10(4) M(-1) s(-1) is deduced for Fe(II)(ox). The reaction of Fe(II) oxalate with dioxygen is strongly pH dependent in a manner which indicates that the reactive species is Fe(II)(ox)2(2-), for which an apparent second order rate constant, k = 3.6 M(-1) s(-1), is deduced. Taken together, these results provide a mechanism for hydroxyl radical production in aqueous systems containing Fe(II) complexed by oxalate. Further ESR studies with DMPO as spin trap reveal that reaction of Fe(II) oxalate with hydrogen peroxide can also lead to formation of the carboxylate radical anion (CO2-), an assignment confirmed by photolysis of Fe(II) oxalate in the presence of DMPO.

Research paper thumbnail of Pathways for production of Fenton's reagent by wood-rotting fungi

FEMS Microbiology Reviews, 1994

Many forms of Fe(II) react with H20 2 to generate hydroxyl radicals (Fenton reaction). There is e... more Many forms of Fe(II) react with H20 2 to generate hydroxyl radicals (Fenton reaction). There is evidence that hydroxyl radicals are important in brown-rot, while they can be formed by secondary reactions during lignin breakdown by white-rot fungi. Their involvement in cellulose breakdown creates a range of oxidized sugars. The two reactants of Fenton's reagent can be generated by Fe(II) autoxidation, or by superoxide in reaction with Fe(III). A rapid autoxidation is not possible for complexes with a high Fe(III)/Fe(II) redox potential. Turning to specific pathways for formation of Fenton's reagent, decomposition of Fe(III)-oxalate is probably solely a photochemical process. Lignin peroxidases can act indirectly as a source of superoxide, either by reactions that lead to a peroxyradical, or by 1-electron oxidation of an aliphatic compound creating a strong reductant. Cellobiose dehydrogenase can provide a direct enzymic source for Fenton's reagent (S.M. Kremer and P.M. Wood (1992) Eur. J. Biochem. 208. 807-814). In the experiments as published, hydroxyl radical production was limited by the slow interaction of cellobiose dehydrogenase with 0 2. This limitation can be removed by the presence of an iron complex with an autoxidizable Fe(lI) state. The successful use of Fenton's reagent by a living organism requires a spatial separation between initiating enzyme(s) and the site of production of hydroxyl radicals. The mobility of the extra electron on Fe(II) by intermolecular transfer may be important for achieving this separation.

Research paper thumbnail of Kinetic and antigenic similarities for cellobiose dehydrogenase from the brown rot fungus Coniophora puteana and the white rot fungus Phanerochaete chrysosporium

FEMS Microbiology Letters, 1996

Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana. Strong cross-... more Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana. Strong cross-reaction was observed with antibodies to cellobiose:quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium. Kinetic measurements were made with cellobiose as electron donor. Ferricyanide and DCPIP both showed a pH optimum close to pH 4, but activity with ferricyanide declined more rapidly when the pH was raised. Dioxygen reduction to hydrogen peroxide was observed, but at a much lower rate than for other acceptors. These properties are similar to those of cellobiose dehydrogenase from P. chrysosporium, despite differences between brown and white rot modes of decay.

Research paper thumbnail of Tri- and tetramethylhydroquinone as electron donors for ammonia monooxygenase in whole cells of Nitrosomonas europaea

FEMS Microbiology Letters, 1986

Ten redox reagents have been tested as electron donors to ammonia monooxygenase in whole ceils of... more Ten redox reagents have been tested as electron donors to ammonia monooxygenase in whole ceils of Nitrosomonas europaea. Positive results were ol~tained with tri-and tetramethylhydroquinone. An earlier study showed that phenol was converted into hydroquinone by the monooxygenase. Cells were therefore incubated with trimethyiphenol, to see if its hydroxylation to trimethylhydroquinone would lead to a self-sufficient conversion of trimethylphenol into trimethylquinone. No trimethylquinone could be detected. The maximal rates of propene epoxidation obtained with triand tetramethylhydroquinone were 1.8 and 4.6 #mol. h-1. mg protein-1, respectively.