Paul M. Wood

Fungal respiration: a fusion of standard and alternative components.

In animals, electron transfer from NADH to molecular oxygen proceeds via large respiratory complexes in a linear respiratory chain. In contrast, most fungi utilise branched respiratory chains. These consist of alternative NADH dehydrogenases, which catalyse rotenone insensitive oxidation of matrix NADH or enable cytoplasmic NADH to be used directly. Many also contain an alternative oxidase that probably accepts electrons directly from ubiquinol. A few fungi lack Complex I. Although the alternative components are non-energy conserving, their organisation within the fungal electron transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally coupled to proton translocation through at least one site. The alternative oxidase enables respiration to continue in the presence of inhibitors for ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase. This may be particularly important for fungal pathogens, since host defence mechanisms often involve nitric oxide, which, whilst being a potent inhibitor of cytochrome c oxidase, has no inhibitory effect on alternative oxidase. Alternative NADH dehydrogenases may avoid the active oxygen production associated with Complex I. The expression and activity regulation of alternative components responds to factors ranging from oxidative stress to the stage of fungal development.

A Mechanism for Production of Hydroxyl Radicals by the Brown-Rot Fungus Coniophora Puteana: Fe(III) Reduction by Cellobiose Dehydrogenase and Fe(II) Oxidation at a Distance from the Hyphae

In timber infested by brown-rot fungi, a rapid loss of strength is attributed to production of hydroxyl radicals (HO.). The hydroxyl radicals are produced by the Fenton reaction [Fe(II)/H2O2], but the pathways leading to Fe(II) and H2O2 have remained unclear. Cellobiose dehydrogenase, purified from cultures of Coniophora puteana, has been shown to couple oxidation of cellodextrins to conversion of Fe(III) to Fe(II). Two characteristics of brown rot are release of oxalic acid and lowering of the local pH, often to about pH 2. Modelling of Fe(II) speciation in the presence of oxalate has revealed that Fe(II)-oxalate complexes are important at pH 4-5, but at pH 2 almost all Fe(II) is in an uncomplexed state which reacts very slowly with dioxygen. Diffusion of Fe(II) away from the hyphae will promote conversion to Fe(II)-oxalate and autoxidation with H2O2 as product. Thus the critical Fe(II)/H2O2 combination will be generated at a distance, enabling hydroxyl radicals to be formed without damage to the hyphae.

Methane oxidation by Nitrosomonas europaea

Incubation of whole cells of the nitrifying bacterium Nitrosomonas europaea with ethylene led to the formation of ethylene oxide. Ethylene oxide production was prevented by inhibitors of ammonium ion oxidation, and showed properties implying that ethylene is a substrate for the ammonia oxidising enzyme, ammonia monooxygenase. Endogenous substrates, hydroxylamine, hydrazine and ammonium ions were compared as sources of reducing power in terms of rates and stoichiometries of ethylene oxidation. The highest rates of ethylene oxide formation (15 μmol h-1 mg protein-1) were obtained with hydrazine as donor. The data suggest that at high concentrations of ethylene the rate of oxidation is limited by the rate at which reducing power can be supplied to the monooxygenase, not by an intrinsic Vmax. Ethylene had an inhibitory effect on the rate of ammonium ion utilisation; an approximate Ki of 80 μM was derived, but the results deviated from simple competitive behaviour. Measurement of relative rates of ethylene oxide formation and ammonium ion utilization led to a kcat/Km value for ethylene of 1.1 relative to NH4+, or 0.04 relative to the true natural substrate, NH3. The effects of higher concentrations of ethylene oxide on oxygen uptake rates were also investigated. The results imply that ethylene oxide is also a substrate for the monooxygenase, but with a much lower affinity than ethylene.

Evidence that cellobiose:quinone oxidoreductase from Phanerochaete chrysosporium is a breakdown product of cellobiose oxidase

Phanerochaete chrysosporium releases two enzymes that oxidize cellobiose and higher cellodextrins: the flavohaemoprotein cellobiose oxidase and the flavoprotein cellobiose:quinone oxidoreductase (CBQase). Partial digestion of these enzymes with Staphylococcal V8 proteinase or cyanogen bromide yielded many identical bands on SDS-polyacrylamide gels. A polyclonal antibody to either purified protein gave cross-reaction. The purification procedure also yielded a haem protein that ran on dodecyl sulphate gels at Mr 31,000, as compared with 91,000 for cellobiose oxidase and 63,000 for CBQase. The 31 kDa haem protein cross-reacted with polyclonal antibody to cellobiose oxidase, but not with antibody to CBQase. Sulphite bleached the flavin of cellobiose oxidase, but gave no reaction with the 31 kDa haem protein, suggesting an absence of flavin. It is proposed that CBQase and the 31 kDa haem protein are formed from cellobiose oxidase by proteolytic cleavage.

A kinetic study of benzene oxidation to phenol by whole cells of Nitrosomonas europaea and evidence for the further oxidation of phenol to hydroquinone

The oxidation of benzene to phenol by whole cells of Nitrosomonas europaea is catalysed by ammonia monooxygenase, and therefore requires a source of reducing power. Endogenous substrates, hydrazine, hydroxylamine and ammonium ions were compared as reductants. The highest rates of benzene oxidation were obtained with 4 mM benzene and hydrazine as reductant, and equalled 6 μmol· h-1·mg protein-1. The specificity of ammonia monooxygenase for benzene as a substrate was determined by measuring kcat/Km for benzene relative to kcat/Km for uncharged ammonia, a value of 0.4 being obtained. Phenol was found to be further hydroxylated to yield hydroquinone. This reaction, like benzene oxidation, was sensitive to the ammonia monooxygenase inhibitor allylthiourea. Catechol and resorcinol were not detected as products of phenol oxidation, implying that at least 88% of the hydroxylation is para-directed.

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

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 --> 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.

The Soluble Cytochrome Oxidase of Nitrosomonas europaea

SUMMARY: 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.

The redox potential for dimethyl sulphoxide reduction to dimethyl sulphide: evaluation and biochemical implications.

One is because this interconversion should be closely analogous to that of methionine sulphoxide and methionine; as Ejiri et al. have commented [l], it is reasonable to assume that cells must have mechanisms both to prevent methionine residues in proteins from becoming oxidised (e.g., by H,Oz) and also to reduce any methionine sulphoxide once formed. Mechanisms for reduction of methionine and biotin sulphoxides are well characterised in microorganisms [ 11. A more direct significance stems from observations implying that certain bacteria can use dimethyl sulphoxide as the electron sink for an energy-conserving electrontransport chain, in a manner similar to anaerobic growth of Escherichia coli on fumarate or nitrate [2]. Thus, Zinder and Brock have isolated a bacterium for which anaerobic growth on dimethyl sulphoxide was inhibited by azide or uncouplers, and found that a c-type cytochrome was prominent in its difference spectra [3]. It has also been shown that some photosynthetic bacteria can grow anaerobically in the presence of dimethyl sulphoxide [4,5], and for certain species transfer to medium containing dimethyl sulphoxide has resulted in synthesis of a characteristic c-type cytochrome ([S]; Jones, 0. T. G. and Ward,

Iron uptake by fungi: Contrasted mechanisms with internal or external reduction

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.

Why do c-type cytochromes exist?

The hypothesis presented is that the different classes of c‐type cytochrome originated as proteins located in the bacterial periplasmic space, or on the periplasmic side of the cytoplasmic membrane. In these locations, covalent bonds between haem and protein prevented the haem from being lost to the surrounding medium. Subsequent evolution has led to internal location of c‐type cytochromes in eucaryotes and cyanobacteria. The covalent links have been retained because of their structural role; a b‐type cytochrome could be created with similar molecular properties, but its formation would require a large evolutionary jump. If this hypothesis is correct, it should be useful in unravelling electron transport chains with unconventional donors or acceptors. Apparent exceptions deserve further investigation.