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M T Wilson

Publications and source records attributed to M T Wilson.

At least 91 records · Page 5Linked to original sources
PubMed1994

Electron transfer and ligand binding in terminal oxidases. Impact of recent structural information.

A consensus structure for the active site of terminal oxidases has been recently proposed by Hosler et al. [(1993) J. Bioenerg. Biomem. 25, 121-135]. We exploit the novel structural information to propose a hypothesis for the large difference in the rate of internal electron transfer found when experiments are started either with the reduced or with the oxidized enzyme. This rationale also allows us to discuss the oxidation state of the prevailing oxygen reacting species with reference to the concentration of the two substrates (oxygen and cytochrome c) and to the structural state of the oxidase.

Amino Acid Sequence↗
PubMed1994

Electron transfer from Phanerochaete chrysosporium cellobiose oxidase to equine cytochrome c and Pseudomonas aeruginosa cytochrome c-551.

The electron-transfer reactions of cellobiose oxidase (CBO) have been investigated by conventional and by rapid-scan stopped-flow spectroscopy at pH 6.0. Analysis of the absorbance/time/wavelength matrix by Singular Value Decomposition (SVD) confirms earlier studies showing that cellobiose rapidly reduces the flavin group (7.7 s-1; cellobiose, 100 microM) which in turn slowly (0.2 s-1) reduces the cytochrome b moiety. In the presence of CBO, cellobiose reduces cytochromes c in a reaction that does not depend on oxygen or superoxide. The rate limit for this process is independent of the source of the cytochromes c and is identical with the rate of cytochrome b reduction. Rapid-mixing experiments show that cytochrome b may donate electrons very rapidly to either mammalian cytochrome c or bacterial cytochrome c-551. The reactions were second-order (kc = 1.75 x 10(7) M-1 x s-1; kc-551 = 4.3 x 10(6) M-1 x s-1; pH 6.0, 21 degrees C and I0.064) and strongly ionic-strength (I)-dependent: kc decreasing with I and kc-551 increasing with I. These results suggest the electron-transfer site near cytochrome b bears a significant negative charge. Equilibrium gel chromatography confirms that CBO oxidase and positively charged mammalian cytochrome c make stable complexes. These results are discussed in terms of a model suggesting an electron-transfer role for cytochrome b in vivo, possibly connected with radical-mediated cellulose breakdown.

Animals↗
PubMed1994

The role of lipid hydroperoxides in the myoglobin-dependent oxidation of LDL.

It has previously been reported that mb in both the iron-oxo ferryl and the ferric oxidation states can promote lipid peroxidation and lead to oxidative modification of low-density lipoprotein. The mechanism of these oxidation reactions is unclear and could involve either lipid hydroperoxide-dependent or independent reactions. In order to ascertain which of the afore-mentioned mechanisms predominates, the effects of exogenous lipid hydroperoxides on the ability of Mb, in its various oxidation states, to oxidize low-density lipoprotein has been investigated. The results suggest that oxidation proceeds through a one-electron redox cycle between met and ferryl myoglobin and that the reactions of both redox forms are at least partially dependent on lipid hydroperoxides within the LDL particle.

Electrophoresis↗
PubMed1993

Kinetic and structural characterization of an intermediate in the biomineralization of bacterioferritin.

The mechanism by which iron-storage proteins take up and oxidise iron(II) is not understood. We show by rapid-kinetic and EPR measurements that iron uptake, in vitro, by a bacterial iron-storage protein, bacterioferritin, involves at least three kinetically distinguishable phases: phase 1, the binding of Fe(II) ions, probably at a dimeric iron ferroxidase centre; phase 2, oxidation of the Fe(II) dimer and production of mononuclear Fe(III); and phase 3, iron core formation.

Bacterial Proteins↗
PubMed1993

The oxidation of alpha-tocopherol in human low-density lipoprotein by the simultaneous generation of superoxide and nitric oxide.

Peroxynitrite is the product of the reaction between nitric oxide and superoxide. It is an oxidant which can also decompose to form the hydroxyl radical and nitrogen dioxide. In this report we show that a powerful oxidant with reactivity similar to that of the hydroxyl radical is formed from the generation of superoxide from xanthine oxidase and nitric oxide from S-nitroso-n-acetylpenicillamine (SNAP). Simultaneous generation of these two radicals by either xanthine oxidase/SNAP or the sydnonimine SIN-1 in the presence of low-density lipoprotein (LDL) results in the depletion of alpha-tocopherol and formation of its oxidised product alpha-tocopheroquinone. The mechanism of oxidation required both the formation of nitric oxide and superoxide. In contrast to the promotion of LDL oxidation by transition metals the oxidation of LDL by SIN-1 was not sensitive to the addition of exogenous lipid hydroperoxide.

Acetaldehyde↗
PubMed1992

The oxygen reactive species of cytochrome-c-oxidase: an alternative view.

In a recent review article Babcok and Wikström (Nature, 1992, 356, 301-309) proposed that the species of cytochrome-c-oxidase which binds molecular oxygen during turnover is the so-called mixed valence enzyme, in which the binuclear center cytochrome a3-CuB is reduced, while the cytochrome a/CuA sites are oxidized. This proposal is based on earlier work (Morgan and Wikström, Biochemistry 1991, 30, 948-958) in which it was found that the steady-state reduction levels of cytochrome c and cytochrome a in respiring rat liver mitochondria (sustained by ascorbate and TMPD) are quite different, the latter being much more oxidized than the former; evaluation of the steady-state reduction levels demanded a large correction due to the optical contribution of oxidized TMPD+ which overlaps with the cytochromes. We report below that application of transient spectroscopy and SVD analysis to respiring rat heart myocytes, under conditions in which the contribution of TMPD+ is very small or absent, allows to show that the steady-state reduction levels of cytochrome c and cytochrome a are comparable at all times accessible to measurement in the rapid-scanning stopped-flow spectrophotometer. Our conclusion, in agreement with previous results, is that mixed valence cytochrome-c-oxidase as defined above is not the prevailing oxygen binding species of cytochrome-c-oxidase, unless electron donation to cytochrome c becomes rate limiting.

Animals↗
PubMed1992

A 1H-NMR study of the interactions between rat tissue kallikrein and two peptide inhibitors.

The 1H-NMR spectra have been obtained for rat submandibular kallikrein in the absence and presence of inhibitors. Two competitive inhibitors were investigated, the tripeptide leupeptin (a potent inhibitor with Ki 0.5 microM) and a hexapeptide (a much weaker, substrate-analogue inhibitor with Ki 380 microM). Analysis of the NMR spectra showed that binding of leupeptin to kallikrein led to a change in the conformation of the enzyme, whereas binding of the substrate analogue to the enzyme produced no such change and may have resulted in a conformational change of the inhibitor.

Amino Acid Sequence↗
PubMed1992

Investigation of the electron-transfer properties of cytochrome c oxidase covalently cross-linked to Fe- or Zn-containing cytochrome c.

Complexes of cytochrome c oxidase and cytochrome c (Fe- or Zn-containing) have been prepared by 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide (EDC) cross-linking. The site to which the cytochrome c covalently binds has been identified as being the same, or close to, the site occupied by cytochrome c in the electrostatic complex which may be formed between the proteins. Stopped-flow experiments, monitored either at a single wavelength or through a rapid wavelength-scan facility, showed that covalently bound Fe-containing cytochrome c cannot donate electrons to cytochrome a. Free Fe-containing cytochrome c was, however, able to transfer electrons to cytochrome a in covalent complexes containing either Fe- or Zn-containing cytochrome c. Turnover experiments showed that the complexed enzyme remains catalytically competent but with decreased (40-80%) activity. The steady-state levels of reduction of both free cytochrome c and cytochrome a in the covalent complex were higher than found in the control (uncomplexed) enzyme. These results are discussed with reference to the structure of the covalent complex and lead us to conclude that cytochrome a may accept electrons directly from free cytochrome c and that cross-linking impairs the redox properties of the CuA site.

Animals↗
PubMed1992

Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae.

Recombinant human HbA, produced by co-expressing alpha-globin and beta-globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N-terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen-binding studies demonstrated that the yeast-derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb-based oxygen carrier.

Amino Acids↗
PubMed1992

Spectroscopic identification of the haem ligands of cellobiose oxidase.

A spectroscopic study of the flavocytochrome b enzyme, cellobiose oxidase, employing optical, NMR, EPR and near infra-red MCD techniques, has identified the axial ligands of the b-type haem. These are a histidine and a methionine, and this ligation set is discussed in relation to the functional role of the haem group.

Carbohydrate Dehydrogenases↗
PubMed1992

Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide.

Both nitric oxide (NO) and superoxide are generated by macrophages, neutrophils and endothelial cells. It has been postulated that the generation of these two radicals under physiological conditions can lead to the formation of peroxynitrite and (as a result of the homolytic lysis of this molecule) the production of hydroxyl radicals. We have used 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), a sydnonimine capable of generating both NO and superoxide simultaneously, to test this hypothesis. SIN-1 (1 mM) generated superoxide and NO at rates of 7.02 microM/min and 3.68 microM/min respectively in phosphate-buffered saline, pH 7.2, at 37 degrees C. Incubation of SIN-1 with both deoxyribose and sodium benzoate resulted in the formation of malondialdehyde (MDA). In addition, the incubation of SIN-1 with sodium benzoate resulted in the production of compounds with fluorescence emission spectra characteristic of hydroxylated products. Both the production of MDA and the generation of fluorescent compounds were inhibited by the hydroxyl radical scavenger mannitol. In all the above respects, SIN-1 mimicked the production of hydroxyl radicals from the ascorbate-driven Fenton reaction. Catalase had no effect on the SIN-1-dependent generation of MDA, and superoxide dismutase was partially inhibitory. SIN-1 produces an oxidant with the properties of the hydroxyl radical by a mechanism clearly different to that of the Fenton reaction. We conclude that the simultaneous production of NO and superoxide from SIN-1 results in the formation of hydroxyl radicals.

Benzoates↗
PubMed1992

The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein.

Oxidation of low density lipoprotein (LDL) has been shown to occur in the artery wall of atherosclerotic lesions in both animal models and human arteries. The oxidant(s) responsible for initiating this process are under intensive investigation and 15-lipoxygenase has been suggested in this context. Another possibility is that nitric oxide and superoxide, generated by cells present in the artery wall, react together to form peroxynitrite which decomposes to form the highly reactive hydroxyl radical. In the present study we have modelled the simultaneous generation of superoxide and nitric oxide by using the sydnonimine, SIN-1 and have investigated its effects on LDL. SIN-1 liberates both superoxide and nitric oxide during autooxidation resulting in the formation of hydroxyl radicals. We have demonstrated that superoxide generated by SIN-1 is not available to take part in a dismutation reaction since it reacts preferentially with nitric oxide. It follows, therefore, that during the autooxidation of SIN-1 little or no superoxide, or perhydroxyl radical will be available to initiate lipid peroxidation. We have shown that SIN-1 is capable of initiating the peroxidation of LDL and also converts the lipoprotein to a more negatively charged form. The SIN-1-dependent peroxidation of LDL is completely inhibited by superoxide dismutase which scavenges superoxide. Neither sodium nitroprusside or S-nitroso-N-acetyl penicillamine, which only produce nitric oxide, are able to modify LDL. These results are consistent with the hypothesis that a product of superoxide and nitric oxide could oxidize lipoproteins in the artery wall and so contribute to the pathogenesis of atherosclerosis in vivo.

Antioxidants↗
PubMed1991

Electron transfer between horse ferritin and ferrihaemoproteins.

Reactions of reduced horse spleen ferritin with horse and Saccharomyces cerevisiae ferricytochromes c, cow ferricytochrome b5, sperm-whale metmyoglobin and Pseudomonas aeruginosa ferricytochrome c-551 were investigated by u.v.-visible spectrophotometry. In all cases the reduced ferritin reduced the ferrihaemoproteins. The rate of reduction varied from less than 0.2 M-1.s-1 for metmyoglobin to 1.1 x 10(3) M-1.s-1 for horse ferricytochrome c (0.1 M-phosphate buffer, pH 7.4, at 25 degrees C). We conclude that the mechanism of ferrihaemoprotein reduction involves long-range electron transfer through the coat of ferritin and that such electron transfer is rapid enough to account for the rates of iron release observed by other workers in reductive release assays.

Animals↗
PubMed1991

Cytochrome oxidase as a proton pump.

The general structure of cytochrome oxidase is reviewed and evidence that the enzyme acts as a redox-linked proton pump outlined. The overall H+/e- stoichiometry of the pump is discussed and results [Wikström (1989), Nature 338, 293] which suggest that only the final two electrons which reduce the peroxide adduct to water are coupled to protein translocated are considered in terms of the restrictions they place on pump mechanisms. "Direct" and "indirect" mechanisms for proton translocation are discussed in the context of evidence for redox-linked conformational changes in the enzyme, the role of subunit III, and the nature of the CuA site.

Electron Transport↗