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M T Wilson

Publications and source records attributed to M T Wilson.

At least 73 records · Page 4Linked to original sources
PubMed1996

Time resolution of binding and membrane insertion of a mitochondrial signal peptide: correlation with structural changes and evidence for cooperativity.

Utilizing a recently developed novel fluorescence technique [Wall et al. (1995) Mol. Membr. Biol. 12, 183-192], it is shown that the interactions of p25, the leader peptide of subunit IV of cytochrome c oxidase, with phospholipid membranes can be identified in real time. p25 is observed to bind following stopped-flow mixing of the peptide with phospholipid membranes with rate constants up to about 700 s-1 and then insert into the membrane with rate constants on the order of 0.4 s-1. Comparison of these processes with similarly time-resolved experiments performed with a stopped-flow CD spectrometer revealed that p25 does not become alpha-helical upon binding to the membrane. Following membrane insertion, however, p25 was observed to adopt an alpha-helical configuration. The temperature dependency of these processes was then found to yield activation energies for the respective components of the p25-membrane interaction.

Cell Membrane↗
PubMed1996

Role of lipid hydroperoxides in the activation of 15-lipoxygenase.

We have used stopped-flow rapid reaction methods, employing both fluorescence and absorbance monitoring, together with HPLC analysis of the products to study the activation of soybean 15-lipoxygenase by 13(S)-hydroperoxy-9, 11(E,Z)-octadecadienoic acid (13-HPOD). When lipoxygenase is mixed with an equimolar concentration of 13-HPOD, the enzyme undergoes a rapid change in fluorescence. The rate of the change of fluorescence is dependent on the concentration of the 13-HPOD (k = 6.7 x 10(6) M-1 s-1) and is accompanied by activation of the enzyme. The fluorescence change is not accompanied by any change in the UV absorbance of the 13-HPOD, suggesting no loss of the conjugated diene during enzyme activation, and HPLC analysis of the products of the reaction confirms that the 13-HPOD can be recovered unchanged following this reaction. In the presence of an inhibitor (BWA4C, a hydroxamate inhibitor) that reduces the active-site iron, the 13-HPOD and the inhibitor are destroyed in a peroxidase-like reaction. On the basis of these observations we propose that 13-HPOD binds to the enzyme and facilitates activation of the enzyme, possibly through the formation of a protein radical, and that the 13-HPOD is not changed chemically in this process.

Arachidonate 15-Lipoxygenase↗
PubMed1996

An EPR investigation of human methaemoglobin oxidation by hydrogen peroxide: methods to quantify all paramagnetic species observed in the reaction.

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglobin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H2O2 was added at 3- and 10-fold molar excess to 100 microM metHb in 50 mM phosphate buffer, pH 7.4, 37 degrees C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H2O2 the EPR signals (-196 degrees C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (-196 degrees C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g parallel = 2.035 and g perpendicular = 2.0053), and the other an isotropic singlet (g = 2.0042, delta H = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic behaviour of both peroxyl (ROO.) and non-peroxyl (P.) free radicals were similar, we concluded that ROO. radicals are not formed from P. radicals by addition of O2. The time courses for both radicals showed a steady state during the time required for H2O2 to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 x 10(-3) s-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H2O2. The formation of the rhombic Fe(III) non-haem centres with g = 4.35 was found. Their yield was proportional to the H2O2 concentration used and the centres were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at -196 degrees C were used.

Electron Spin Resonance Spectroscopy↗
PubMed1996

Redox cycling of human methaemoglobin by H2O2 yields persistent ferryl iron and protein based radicals.

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H2O2, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H2O2 resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H2O2 showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H2O2. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalytic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10(4) fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: approximately 20 mol H2O2 and abolished the protein radical steady state. The biological implications for these results are discussed.

Catalase↗
PubMed1996

Formation of oxysterols during oxidation of low density lipoprotein by peroxynitrite, myoglobin, and copper.

Oxidation of low density lipoprotein (LDL) in the artery wall leads to the formation of cholesterol oxidation products that may result in cytotoxicity. Different mechanisms could contribute to LDL oxidation in vivo resulting in characteristic and specific modification of the cholesterol molecule. Alternatively, attack on cholesterol by chain propagating peroxyl radicals could result in the same distribution of oxidation products irrespective of the initial pro-oxidant mechanism. To distinguish between these possibilities we have monitored the formation of nine oxysterols during LDL oxidation, promoted by copper, myoglobin, peroxynitrite, or azo bis amidino propane. Regardless of the oxidant used, the pattern of oxysterol formation was essentially the same. The yields of products identified decreased in the order 7-oxocholesterol > 7 beta-hydroxycholesterol > 7 alpha-hydroxycholesterol > 5,6 beta-epoxycholesterol > 5,6 alpha-epoxycholesterol except in the case of peroxynitrite in which case a higher yield of 5, 6 beta-epoxycholesterol relative to 7-oxocholesterol was found. No formation of cholestane 3 beta, 5 alpha, 6 beta-triol, or the 24-,25-,27-hydroxycholesterols was seen. Concentration of 7-oxocholesterol levels in LDL was positively correlated with the degree of protein modification. Endogenous alpha-tocopherol in LDL or supplementation with butylated hydroxytoluene prevented oxysterol formation. Taken together these data indicate that the oxidation of cholesterol and protein in LDL occur as secondary oxidation events consequent on the attack of fatty acid peroxyl/alkoxyl radicals on the 7-position of cholesterol, and with amino acids on apoB. Furthermore, oxidant processes with atherogenic potential, such as peroxynitrite, copper, and myoglobin are capable of producing oxidized LDL containing cytotoxic mediators.

Amidines↗
PubMed1995

Inhibition of cytochrome c oxidase in turnover by nitric oxide: mechanism and implications for control of respiration.

Binding of nitric oxide (NO) to isolated cytochrome c oxidase in turnover was investigated by static and kinetic spectroscopic methods. These studies indicate that cytochrome c oxidase rapidly binds NO when the enzyme enters turnover. Our results show that NO binds to ferrocytochrome a3, competing with oxygen for this binding site. However, the main features of the binding process, in particular the rapid onset of inhibition, cannot be fully explained on this basis. We suggest, therefore, that there is a second binding site for NO, which has lower affinity but nevertheless plays an important role in the inhibitory process. A likely possibility is that CuB+ constitutes this second binding site. The fast onset of inhibition observed in the presence of NO, along with the dependence on the oxygen concentration, suggests that under physiological conditions, where the oxygen concentration is low, nanomolar concentrations of NO can effectively act as a regulator of the mitochondrial respiratory chain.

Animals↗
PubMed1995

Pro-oxidant effects of cross-linked haemoglobins explored using liposome and cytochrome c oxidase vesicle model membranes.

The therapeutic use of cell-free haemoglobin as a blood substitute has been hampered by toxicological effects. A model asolectin (phosphatidylcholine/phosphatidylethanolamine) liposome system was utilized to study the pro-oxidant efficiency of several chemically modified haemoglobins on biological membranes. Lipid peroxidation, resulting from the interactions between haemoglobin and liposomes, was measured by conjugated diene formation and the maximal rates of oxygen uptake. Spectral changes gave insight into the occurrence of the ferryl iron species. The residual reactivity of oxidatively damaged haemoglobins with ligands during incubation with liposomes was assessed from rapid kinetic carbon monoxide-binding experiments. Liposomes in which cytochrome c oxidase was embedded show both haemoglobin and the enzyme to be oxidatively damaged during incubation. The functional state of cytochrome c oxidase was monitored in the presence and absence of a free radical scavenger. Once in contact, both unmodified and modified haemoglobins triggered and maintained severe radical-mediated membrane damage. Differences in the pro-oxidant activities among haemoglobins may be explained by either the differential population of their ferryl intermediates or disparate dimerization and transfer of haem into the membrane with subsequent haem degradation. This study may contribute to a better understanding of the molecular determinants of haemoglobin interactions with a variety of biological membranes.

Animals↗
PubMed1995

Measurement of the spin concentration of metalloprotein samples from saturation-magnetization data with particular reference to cytochrome c oxidase.

A protocol for obtaining high-quality saturation-magnetization data from metalloprotein samples, employing a superconducting quantum interference device (SQUID) magnetometer, has previously been reported [E. P. Day, T. A. Kent, P. A. Lindahl, E. Münck, W. H. Orme-Johnson, H. Roder and A. Roy (1987) Biophys. J. 52, 837-853 and E. P. Day (1993) Methods Enzymol. 227, 437-463]. Following studies of several dozen different metalloprotein derivatives, the methodology has been further refined, particularly in the area of sample preparation. The details of the sample-handling procedures now in use are described, and moreover, the critical issue of verifying that contamination by paramagnetic impurities remains insignificant is considered. Importantly, it is shown that an independent determination of the quantity of paramagnetic sample present in the magnetometer is undesirable. Much more reliable parameters concerning the ground-state magnetic properties of the system under study are obtained if enough saturation-magnetization data are collected to enable the spin concentration to be determined during the subsequent fitting procedure. As proof of the validity of this method, the results of magnetization studies on ferricytochrome c, ferrocytochrome c and the benzohydroxamic acid adduct of horseradish peroxidase are presented. The ability of saturation-magnetization measurements to routinely determine spin concentration to within +/- 4% of accepted values is firmly established. In addition, a saturation-magnetization study has been performed on resting and fully reduced derivatives of cytochrome c oxidase. These results provide an illustration of the usefulness of the technique in probing some systems which have proved difficult to study by other methods. The increased difficulties inherent in obtaining meaningful data from these cytochrome c oxidase and other integer spin systems are delineated.

Animals↗
PubMed1995

Electron transfer and proton pumping in cytochrome oxidase.

This article presents an outlook on the structure and function of terminal oxidases, the respiratory enzymes which catalyze the reduction of dioxygen to water in aerobic organisms. The structure of the redox active metals, their interactions with the protein matrix, and their role in electron transfer ligand binding and proton pumping are briefly reviewed.

Amino Acid Sequence↗
PubMed1995

Triplet-state quenching in complexes between Zn-cytochrome c and cytochrome oxidase or its CuA domain.

The quenching of the triplet state of Zn-cytochrome c in electrostatic complexes with cytochrome oxidase and its soluble CuA domain has been studied by laser flash photolysis. The triplet state of free Zn-cytochrome c decayed with a rate of about 200 s-1. With the oxidase, biphasic decay with rate constants of 2 x 10(5) and 2 x 10(3) s-1, respectively, was observed. At high ionic strength (I = 0.2) the decay was the same as with free Zn-cytochrome c. The quenching was also eliminated by reduction of the oxidase. The decay rate in the complex with the CuA domain was 4 x 10(4) s-1. The results are interpreted in terms of rapid electron transfer to CuA and a slower one to cytochrome a. No electron transfer products were detected, because the backward reaction is faster than the forward one. This can be explained by the high driving force (1.1 eV) for the forward electron transfer, taking the system into the inverted Marcus region. The distance in the electrostatic complex between cytochrome c and the electron acceptor, presumed to be CuA, is calculated to be 16 A.

Animals↗
PubMed1994

Probing the oxygen binding site of cytochrome c oxidase by cyanide.

Cyanide binding to cytochrome c oxidase has been investigated by sequential mixing and rapid scan stopped flow spectroscopy. Double mixing experiments confirm earlier reports that cyanide binds rapidly to partially reduced enzyme species formed in turnover. The absorbance/time/wavelength matrices, captured during the onset of cyanide inhibition of cytochrome c oxidase by rapid scan stopped flow, were analyzed by singular value decomposition and the spectral contributions of the chromophores separated. Examination of the time courses and amplitudes of the spectral signals provide evidence that entry of a single (1-1.3) electron into the enzyme is sufficient to trigger rapid cyanide binding. This electron resides predominantly on the cytochrome a/CuA pair in the inhibited enzyme. In addition, although cytochrome a3 remains oxidized, it does not appear to be the site for the initial inhibitory binding of cyanide. Our data suggest that CuB2+ is the initial binding site.

Cyanides↗
PubMed1994

The oxidation of cytochrome-c oxidase vesicles by hemoglobin.

Human hemoglobin has been used as a pro-oxidant for artificial unilamellar phospholipid vesicles, containing cytochrome-c oxidase inserted into the bilayer. This experimental system was suitable to follow directly the kinetics of lipid oxidation and the effects on both the vesicle membrane permeability and the functional state of cytochrome-c oxidase. Following mixing of vesicles with hemoglobin, an oxygen dependent, peroxyl radical mediated, rapid oxidation (taking a few minutes) of the lipid was found to occur. On a similar time scale the membrane became ion-leaky and cytochrome-c oxidase damaged. The pro-oxidant effects of hemoglobin in various oxidation and ligation states were studied and a mechanism, based on a ferric/ferryl redox cycle of the heme-iron is proposed to account for these observations.

Cell Membrane Permeability↗