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M T Wilson

Publications and source records attributed to M T Wilson.

157 records · Page 9Linked to original sources
PubMed1975

A temperature-jump study of the reaction between azurin and cytochrome c oxidase from Pseudomonas aeruginosa.

The electron-transfer reaction between azurin and the cytochrome oxidase from Pseudomonas aeruginosa was investigated by temperature-jump relaxation in the absence of O2 and in the presence of CO. The results show that: (i) reduced azurin exists in two forms in equilibrium, only one of which is capable of exchanging electrons with the Pseudomonas cytochrome oxidase, in agreement with M. T. Wilson, C. Greenwood, M. Brunori & E. Antonini (1975) (Biochem. J. 145, 449-457); (ii) the electron transfer between azurin and Pseudomonas cytochrome oxidase occurs within a molecular complex of the two proteins; this internal transfer becomes rate-limiting at high reagent concentrations.

Azurin↗
PubMed1974

Studies on partially reduced mammalian cytochrome oxidase. Reactions with carbon monoxide and oxygen.

A number of methods were used to prepare a species of mammalian cytochrome oxidase (EC 1.9.3.1, ferrocytochrome c-oxygen oxidoreductase) in which only cytochrome a(3) is reduced and in combination with CO. The kinetics of CO binding by cytochrome a(3) (2+) in this species is significantly different from that exhibited by cytochrome a(3) (2+) in the fully reduced enzyme. The second-order rate constant for combination was 5x10(4)m(-1).s(-1) and the ;off' constant was 3x10(-2)s(-1). The kinetic difference spectra cytochrome a(3) (2+)-cytochrome a(3) (2+)-CO reveal further differences between the mixed-valence and the fully reduced enzyme. The reaction between cytochrome a(3) (2+) and oxygen in the mixed-valence species was followed in flow-flash experiments and reveals a fast, oxygen-dependent (8x10(7)m(-1).s(-1) at low oxygen) rate followed by a slow process, whose rate is independent of oxygen but whose amplitude is dependent on [O(2)]. The fast oxygen-dependent reaction yields as the first product the so-called ;oxygenated' enzyme. We conclude from these experiments that the ligand-binding behaviour of cytochrome a(3) depends on the redox state of its partners, a fact which represents clear evidence for site-site interaction in this enzyme. The fact that oxygen reacts rapidly with this enzyme species in which only one component, namely cytochrome a(3), is reduced represents clear and unequivocal evidence that this is indeed the O(2)-binding site in cytochrome oxidase and may indicate that reduction of oxygen can proceed via single electron steps.

Binding Sites↗
PubMed1974

Kinetics of carbon monoxide binding and electron transfer by cytochrome c polymers.

Studies on horse heart cytochrome c polymers were carried out by stopped-flow and photolysis techniques, to investigate the properties of the CO complex and the kinetics of electron transfer, mainly of the dimeric and tetrameric forms. CO binding, which does not occur with native monomers, proceeds at both pH7.0 and pH9.6, and appears to follow complex kinetics: an initial phase is observed, which is CO-concentration-dependent, followed by a very slow monomolecular phase (k~2x10(-3)s(-1) at pH7) before establishment of equilibrium. Photodissociation of the CO complex has a very low quantum yield, probably less than 0.1. Static titration data of the dimer gave an ;n' value of 0.4. These data strongly suggest heterogeneity of the population of binding sites, and have been interpreted in terms of the existence of different structures, probably owing to the non-unique type of binding of monomers during polymerization. Polymers of cytochrome c carboxymethylated on the methionine residue normally ligated to iron show simple CO recombination kinetics after photolytic removal (k(on)=1.5x10(6)m(-1).s(-1) at pH6). We therefore suggest that, for native cytochrome c, polymerization has an effect on the lability of the haem crevice, rendering the iron available for binding ligands, without, however, forming the structure of a truly open crevice. Electron transfer is, on the other hand, a simple process, and no gross differences are observed between monomer and polymers. A simple model, taking into account all these data, is suggested.

Animals↗
PubMed1974

The reduction of carboxymethyl-cytochrome c by chromous ions.

The reduction of ferricytochrome c and ferricytochrome c carboxymethylated at the haem-linked methionine (residue 80) by Cr(2+) ions was studied by stopped-flow techniques. At pH6.2 the kinetics of reduction of ferricytochrome c are simple and correspond to a second-order rate constant of 1.21x10(3)m(-1).s(-1). Under identical conditions the kinetics of reduction of the carboxymethyl derivative, carboxymethyl-cytochrome c, are complex; two Cr(2+)-concentration-dependent processes (1.5x10(4)m(-1).s(-1) and 1.3x10(3)m(-1).s(-1)) lead to the formation of an intermediate which decays in monomolecular fashion (0.15s(-1)) to form the normal fully reduced material. The kinetic difference spectrum for the overall process corresponds to that found statically, whereas the kinetic difference spectrum of the intermediate minus the oxidized form resembles that of the low-spin ferrous form of carboxymethyl-cytochrome c minus oxidized carboxymethyl-cytochrome c. A model is proposed in which the reduction of low-spin ferric carboxymethyl-cytochrome c to high-spin ferrous carboxymethyl-cytochrome c involves a low-spin ferrous intermediate. The monomolecular step involving the decay of this low-spin ferrous intermediate is associated with an activation energy of approx. 126kJ.mol(-1) and is thought to involve both a change of spin state and a protein-conformational event. Although carboxymethyl-cytochrome c represents a mixture of species separable on a charge basis, the above observations were independent of which species was chosen for study.

Carboxylic Acids↗
PubMedDate not supplied

Stopped-flow studies of cytochrome oxidase reconstituted into liposomes: proton pumping and control of activity.

The transient kinetics of proton pumping and the electron transfer properties of cytochrome oxidase inserted into small unilamellar vesicles have been investigated by stopped-flow spectrophotometry. In the presence of valinomycin, proton pumping and cytochrome c oxidation by cytochrome oxidase are synchronous up to rate constants of approximately 9 sec-1. Moreover, the enzyme depleted of subunit III ("three-less oxidase") was also shown to pump protons, although with a significantly smaller stoichiometry. Thus, subunit III is not the only (or even the main) proton channel, although it may be involved in the regulation of activity. The kinetics of cytochrome c oxidation by COV in the absence and in the presence of ionophores have been investigated. Analysis of the time course of the process in the transient and steady state phases indicates that the onset of control by the electrochemical gradient follows the transfer of four electrons, i.e., one complete turnover of the oxidase. Two possible alternative interpretations for the control of the turnover phase are presented and discussed.

Animals↗
PubMedDate not supplied

Evaluation of latex agglutination and dot immunobinding assay for the detection of cytomegalovirus antibody.

The Virogen CMV Antibody Test and Difco CMV-Cube were compared with three other available methods, enzyme immunoassay, immunofluorescence and latex agglutination, for the detection of cytomegalovirus (CMV) antibody in 126 random sera submitted to the clinical laboratory. Based on concordance of three or more methods, 72% of the samples were positive for CMV antibody. The sensitivities and specificities of the assays were Virogen, 98% and 97%, and CMV-Cube 97% and 97%, respectively.

Antibodies, Viral↗