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D J Lowe

Publications and source records attributed to D J Lowe.

At least 73 records · Page 4Linked to original sources
PubMed1978

Simulation of the electron-paramagnetic-resonance spectrum of the iron-protein of nitrogenase. A prediction of the existence of a second paramagnetic centre.

The e.p.r. spectra of the Fe-proteins of nitrogenase from all sources studied have unusual features in that they have very anisotropic linewidths and low integrated intensities. These characteristics can be explained by assuming that one of the two electrons accepted by these proteins is located at a rapidly relaxing paramagnetic centre that is unobservable by e.p.r., but causes anisotropic broadening of the e.p.r. signal of the other electron. Complex-formation between Fe-proteins and MgATP is described in terms of a 50-60 degrees rotation of the e.p.r.-observable centre.

Chemical Phenomena↗
PubMed1978

Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes.

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo--Fe protein showed that they arose from Fe--S clusters in the Mo--Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the --1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme--product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe--S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO.

Acetylene↗
PubMed1978

Electron paramagnetic resonance in biochemistry. Computer simulation of spectra from frozen aqueous samples.

Two computer programs are described; they can be used to simulate e.p.r. spectra of effective spin-1/2 systems in frozen aqueous samples. One program is written in BASIC and the other in FORTRAN IV. Both programs are deposited as a Supplementary Publication (SUP 50082; 27 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K. References are given to the applications of these programs in biochemical work.

Computers↗
PubMed1978

Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid.

Molybdenum(V) e.p.r. signals from reduced functional milk xanthine oxidase molecules (the Rapid signals), obtained in the presence of purine substrates and products, were further investigated [cf. Bray & Vänngård, (1969) Biochem. J. 114, 725-734; Pick & Bray (1969) Biochem. J. 114, 735-742]. Xanthine forms two complexes with the enzyme that are believed to correspond to different orientations of the substrate molecule in the active site. Only one complex appears to undergo the catalytic reaction. Non-productive complexes, analogous to theone with xanthine, are not formed by 1-methylxanthine or purine. Uric acid forms more than one e.p.r.-detectable complex, one of which is analogous to the non-productive xanthine complex. The computer program used for handing the e.p.r. data is described briefly.

Animals↗
PubMed1978

A precursor in the photolytic cleavage of the Co (III)-C bond of coenzyme B-12 unobservable by electron paramagnetic resonance.

Photolysis of a frozen (80--200 K) anaerobic solution of 5'-deoxyadenosyl-cobalamin in aqueous propan-1,2-diol produces only a small Co(II) signal detectable by electron paramagnetic resonance (EPR). Upon warming to room temperature and refreezing without further irradiation the Co(II) signal increases many-fold. The interpretation is that at low temperature there is an EPR-undetectable "incipient" homolysis of the Co-C bond of the coenzyme which is revealed at higher temperature. The possible implications of this observation for the coenzyme B-12-dependent enzymes are noted.

Chemical Phenomena↗
PubMed1978

Magnetic coupling of the molybdenum and iron-sulphur centres in xanthine oxidase and xanthine dehydrogenases.

Magnetic interaction between molybdenum and one of the iron-sulphur centres in milk xanthine oxidase [Lowe, Lynden-Bell & Bray (1972) Biochem. J. 130, 239-249] was studied further, with particular reference to the newly discovered Mo(V) e.p.r.(electron-paramagnetic-resonance) signal, Resting II [Lowe, Barber, Pawlik & Bray (1976) Biochem. J. 155, 81-85]. E.p.r. measurements at 35GHz near to 4.2K showed that the interaction has the same sign at all molybdenum orientations and is ferromagnetic. The predicted splitting of the e.p.r. signal from the reduced iron-sulphur centre, Fe/S I, was observed, Providing positive identification of this as the other interacting species. Chemical modification of the molybdenum environment in xanthine oxidase can change the size of the interaction severalfold, but interaction always remains approximately isotropic. The interaction in turkey liver xanthine dehydrogenase is indistinguishable from that in the oxidase. However, a bacterial xanthine dehydrogenase with different iron-sulphur centres shows rather larger interaction. Guanidinium chloride disturbs the iron-sulphur centres of the oxidase, and when this occurs there is a parallel and relatively small change in the interaction. Removal of flavin from the molecule, or raising the pH to 12.0, changes the interaction slightly without affecting the chromophores themselves. It is concluded that the Fe/S I centre and the Mo are at least 1.0nm and probably nearer 2.5nm apart, and that the conformation of the protein between them is relatively stable up to pH 12.

Animals↗
PubMed1976

A new non-functional form of milk xanthine oxidase containing stable quinquivalent molybdenum.

A new non-functional modified form of milk xanthine oxidase is described. This contains molybdenum in a quinquivalent state, which is resistant to both oxidation and reduction. The new species is derived from the native enzyme in a two-step process. The first step is the conversion into the desulpho form, via loss of the 'persulphide' sulphur, and the second involves reaction with ethylene glycol or other reagents. The species gives a characteristic Mo(V) electron-paramagnetic-resonance signal, without proton splittings, designated Resting II. This is virtually identical with signals reported previously from resting turkey liver xanthine dehydrogenase and rabbit liver aldehyde oxidase. The possibility is discussed that species Resting II, prepared with ethylene glycol, contains a -COCH2OH residue bound to a nitrogen ligand of molybdenum.

Aldehydes↗
PubMed1976

Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite.

The kinetics of the reduction of oxidized Fe-protein of nitrogenase from Azotobacter chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing e.p.r. (electron-paramagnetic-resonance) spectroscopy. The appearance of the gav. = 1.94 e.p.r. signal (0.24 electron integrated intensity/mol) was associated with a one-electron reduction by SO2--with k greater than 10(8)M-1-S-1 at 23 degrees C. A value of k = 1.75s-1 was obtained for the rate of dissociation of S2O42- into 2SO2-- at 23 degrees C. Further reductions by SO2-- occurred in three slower phases with rate constants in the range 10(4) -10(6)M-1-S-1. These latter phases have no corresponding e.p.r. signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2-- of the Fe-protein alone (k greater than 10(8)M-1-S-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 X 1O(2)s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover.

Adenosine Triphosphate↗
PubMed1976

Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12.

Studies on the respiratory nitrate reductase (EC 1.7.99.4) from Escherichia coli K12 by electron-paramagnetic-resonance spectroscopy indicate that its molybdenum centre is comparable with that in other molybdenum-containing enzymes. Two Mo(V) signals may be observed; one shows interaction of Mo(V) with a proton exchangeable with the solvent and has: A (1H) 0.9-1.2mT; g1 = 1.999; g2=1.985; g3 = 1.964; gav. = 1.983. Molybdenum of both signal-giving species may be reduced with dithionite and reoxidized with nitrate.

Electron Spin Resonance Spectroscopy↗
PubMed1976

Studies by electron-paramagnetic-resonance spectroscopy on the mechanism of action of xanthine dehydrogenase from Veillonella alcalescens.

E.p.r- (electron-paramagnetic-resonance) spectroscopy was used to compare chemical environment and reactivity of molybdenum, flavin and iron-sulphur centres in the enzyme xanthine dehydrogenase from Veillonella alcalescens (Micrococcus lactilyticus) with those of the corresponding centres in milk xanthine oxidase. The dehydrogenase is frequently contaminated with small but variable amounts of a species resistant to oxidation and giving a new molybdenum (V) e.p.r. signal, "Resting I". There is also a "desulpho" form of the enzyme giving a Slow Mo(V) signal, indistinguishable from that of the milk enzyme. Molybdenum of the active enzyme behaves in a manner analogous to that of the milk enzyme, giving a Rapid Mo(V) signal on partial reduction with substrates or dithionite. Detailed comparison shows that molybdenum in each enzyme must have the same ligand atoms arranged in the same manner. As with the milk enzyme, complex-formation between reduced dehydrogenase and purine substrate molecules, presumably interacting at the normal substrate-binding site, modifies the Rapid signal, confirming that such substrates interact near molybdenum. The dehydrogenase-flavin semiquinone signal is identical with that of the oxidase but, in contrast, there is only one iron-sulphur signal. The latter gives an e.p.r. spectrum similar to that of aldehyde oxidase.

Animals↗
PubMed1976

Studies by electron-paramagnetic-resonance spectroscopy and stopped-flow spectrophotometry on the mechanism of action of turkey liver xanthine dehydrogenase.

Studies by e.p.r. (electron-paramagnetic-resonance) spectroscopy and by stopped-flow spectrophotometry on turkey liver xanthine dehydrogenase revealed strong similarities to as well as important differences from the Veillonella alcalescens xanthine dehydrogenase and milk xanthine oxidase. The turkey enzyme is contaminated by up to three non-functional forms, giving molybdenum e.p.r. signals designated Resting I, Resting II and Slow. Slow and to a lesser extent Resting I signals are like those from the Veillonella enzyme, whereas Resting II is very like a resting signal described by K. V. Rajagopolan, P. Handler, G. Palmer & H. Beinert (1968) (J. Biol. Chem. 243, 3784-3796) for aldehyde oxidase. Another non-functional form that gives the Inhibited signal is produced on treatment of the enzyme with formaldehyde. Stopped-flow measurements at 450 nm show that, as for the milk enzyme, reduction by xanthine is rate-limiting in enzyme turnover. The active enzyme gives rise to Very Rapid and Rapid molybdenum(V) e.p.r. signals, as well as to an FADH signal. That these signals are almost indistinguishable from those of the milk enzyme, confirms the similarities between the active sites. There are two types of iron-sulphur centres that give signals like those in the milk enzyme, though with slightly different parameters. Quantitative reduction titration of the functional enzyme with xanthine revealed two important differences between the turkey and the milk enzymes. First, the turkey enzyme FADH/FADH2 system has a redox potential sufficiently low that xanthine is incapable of reducing the flavin completely. This finding presumably explains the very low oxidase activity. Secondly, whereas the Fe/S II chromophore in the milk enzyme has a relatively high redox potential, for the turkey enzyme the value of this potential is lower and similar to that of its Fe/S I chromophore.

Animals↗
PubMed1975

Electron-electron double resonance measurements on xanthine oxidase.

Electron-electron double resonance measurements were carried out on milk xanthine oxidase (xanthine:oxygen oxidoreductase EC 1.2.3.2) and the spectra obtained supported a previous model, based on EPR data, proposing a spin-spin interaction between unpaired electrons associated with Fe-S and Mo. The technique demonstrated that the additional apparently isotropic, splitting in the Mo EPR spectra observed at low temperature is produced by a single site giving two spectra interconverting at a rate consistent with the Fe-S spin lattice relaxation time. Other data concerning the model and the relaxation behaviour of the species are discussed.

Animals↗