scholarly journals Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases

1977 ◽  
Vol 163 (2) ◽  
pp. 279-289 ◽  
Author(s):  
M J Barber ◽  
R C Bray ◽  
R Cammack ◽  
M P Coughlan
Keyword(s):  
Uric Acid ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Xanthine Dehydrogenase ◽  
Resonance Spectroscopy ◽  
Redox Potentials ◽  
Paramagnetic Resonance ◽  
Reduction Potentials ◽  
Oxidation Reduction ◽  
Temperature Electron ◽  
Full Reduction

Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.

scholarly journals Oxidation-reduction potentials of molybdenum, flavin and iron-sulphur centres in milk xanthine oxidase

1976 ◽  
Vol 157 (2) ◽  
pp. 469-478 ◽  
Author(s):  
R Cammack ◽  
M J Barber ◽  
R C Bray
Keyword(s):  
Xanthine Oxidase ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Bovine Milk ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Reduction Potentials ◽  
Point Reduction ◽  
Oxidation Reduction ◽  
Mediator System ◽  
Electron Paramagnetic

1. The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by e.p.r. (electron-paramagnetic-resonance) spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH8.2, are: Fe/S centre I, −343 +/- 15mV; Fe/S II, −303 +/- 15mV; FAD/FADH-; −351 +/- 20mV; FADH/FADH2, −236 +/-mV; Mo(VI)/Mo(V) (Rapid), −355 +/- 20mV; Mo(V) (Rapid)/Mo(IV), −355 +/- 20mV. 2. Behaviour of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V) (Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with six other buffers. 3. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow molybdenum signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centres. The potentials associated with the Slow signal are: Mo(VI)/Mo(V) (Slow), −440 +/- 25mV; Mo(V) (Slow)/Mo(IV), −480 +/- 25 mV. This signal exhibits very sluggish equilibration with the mediator system. 4. The deviations from ideal behaviour are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centres.

Defect induced p-type conductivity in zinc oxide at high temperature: electron paramagnetic resonance spectroscopy

Nanoscale ◽  
2017 ◽  
Vol 9 (31) ◽  
pp. 10983-10986 ◽  
Author(s):  
Emre Erdem
Keyword(s):  
Surface Defects ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Type Conductivity ◽  
Zno Nanocrystals ◽  
Defect Centers ◽  
Temperature Electron ◽  
P Type ◽  
Electron Paramagnetic

It has been observed that the defect centers on the surface play a crucial role in the conductivity behavior of ZnO. Above 300 °C only surface defects can be visible in EPR spectra for ZnO nanocrystals which indicate p-type conductivity.

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

1976 ◽  
Vol 153 (2) ◽  
pp. 297-307 ◽  
Author(s):  
M J Barber ◽  
R C Bray ◽  
D J Lowe ◽  
M P Coughlan
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Redox Potential ◽  
Active Sites ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Xanthine Dehydrogenase ◽  
Resonance Spectroscopy ◽  
Stopped Flow ◽  
Paramagnetic Resonance ◽  
Flow Measurements ◽  
Electron Paramagnetic

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.

scholarly journals Midpoint redox potentials of plant and algal ferredoxins

1977 ◽  
Vol 168 (2) ◽  
pp. 205-209 ◽  
Author(s):  
R Cammack ◽  
K K Rao ◽  
C P Bargeron ◽  
K G Hutson ◽  
P W Andrew ◽  
...  
Keyword(s):  
Green Algae ◽  
Higher Plants ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Photosynthetic Electron Transport ◽  
Resonance Spectroscopy ◽  
Redox Potentials ◽  
Higher Plant ◽  
Paramagnetic Resonance ◽  
Blue Green Algae ◽  
Nostoc Strain

Midpoint potentials of plant-type ferredoxins from a range of sources were measured by redox titrations combined with electron-paramagnetic-resonance spectroscopy. For ferredoxins from higher plants, green algae and most red algae, the midpoint potentials (at pH 8.0) were between —390 and —425 mV. Values for the major ferredoxin fractions from blue-green algae were less negative (between —325 and —390 mV). In addition, Spirulina maxima and Nostoc strain MAC contain second minor ferredoxin components with a different potential, —305 mV (the highest so far measured for a plant-algal ferrodoxin) for Spirulina ferrodoxin II, and —455 mV (the lowest so far measured for a plant-algal ferredoxin) for Nostoc strain MAC ferredoxin II. However, two ferredoxins extracted from a variety of the higher plant Pisum sativum (pea) had midpoint potentials that were only slightly different from each other. These values are discussed in terms of possible roles for the ferredoxins in addition to their involvement in photosynthetic electron transport.

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

1976 ◽  
Vol 153 (2) ◽  
pp. 287-295 ◽  
Author(s):  
H Dalton ◽  
D J Lowe ◽  
R T Pawlik ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Aldehyde Oxidase ◽  
Detailed Comparison ◽  
Xanthine Dehydrogenase ◽  
Resonance Spectroscopy ◽  
Enzyme Complex ◽  
Partial Reduction ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

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.

scholarly journals Engineered holocytochrome c synthases that biosynthesize new cytochromes c

2017 ◽  
Vol 114 (9) ◽  
pp. 2235-2240 ◽  
Author(s):  
Deanna L. Mendez ◽  
Shalon E. Babbitt ◽  
Jeremy D. King ◽  
John D’Alessandro ◽  
Michael B. Watson ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Axial Ligand ◽  
Heme Iron ◽  
Resonance Spectroscopy ◽  
Redox Potentials ◽  
Hydrogen Electrode ◽  
Paramagnetic Resonance ◽  
Axial Ligands ◽  
Circular Dichroïsm

Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c. In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli. Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; −19 mV, M81A; −69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these “release mutants.”

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