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M T Stankovich

Publications and source records attributed to M T Stankovich.

43 records · Page 3Linked to original sources
PubMed1986

Oxidation-reduction potentials of butyryl-CoA dehydrogenase.

In order to obtain butyryl-CoA dehydrogenase from Megasphaera elsdenii in pure enough form to perform redox studies, the existing purification procedures first had to be modified and clarified [Engel, P. (1981) Methods Enzymol. 71, 359-366]. These modifications are described, and the previously unpublished spectral properties of the electrophoretically pure CoA-free butyryl-CoA dehydrogenase are presented. In our spectral reductive titration of pure enzyme, we show that although blue neutral flavin radical is stabilized in nonquantitative amounts in dithionite titrations (19%) or in electrochemical reductions mediated by methylviologen (5%), it is not thermodynamically stabilized; therefore, only a midpoint potential for butyryl-CoA dehydrogenase is obtained. The electron-transfer behavior from pH 5.5 to pH 7.0 indicates reversible two-electron transfer accompanied by one proton: EFlox + 2e- + H+ = EFlredH- Em7 = -0.079 V vs. SHE where EFlox is oxidized butyryl-CoA dehydrogenase, EFlredH- is two electron reduced enzyme, and Em7 is the midpoint potential at pH 7.0 at 25 degrees C. Redox data and activity data both indicate that the enzyme loses activity rapidly at pH values above 7.0. The Em7 of the butyryl-CoA dehydrogenase is 40 mV positive of the Em7 of the butyryl-CoA/crotonyl-CoA couple [Gustafson, W. G., Feinberg, B. A., & McFarland, J. T. (1986) J. Biol. Chem. 261, 7733-7741]. Binding of substrate analogue acetoacetyl-CoA caused the potential of butyryl-CoA dehydrogenase to shift 100 mV negative of the free enzyme. The negative shift in potential makes electron transfer from enzyme to substrate more probable, which is consistent with the direction of electron transfer in the bacterial system.(ABSTRACT TRUNCATED AT 250 WORDS)

Butyryl-CoA Dehydrogenase↗
PubMed1985

Thermodynamic control of D-amino acid oxidase by benzoate binding.

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.

Animals↗
PubMed1984

Measurement of the oxidation-reduction potentials for one-electron and two-electron reduction of electron-transfer flavoprotein from pig liver.

Potentiometric titrations of pig liver electron-transfer flavoprotein (ETF) were performed at pH 7.5 and 4 degrees C, both in the reductive and oxidative directions. Reduction of ETF to the hydroquinone form required a total of two reducing equivalents/mol of ETF with the formation of sub-stoichiometric amounts of anionic semiquinone as an intermediate. The oxidation-reduction potentials for the two one-electron couples, oxidized ETF/ETF semiquinone and ETF semiquinone/fully reduced ETF, are +4 mV and -50 mV respectively. The overall midpoint potential for the two-electron couple (oxidized ETF/fully reduced ETF) is -23 mV.

Animals↗
PubMed1984

Redox potential-pH properties of the flavoprotein L-amino-acid oxidase.

The redox potential-pH characteristics of the enzyme L-amino-acid oxidase (L-amino-acid: oxygen oxidoreductase (deaminating), EC 1.4.3.2) have been measured in the pH range 6.2 to 8.3 at 4 degrees C. All potentials are reported versus the standard hydrogen electrode. Consistent with the protonation states proposed for the anionic red semiquinone and anionic dihydroquinone forms of the flavin coenzyme, the first electron potential is independent of pH (-0.056 +/- 0.006 V), while the second electron transfer potential is pH-dependent, exhibiting a 0.060 V/pH unit slope. At all pH values investigated, the percentage of semiquinone species observed matches closely that calculated from measured potential separations. The semiquinone species is thermodynamically stable, as indicated by formation of semiquinone, similarity of redox potentials in oxidative and reductive directions, and by the slope of Nernst plots.

Amino Acid Oxidoreductases↗
PubMed1978

Determination of glucose oxidase oxidation-reduction potentials and the oxygen reactivity of fully reduced and semiquinoid forms.

The oxidation-reduction potential values for the two electron transfers to glucose oxidase were obtained at pH 5.3, where the neutral radical is the stable form, and at pH 9.3, where the anion radical is the stable form. The midpoint potentials at 25 degrees were: pH 5.3 EFl1ox + e- H+ equilibrium EFlH. Em1 = -0.063 +/- 0.011 V EFlH. + e- + H+ equilibrium EFlredH2 Em2 = -0.065 +/- 0.007 V pH 9.3 EFlox + e- EFi- Em1 = -0.200 +/- 0.010 V EFi- + e- + H+ equilibrium EFlredH- Em2 = -0.240 +/- 0.005 V All potentials were measured versus the standard hydrogen electrode (SHE). The potentials indicated that glucose oxidase radicals are stabilized by kinetic factors and not by thermodynamic energy barriers. The pK for the glucose oxidase radical was 7.28 from dead time stopped flow measurements and the extinction coefficient of the neutral semiquinone was 4140 M-1 cm-1 at 570 nm. Both radical forms reacted with oxygen in a second order fashion. The rate at 25 degrees for the neutral semiquinone was 1.4 X 10(4) M-1 s-1; that for the anion radical was 3.5 X 10(4) M-1 s-1. The rate of oxidation of the neutral radical changed by a factor of 9 for a temperature difference of 22 degrees. For the anion radical, the oxidation rate changed by a factor of 6 for a 22 degrees change in temperature. We studied the oxygen reactivity of the 2-electron reduced form of the enzyme over a wide wavelength range and failed to detect either oxygenated flavin derivatives or semiquinoid forms as intermediates. The rate of reoxidation of fully reduced glucose oxidase at pH 9.3 was dependent on ionic strength.

Aspergillus niger↗
PubMed1976

Determination of the redox potential of deazariboflavin by equilibration with flavins.

The redox potential of deazariboflavin has been determined for pH values from 5.5 to 9.2 by equilibration with riboflavin and lumiflavin 3-acetate. The position of the equilibrium with riboflavin was measured spectrophotometrically and fluorimetrically; the equilibrium potential with lumiflavin 3-acetate was measured spectrophotometrically and potentiometrically. The Em7 for deazariboflavin was found to be--0.273 +/- 0.003 V against the standard hydrogen electrode. Equilibrium with flavodoxin at pH 9.5 and 10.0 was also used to determine the redox potential of deazariboflavin at high pH values. The pK of dihydrodeazariboflavin was found from the break in the potential vs. pH diagram and from spectrophotometric pH titration. The pK value obtained by both methods is 7.00 +/- 0.05. We found that borate, a product of the reducing agent borohydride, complexed with the ribityl sidechain of deazariboflavin, causing a shift in the pK for the reduced form to values of about 8.

Flavins↗