Search PubMed⌕ Search

Biomedical subjects

M T Stankovich

Publications and source records attributed to M T Stankovich.

At least 37 records · Page 2Linked to original sources
PubMed1993

Influence of two substrate analogues on thermodynamic properties of medium-chain acyl-CoA dehydrogenase.

The objective of this work was to identify the key structural functionalities of substrate or product that modulate the thermodynamic properties of medium-chain acyl-CoA dehydrogenase (MCAD). In order to achieve this, two classes of substrate analogues, acetyl-CoA and thioether-CoAs, were complexed with MCAD and their effects on the redox properties of MCAD were measured. A pH dependence study of the redox potential of uncomplexed MCAD allowed us to compare redox properties between complexed and uncomplexed MCAD and to calculate the dissociation constants of the analogues to the three redox states of MCAD. The results from this work indicate that these analogues are not influencing the thermodynamic behavior of MCAD in the same way as natural substrate. Thus, we propose that the following two key structural features of the binding ligand are necessary for mimicking the thermodynamic effects natural substrate has on MCAD: a thioester carbonyl on carbon 1 and a fatty acyl-CoA chain length around 8 carbon units. Furthermore, with the advent of structural knowledge, insights into the interactions of these structural features with MCAD and their influence on MCAD's highly regulated dehydrogenation mechanism are discussed.

Acetyl Coenzyme A↗
PubMed1992

Redox properties of electron-transfer flavoprotein ubiquinone oxidoreductase as determined by EPR-spectroelectrochemistry.

We have determined the formal potential values for each electron transfer to electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), in order to further characterize the thermodynamics of electron transport from various acyl-CoA thioesters to the mitochondrial ubiquinone pool. ETF-QO contains one [4Fe-4S]2+,1+ cluster and one FAD prosthetic group. A preliminary visible-spectroelectrochemical titration showed that the two redox centers were reduced almost simultaneously. Since the visible spectra of the chromophores overlap, it was not possible to resolve the formal potential value for each electron transfer to the protein using this method. Accordingly, an EPR-spectroelectrochemical cell was designed so that each formal potential value could be resolved by EPR quantitation of the flavin semiquinone and the reduced iron-sulfur cluster during the titration. The formal potential values for electron transfer to ETF-ubiquinone oxidoreductase at pH 7.5 and 4 degrees C were E1 degrees' = +0.028 V and E2 degrees' = -0.006 V for the first and second electron transfers, respectively, to the FAD and E degrees' = +0.047 V for the iron-sulfur cluster. The thermodynamics of electron transport from the acyl-CoA substrates of beta-oxidation to the mitochondrial electron transport chain have been fully resolved with completion of this work. The results are discussed in terms of their significance to the overall electron transport process from beta-oxidation.

Acyl Coenzyme A↗
PubMed1991

Electrochemical properties of the diiron core of uteroferrin and its anion complexes.

The reduction potentials (Em) of the purple acid phosphatase from porcine uterus, uteroferrin (Uf), and its phosphate, arsenate, and molybdate complexes were determined by coulometric methods at various pH values. The midpoint potential of Uf at the pH value for optimal enzyme activity (pH 5) was found to be +367 mV versus a normal hydrogen electrode (NHE), while at pH 6.01 Uf exhibits a reduction potential of +306 mV. At pH 6.01 molybdate was found to shift the potential of Uf more positive by 192 mV, while phosphate and arsenate shift the potential of Uf more negative by 193 and 89 mV, respectively. These shifts are consistent with the different susceptibilities of Uf to aerobic oxidation in the presence of these anions. Comparison of the reduction potential of Uf at pH 7.0 with those reported for other dinuclear non-heme iron enzymes and various (mu-oxo)diiron model complexes suggest that the potential of Uf is too positive to be consistent with a mu-oxo-bridge in Ufo. The pH dependence of the reduction potentials of Uf (60 mV/pH unit) and the fact that the electron transfer rate increases with decreasing pH indicate a concomitant participation of a proton during the oxidation-reduction process. This process was assigned to the protonation of a terminally bound hydroxide ligand at the Fe(II) center upon reduction of Ufo. Structural implications provided by the electrochemical data indicate that molybdate affects the dinuclear core in a manner that differs from that of phosphate and arsenate. This observation is consistent with previous spectroscopic and biochemical studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1991

Oxidation-reduction properties of trimethylamine dehydrogenase: effect of inhibitor binding.

The redox potentials of trimethylamine dehydrogenase from the bacterium W3A1 have been determined by means of uv-visible spectroelectrochemistry. In the presence of the inhibitor tetramethylammonium chloride a shift of +0.2 V was observed in the midpoint redox potential for conversion of the oxidized 6-S-cysteinyl-FMN to the flavin radical form. The pH-independent value was +0.23 V vs the standard hydrogen electrode. The pH-dependent conversion of this radical to fully reduced flavin was shifted negative by 0.1 V in the presence of the inhibitor to -0.05 V at pH 7.0 and -0.15 V at pH 8.4. Tetramethylammonium chloride also caused moderate negative shifts (0.03-0.05 V) in the midpoint redox potential for the Fe4S(+2)4/Fe4S(+1)4 couple of trimethylamine dehydrogenase. The midpoint potentials are +0.06 V at pH 7.0 and +0.04 V at pH 8.4. Therefore, in the presence of tetramethylammonium chloride, electron transfer from the flavin radical to the Fe4S(+2)4 group is energetically unfavorable and trimethylamine dehydrogenase is trapped in the flavin radical state. The redox potential changes provide a thermodynamic basis for inhibition by tetramethylammonium chloride. Spectroelectrochemical titrations of trimethylamine dehydrogenase which had been inactivated by phenylhydrazine revealed heterogeneity in the redox behavior which had not been observed in other laboratories. The reason for this heterogeneity was not determined, but the midpoint redox potential for the Fe4S(+2)4/Fe4S(+1)4 couple of the main fraction of the inactivated enzyme was the same as that of active trimethylamine dehydrogenase.

Bacteria↗
PubMed1990

Spectral and electrochemical properties of glutaryl-CoA dehydrogenase from Paracoccus denitrificans.

Studies of the spectral (UV/vis and resonance Raman) and electrochemical properties of the FAD-containing enzyme glutaryl-CoA dehydrogenase (GCD) from Paracoccus denitrificans reveal that the properties of the oxidized enzyme (GCDox) appear to be invariant from those properties known for other acyl-CoA dehydrogenases such as mammalian general acyl-CoA dehydrogenase (GACD) and butyryl-CoA dehydrogenase (BCD) from Megasphaera elsdenii. However, when either free or complexed GCD is reduced, its spectral and electrochemical behavior differs from that of both GACD and BCD. Free GCD does not stabilize any form of one-electron-reduced GCD, but when GCD is complexed to its inhibitor, aceto-acetyl-CoA, the enzyme stabilizes 20% of the blue neutral radical form of FAD (FADH.) upon reduction. Like GACD, when crotonyl-CoA- (CCoA) bound GCD is reduced, the red anionic form of FAD radical (FAD.-) is stabilized, and excess reduction equivalents are necessary to effect full reduction of the complex. A comproportionation reaction is proposed between fully reduced crotonyl-CoA-bound GCD (GCD2e-CCoA) and GCDox-CCoA to partially explain the stabilization of GCD-bound FAD.- by CCoA. When GCD is reduced by its optimal substrate, glutaryl-CoA, a two-electron reduction is observed with concomitant formation of a long-wavelength charge-transfer band. It is proposed that the ETF specific for GCD abstracts one electron from this charge-transfer species and this is followed by the decarboxylation of the oxidized substrate. At pH 6.4, potential values measured for free GCD and GCD bound to acetoacetyl-CoA are -0.085 and -0.129 V, respectively. Experimental evidence is given for a positive shift in the reduction potential of GCD when the enzyme is bound to a 1:1 mixture of butyryl-CoA and CCoA. However, significant GCD hydratase activity is observed, preventing quantitation of the potential shift.

Chemical Phenomena↗
PubMed1990

Regulation of the redox potential of general acyl-CoA dehydrogenase by substrate binding.

Significant thermodynamic changes have been observed for general acyl-CoA dehydrogenase (GAD) upon substrate binding. Spectroelectrochemical studies of GAD and several of its substrates have revealed that these substrates are essentially isopotential for chain lengths of C-4 to C-16 (E 0' =-0.038 to -0.045 V vs SHE). When GAD is bound by these substrates, a dramatic shift in the midpoint potential of the enzyme is observed (E 0' = -0.136 V for ligand-free GAD and -0.026 V for acyl-CoA-bound GAD), thus allowing a thermodynamically favorable transfer of electrons from substrate to enzyme. This contrasts with values reported elsewhere. From these data an isopotential scheme of electron delivery into the electron-transport chain is proposed.

Acyl-CoA Dehydrogenases↗
PubMed1990

Redox and spectral properties of flavodoxin from Anabaena 7120.

We report here on the spectrophotometric and electrochemical properties of the flavodoxin from Anabaena 7120 and compare these properties with those of flavodoxins that have been studied previously. Molar absorption coefficients have been determined for all three oxidation states of this protein, at various wavelengths. For oxidized flavodoxin, molar absorption coefficients for the absorption maxima at 464 and 373 nm were 9200 and 8500 M-1 cm-1, respectively. Reduction by the first electron produced a neutral blue semiquinone which exhibited an absorption maximum at 575 nm. The molar absorption coefficients at 575 nm were 200 M-1 cm-1 for the oxidized form, 5100 M-1 cm-1 for the semiquinone form, and 250 M-1 cm-1 for the hydroquinone form. Redox potentials have been determined, in the pH range of 6.0 to 8.5, for both electron transfers. At pH 7.0, the midpoint potential values for the first and second electron transfers were -0.196 and -0.425 V, respectively. We determined that the first electron transfer is pH dependent and that a proton transfer accompanies this one electron transfer. It was also determined that the second electron transfer is pH independent in the pH range of 6.0 to 8.5.

Cyanobacteria↗
PubMed1989

Oxidation-reduction potentials and ionization states of extracellular peroxidases from the lignin-degrading fungus Phanerochaete chrysosporium.

The oxidation-reduction potentials of lignin peroxidase isozymes H1, H2, H8, and H10 as well as the Mn-dependent peroxidase isozymes H3 and H4 are reported. The potentiometric titrations involving the ferrous and ferric states of the enzyme had Nernst plots indicating single-electron transfer. The Em7 values of lignin peroxidase isozymes H1, H2, H8, and H10 are -142, -135, -137, and -127 mV versus standard hydrogen electrode, respectively. The Em7 values for the Mn-dependent peroxidase isozymes H3 and H4 are -88 and -93 mV versus standard hydrogen electrode, respectively. The midpoint potential of H1, H8, and H4 remained unchanged in the presence of their respective substrates, veratryl alcohol and Mn(II). The midpoint potential between the ferric and ferrous forms of isozymes H1 and H4 exhibited a pH-dependent change between pH 3.5 and pH 6.5. These results indicate that the reductive half-reaction of the enzymes is the following: ferric peroxidase + le- + H+----ferrous peroxidase. Above pH 6.5, the effect of pH on the midpoint potential is diminished and indicates that an ionization with an apparent pKa equal to approximately 6.6-6.7 occurs in the reduced form of the enzymes. A heme-linked ionization group in the ferrous form of the enzymes was confirmed by studying the effect of pH on the absorption spectra of isozymes H1 and H4. These spectrophotometric pH titration experiments confirmed the electrochemical results indicating pKa values of 6.59 and 6.69 for reduced isozymes H1 and H4, respectively. These results indicate the presence of a heme-linked ionization of an amino acid in the reduced form of the lignin peroxidase isozymes similar to that of other plant peroxidases.

Basidiomycota↗
PubMed1989

Unusual redox properties of electron-transfer flavoprotein from Methylophilus methylotrophus.

The most positive redox potential ever recorded for a flavin adenine dinucleotide (FAD) containing protein has been measured for an electron-transfer flavoprotein (ETF) synthesized by Methylophilus methylotrophus. This potential value, 0.196 V versus the standard hydrogen electrode (vs SHE), was measured at pH 7.0 for the one-electron reduction of fully oxidized ETF (ETFox) to the red anionic semiquinone form of ETF (ETF.-). Quantitative formation of ETF.- was observed. The first successful reduction of ETF from M. methylotrophus to its two-electron fully reduced form was also achieved. Although addition of the second electron to ETF.- was extremely slow, the potential value measured for this reduction was -0.197 V vs SHE, suggesting a kinetic rather than thermodynamic barrier to two-electron reduction. These data are believed to be consistent with the postulated catalytic function of ETF to accept one electron from the iron-sulfur cluster of trimethylamine dehydrogenase (TMADH). The second electron reduction appears to have no catalytic function. The very positive potential measured for this ETF and the wide separation of potentials for the two electron reduction steps show that this ETF is a unique and interesting flavoprotein. In addition, this work highlights that while ETFs exhibit similar structural and spectral properties, they display wide variations in redox properties.

Bacteria↗
PubMed1989

Regulation of oxidation-reduction potentials of anthranilate hydroxylase from Trichosporon cutaneum by substrate and effector binding.

The pH dependence of the redox behavior of anthranilate hydroxylase from Trichosporon cutaneum in its uncomplexed and anthranilate-complexed forms, as well as the effects on the reduction potential, at pH 7.4, of enzyme in complex with 3-methylanthranilate, salicylate, 3-acetylpyridine adenine dinucleotide phosphates, and azide plus anthranilate, is described. At pH 7.4 the midpoint potential of uncomplexed enzyme (EFlox/EFlredH-) is -0.229 V vs SHE, close to that of free flavin. The aromatic substrates and effector all shift the midpoint potential value in a positive direction by 0.068-0.100 V. This shift results in thermodynamically more favorable reduction of the substrate/effector-complexed enzyme by NADPH. Consistent with thermodynamic considerations, the aromatic substrates (or effector) are bound to the reduced enzyme 2-4 orders of magnitude more tightly than to the oxidized enzyme. The tighter binding of the substrate to the two-electron-reduced enzyme may be related to the double hydroxylation reaction performed by this enzyme, which is a more complex reaction than is carried out by typical flavoprotein hydroxylases. The acetylpyridine nucleotides appear to have no significant regulatory role.

Azides↗
PubMed1988

Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding.

The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzoates↗
PubMed1987

Regulation of the butyryl-CoA dehydrogenase by substrate and product binding.

Until now, workers in the field of fatty acid metabolism have suggested that the substrates are isopotential with the enzymes and that the reactions are forced to completion by the formation of charge-transfer complexes [Gustafson, W. G., Feinberg, B. A., & McFarland, J. T. (1986) J. Biol. Chem. 261, 7733-7741]. To date, no experimental evidence for this hypothesis exists. The work presented here shows that the butyryl-CoA/crotonyl-CoA couple is not isopotential with the enzymes with which it interacts. The potential of the butyryl-CoA/crotonyl-CoA couple (E ' = -0.013 V) is significantly more positive than the potential of either of the enzymes with which it interacts, bacterial butyryl-CoA dehydrogenase (E ' = -0.079 V) and mammalian general acyl-CoA dehydrogenase (E ' = 0.133 V). These data imply that the regulation of enzyme potential is essential for any electron transfer from substrate to enzyme to occur in mammalian or bacterial systems. In support of this assertion, a significant shift in potential for bacterial butyryl-CoA dehydrogenase (an analogue of the mammalian enzyme) in the presence of butyryl-CoA and crotonyl-CoA is reported. The potential is shifted positive by 60 mV. Larger potential shifts will undoubtedly be observed with the mammalian enzyme, which would be consistent with the catalytic direction of electron transfer.

Acyl Coenzyme A↗
PubMed1987

Redox properties of electron-transferring flavoprotein from Megasphaera elsdenii.

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii catalyzes electron transfer from NADH or D-lactate dehydrogenase to butyryl-CoA dehydrogenase. As a basis for understanding the interactions of ETF with its substrates, we report here on the redox properties of ETF alone. ETF exhibited reversible, two-electron transfer during electrochemical reduction in the presence of mediator dyes. The midpoint redox potentials of the FAD cofactor were -0.185 V at pH 5.5, -0.259 V at pH 7.1 and -0.269 +/- 0.013 V at pH 8.4, all versus the standard hydrogen electrode In the presence of the indicator dye 1-deazariboflavin, the Nernst slopes were 0.029 V and 0.026 V at pH 5.5 and pH 7.1, respectively, compared with an expected value of 0.028 V at 10 degrees C. At pH 8.4, in the presence of 2-hydroxy-1,4-naphthoquinone or phenosafranine, the Nernst slope varied from 0.021 V to 0.041 V. In the experiments at pH 8.4, equilibration was very slow in the reductive direction and a difference of as much as 30 mV was observed between reductive and oxidative midpoints. ETF exhibited no thermodynamic stabilization of the radical form of the FAD cofactor during electrochemical reduction at pH 5.5, 7.1 or 8.4. However, up to 93% of kinetically stable, anionic radical was produced by dithionite titration at pH 8.5. Molar absorptivities of ETF radical were 17,000 M-1 X cm-1 at 365 nm and 5100 M-1 X cm-1 at 450 nm. The four ETF preparations used here contained less than 7% 6-OH-FAD. However, two of the preparations contained significant amounts (up to 30%) of flavin which stabilized radical and reduced at potentials 0.2 V more positive than those required for reduction of the major form of ETF. This is referred to as the B form of ETF. The proportion of ETF-FAD in the B form was increased by incubation with free FAD or by a cycle of reduction and reoxidation. These treatments caused marked changes in the absorption spectrum of oxidized ETF and decreases of 20-25% in ETF units/A450.

Butyryl-CoA Dehydrogenase↗
PubMed1987

The effects of reversible freezing inactivation and inhibitor binding on redox properties of L-amino-acid oxidase.

We measured the redox potentials of frozen inactivated L-amino-acid oxidase (L-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) and inhibitor-bound (anthranilic acid) enzyme, and compared these redox properties to those of active L-amino-acid oxidase and benzoate-bound D-amino-acid oxidase (EC 1.4.3.3), respectively. The redox properties of the inactive enzyme are similar to the properties of free flavin; the potential is within 0.015 V of free flavin and no radical stabilization is seen. This corresponds to the loss of most interactions between apoprotein and flavin. In contrast, the anthranilic acid lowers the amount of radical stabilized from 85% to 35%. The potentials are still 0.150 V positive of free flavin, indicating that in the presence of inhibitor, many flavin-protein interactions remain intact. The difference between this behavior and that of D-amino-acid oxidase bound to benzoate, where the amount of radical declined from 95% to 5%, is explained on the basis of the relative tightness of binding of apoprotein to FAD. D-Amino-acid oxidase apoprotein has a relatively low Ka (10(6)) for FAD, and benzoate has a relatively high Ka (10(5)) for the enzyme. Therefore, the binding of benzoate increases the tightness of FAD binding to apo-D-amino-acid oxidase (10(11)), indicating significant changes in flavin-protein interactions. In contrast, apo-L-amino-acid oxidase binds flavin tightly (the Ka is greater than 10(7)) and the enzyme binds to anthranilate much less tightly, with a Ka of 10(3). The L-amino-acid oxidase apoprotein binding to FAD is tight initially, and the binding of anthranilate changes it only slightly.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗