Marian T. Stankovich

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.

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.

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.

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.

Activation of substrate/product couples by medium-chain acyl-CoA dehydrogenase☆

Natural substrate/product binding activates medium-chain acyl-CoA dehydrogenase (MCAD) to accept electrons from its substrate by inducing a positive flavin midpoint potential shift. The energy source for this activation has never been fully elucidated. If ground-state alterations of the ligand, such as polarization, are entirely responsible for enzyme activation, the ligand potential should shift equally to that of the flavin but in the opposite direction. Ligand polarization is likely responsible for only a small portion of this activation. Here, thiophenepropionoyl- and furylpropionoyl-CoA analogs were used to directly measure the redox modulations of several ligand couples upon binding to MCAD. These measurements identified the thermodynamic contribution of ligand polarization to enzyme activation. Because the ligand potential alterations are significantly smaller than modulations in the flavin potential due to binding, other phenomena such as pK(a) changes, desolvation, and charge alterations are likely responsible for the thermodynamic modulations required for MCADs activity.

Redox properties of cytochrome c3 from Desulfovibrio desulfuricans NCIMB 8372 : effects of electrode materials and sodium chloride

Abstract Direct electron transfer between cytochrome c 3 from Desulfovibrio desulfuricans NCIMB 8372 and electrodes made of different materials was studied using cyclic voltammetry and differential pulse voltammetry. The mid-point potentials of the four oxidation-reduction states were calculated by simulations using two models. The values have been compared with corresponding values from the mediated spectropotentiometric titration. It was found that the electrode material influenced the value of the potentials except for the first reduction step of the cytochrome. With basal plane graphite and glassy carbon, the most negative reduction step was shifted to a value 30 mV more negative than the value from the potentiometric titration experiment. With gold, the shift was only 10 mV and with dipyridyl-modified gold no shift was observed. When NaCl was included in the electrolyte, the potentials of all four steps shifted to values approximately 100 mV more negative in tests with basal plane graphite, glassy carbon and gold electrodes alike. The mid-point potentials of the four hemes were determined from two different simulation models. The mutual conversion of these two series of data is discussed. One of the four hemes is found to be independent of the other hemes. No strong interaction between the remaining hemes is proposed. The binding pattern of the ferri- and ferrocytochrome to anion and cation exchangers suggests that, upon reduction, the isoelectric point changes from pH 9.5–10 to pH 6.5–7.

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)

Comparison of ligand polarization and enzyme activation in medium- and short-chain acyl-coenzyme A dehydrogenase-novel analog complexes.

Spectroelectrochemical and off-resonance Raman indicate that substrate/product binding to medium-chain acyl-coenzyme A (CoA) dehydrogenase (pMCAD) results in ligand polarization and positive flavin potential shifts, which activate the enzyme for electron transfer. Bacterial short-chain acyl-CoA dehydrogenase (bSCAD) typically exhibits smaller potential shifts upon substrate/product binding that have not been linked to ligand polarization. To further investigate the roles of ligand binding and polarization in activation, several novel aromatic carboxyloyl-CoAs were designed. These analogs allowed for the first direct comparison of pMCAD and bSCAD mechanisms. The results indicate that pMCAD activation can occur without perceptible analog polarization. bSCAD data provide the first spectral evidence of ligand polarization. The potential alterations exhibited by ligand-bound bSCAD are smaller than those of pMCAD, while their directionality and magnitude suggest differing enzyme-analog interactions. Such data provide the first indication of variations in the activation mechanism of these enzymes, which were thought to be comparable in both structure and function.