Benjamin A. Feinberg

Redox properties of 2[4Fe4S] ferredoxins

Abstract The redox properties of the 2[4Fe4S] ferredoxin isolated from Clostridium pasteurianum were examined directly at an edge pyrolytic graphite electrode using square-wave voltammetry. The direct electrochemical approach was compared with other techniques that have been used to determine the equilibrium reduction potential of this protein. A microcell is described that permits routine electrochemical experiments on small sample volumes (25–50 μl). The electrostatic potentials of a homologous ferrodoxin with a known x-ray crystal structure, which includes the first treatment of net atomic charges in the iron—sulfur clusters, was determined using a macroscopic electrostatic model.

A spectroelectrochemical cell designed for low temperature electron paramagnetic resonance titration of oxygen-sensitive proteins

In this paper we describe an anaerobic titrator made virtually from glass with a small amount of high vacuum epoxy mounted directly to a quartz EPR tube. A complete titration may be carried out with as little as 600 microliters of sample. This cell features the anaerobic manipulation of an electrochemically poised solution from an electrochemical pouch to an EPR tube. The cell uses a gold foil working electrode and Ag/AgCl reference and counter electrodes. The reference and counter electrodes are isolated from the sample by leached Vycor glass. In the work reported here, we used this cell to determine the equilibrium redox potential of methyl viologen in an EPR titration. With methyl viologen as an indicator we found that the cell has a residual oxygen level of 1.5 microM with a leak rate of 0.005 nmol/min. After moving the solution into the EPR tube, freezing, performing EPR, and thawing, the potential of the methyl viologen solution drifted only 2 mV. During the titration, the poised potentials were stable, drifting only 1 mV/min. Formal potentials as low as -630 mV in a vitamin B12-type protein have been determined with this cell (S. R. Harder, W.-P. Lu, B. A. Feinberg, and S. W. Ragsdale (1989) Biochemistry, in press).

Interaction between organic and inorganic pollutants in the clay interlayer

A synergistic mechanism for the retention of organic and inorganic pollutants in clays is discussed in this paper. The mechanism of adsorption of cis- or trans- 1,2-dichloroethylene vapor (CDE or TDE, respectively) by hydrated smectite clay (hectorite) exchanged with Pb2+, Hg2+, Cd2+, Ca2+, Ag+, or Na+ has been investigated by simultaneously measuring chlorohydrocarbon uptake and water desorption isotherm and by recording the infrared (IR) spectrum of the adsorbed phase. Hydrated hectorite saturated with divalent cations adsorbs about 55% more CDE or 35% more TDE than those saturated with monovalent cations. The quantity of chlorohydrocarbon adsorbed is also a function of the hydration of the clay interlayer space. When dehydrated, hectorite does not adsorb CDE or TDE. Upon long outgassing at room temperature or even at 100°C, the characteristic IR bands of clays with adsorbed chlorohydrocarbon, although much weakened, are still observable. The ratio of the amount of water desorbed to the amount of chlorohydrocarbon adsorbed varied from about 0.22 to 0.34. A shift of the center of gravity of the hydration water OH stretching frequency towards a higher wavenumber and of the asymmetric CC1 stretching vibration toward a lower frequency suggest that the formation of hydrogen bonds between CDE or TDE and water is the driving force for adsorption and that the cation-dipole interaction does not play a major role.

Reduction potentials in relation to physiological activities of benzenoid and heterocyclic nitroso compounds: Comparison with the nitro precursors

Abstract Reduction potentials were determine for various physiologically active benzenoid and heterocyclic nitroso compounds, namely, substituted nitrosobenzenes, 1-nitrosopyrene, and 1-methyl-2-nitrosoimidazole. The values, favorable for biological activity, ranging from 0.2 to −0.2 V, increased in acidic medium. These potentials were appreciably higher than those for the corresponding nitro parents. In most cases, the nitroso form was more biologically active than the nitro counterpart. Catalytic electron transfer processes may play a role in vivo , along with other actions, in the observed responses from the nitroso category.

The chronoamperometric determination of homogeneous small molecule-redox protein reaction rates

Abstract An essentially new application of chronoamperometry is presented for the determination of homogeneous second-order rate constants for the reactions between small molecule reductants and redox proteins. The first part of the work is a comparison between stopped-flow kinetics and chronoamperometric kinetics for the reaction of ferrous-EDTA with horse cytochrome c . The reaction was demonstrated to be first order in both ferrous-EDTA and cytochrome c and the effect of ionic strength was also studied. All of the chronoamperometric results compared well with the stopped-flow work which had been done previously. Chronoamperometry was then used to study several other reactions which have not been previously examined, including the reaction of ferrous-diethylenetriamine pentaacetic acid with cytochrome c . The reaction was slower than the ferrous-EDTA reaction but was more sensitive to ionic strength because of the greater charge (−3) on the complex. The second study was the reaction of ferrous-EDTA with Rhodospirillum rubrum cytochrome c 2 as a function of ionic strength. This novel application of chronoamperometry to small molecule-redox protein reactions represent a new and relatively easy alternative to anaerobic stopped-flow kinetics.

Intermediates during the fatty acyl CoA dehydrogenase catalyzed reduction of electron transfer flavoprotein (ETF) by fatty acyl CoA esters

Abstract The reaction kinetics for the reduction of ETF by saturated fatty acyl CoA esters has traditionally been studied by dye assays utilizing transfer of electrons from the reduced FAD of ETF to 2,6-Dichloroindophenol (DCI). We have found that it is preferable to use the natural fluorescence and absorbance properties of ETF itself in order to investigate both the steady state and single turnover reduction of ETF. Our investigations indicate formation of a red anionic semiquinone as an intermediate in the two electron reduction of ETF under steady state kinetic condition using butyryl CoA as substrate. Furthermore, V max E ∼- 6.0 sec −1 calculated from steady state kinetic data for formation of this semiquinone observed from the increase in 370 nm absorbance or decrease in ETF fluorescence emission at 510 nm (the dehydrogenase catalyst does not fluoresce) is much faster than V max E ∼- 1.0 sec −1 for hydroquinone formation. Even though DCI is a two electron acceptor, V max E ∼- 4.0 sec −1 for butyryl CoA is limited by the rate of formation of ETF semiquinone, which must, therefore, transfer its single electron to DCI. Investigation of the extent of ETF reduction to semiquinone (measured from fluorescence) by a substrate which transfers electrons quantitatively to the dehydrogenase catalyst indicates that the overall stoichiometry is two electrons transferred to the two FAD flavins on the dimeric ETF molecule. Furthermore, hydroquinone is not formed from disproportionation of the ETF semiquinone dimer since formation of hydroquinone is not accompanied by the fluorescence increase predicted from such disproportionation.

Are Reduction Potentials of Antifungal Agents Relevant to Activity

Cyclic voltammetry data were obtained for several categories of fungicidal agents including quinones (akrobomycin, podosporin A), iminium ions and precursors (pyridazines, 15-azahomosterol, griseofulvin-4′-oxime), and metal derivatives of chelators (pyridine-2-aldehyde thiosemicarbazones). The reductions usually occurred in the range of −0.7 to +0.3 V. Reduction potentials provide information on the feasibility of electron transfer in vivo. Catalytic production of oxidative stress from redox cycling is a possible mode of action. Alternatively, there may be interference with normal electron transport chains.

The redox couple of the cytochrome c cyanide complex: The contribution of heme iron ligation to the structural stability, chemical reactivity, and physiological behavior of horse cytochrome c

Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c–cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0′ was −240 mV versus SHE, equivalent to −23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0′, the binding constant of cyanide to the reduced protein was estimated to be 4.7 × 10−3 LM−1 or 13.4 kJ/mol (3.2 kcal/mol), which is 48.1 kJ/mol (11.5 kcal/mol) less favorable than the binding of cyanide to ferricytochrome c. For coordination of cyanide to ferrocytochrome c, the entropy change was earlier experimentally evaluated as 92.4 Jmol−1K−1 (22.1 e.u.) at 25 K, and the enthalpy change for the same net reaction was calculated to be 41.0 kJ/mol (9.8 kcal/mol). By taking these results into account, it was discovered that the major obstacle to cyanide coordination to ferrocytochrome c is enthalpic, due to the greater compactness of the reduced molecule or, alternatively, to a lower rate of conformational fluctuation caused by solvation, electrostatic, and structural factors. The biophysical consequences of the large difference in the stabilities of the closed crevice structures are discussed.

Comparative Kinetic-Ionic Strength Study of Two Differently Charged Cytochromes C : Effects are Limited to Overall Charge

The reduction kinetics of two differently charged cytochromes c, horse cytochrome c and Rhodosprillum rubrum cytochrome c2, by ferrous EDTA2− were studied as a function of ionic strength. Since both proteins have nearly the same heme edge region, but have very different overall surface charge, this comparative study served as a direct test of the utility of small nonbinding non-physiological redox agents in the study of the charge of electron transfer sites of redox proteins. Calculations based on the ionic strength-kinetic data yielded protein charges of +10 and +2.3 for cytochrome c and cytochrome c2 respectively and compared well with values of +9 and +3 for the overall charge of the proteins based on acidic and basic amino acid residues. It is concluded that ionic strength effects upon the redox kinetics with such nonbinding nonphysiological redox agents reflect the influence of the overall protein charge and not the localized charge of the presumed site of electron transfer.

Cyanide reactivity of cytochrome c derivatives.

The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility. The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome. Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide. The derivative demonstrate markedly different kinetics which indicate the following conclusions. (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction. (2) Compared to native cytochromes, reaction rates are 10-20 fold greater. This is in accord with a more accessible heme crevice, but not a completely opened crevice. For the completely opened case, rate increases are expected to be between three and five orders of magnitude. (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation. A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step. This conformation change has a limiting rate of 6 . 10(-2) s-1.