Lisandra L. Martin

Probing the stability of the disulfide radical intermediate of thioredoxin using direct electrochemistry

Thioredoxin, a redox active disulfide protein, has been specifically immobilized at a modified gold electrode. The thioredoxin is uniquely oriented relative to the electrode surface via a histidine tag thereby enabling the redox mechanism of protein to be examined. When scanning the applied potential in the negative direction (cathodic), two one-electron reduction waves can be observed. The first of these redox waves occurs at −90xa0mV and is electrochemically reversible at all scan rates whereas the second wave occurs at −433xa0mV is irreversible. These two processes are interpreted as the initial reduction of the disulfide form of the protein to a stable (reversible) semi-reduced radical anion intermediate, followed by an electrochemically irreversible process to form a fully reduced thioredoxin. These electron transfer characteristics suggest that a radical intermediate retaining the sulfur-sulfur bond is thermodynamically stable but the addition of a second electron results in bond scission.

Electrochemical behaviour of human adrenodoxin on a pyrolytic graphite electrode

Adrenodoxin (Adx) functions as a redox protein in the delivery of electrons to all mitochondrial cytochromes P450. In order to further characterize the human form of this protein, direct electrochemistry of human adrenodoxin (Hadx) has been observed for the first time on a pyrolytic graphite electrode (PGE) modified with poly-L-lysine. A single well-defined redox wave was observed with a midpoint potential of -448+/-3 mV vs. Ag/AgCl (sat. KCl) at scan rates of 10 mV/s and over the pH range 4.0-8.0. At slow scan rates, the reduction process was close to being electrochemically reversible whereas, at faster scan rates, only quasi-reversibility was observed. A correlation was observed between the peak separation (DeltaE) for the cyclic voltammograms and pH over a wide range of scan rates. The variation of DeltaE with pH was at a minimum (optimum reversibility) at pH 7.0 for all scan rates tested. This correlation may suggest that the direct electrochemistry method could possibly provide a means for determining protein or enzyme activity. The electron transfer rate constant, k(s), was determined to be 0.28 s(-1) at pH 7.0 and a small pH dependence was observed. The results obtained in this study demonstrate the facile nature of direct electron transfer for human adrenodoxin, and provide an estimate of the midpoint reduction potential at a pyrolytic graphite electrode via electrostatic immobilisation.

The influence of promoter and of electrode material on the cyclic voltammetry of Pisum sativum plastocyanin

The reversible cyclic voltammetry of pea plastocyanin (Pisum sativum) was studied with a wide range of electrodes: edge-oriented pyrolytic graphite (PGE), glassy carbon (GCE), gold (Au) and platinum (Pt) electrodes. Plastocyanin was coated onto the electrode surface by exploiting the electrostatic interaction between the negatively charged protein and a wide range of positively charged promoters. The effect of the redox response with an extended range of promoters, including poly-L-lysine, polymyxin B, neomycin, tobramycin, geneticin, spermine and spermidine, were included in this study. The resulting cyclic voltammograms reveal that the observed midpoint potential for plastocyanin can be shifted significantly depending on the choice of promoter. The stability of the negatively charged plastocyanin-promoter layer on an electrode was gauged by the rate of bulk diffusion of the protein from the immobilised film into the solution. Reversible cyclic voltammograms were obtained using edge-oriented pyrolytic graphite (PGE) and glassy carbon electrodes (GCE) with all promoters; however, platinum and gold electrodes were unable to sustain a defined redox response. The combination of pyrolytic graphite electrode/poly-L-lysine/plastocyanin was found to be the most stable combination, with a redox response which remained well defined in solution for more than 1 h at pH 7.0. The midpoint potentials obtained in this manner differed between the two graphite electrodes PGE and GCE using poly-L-lysine as the promoter. This effect was in addition to the expected pH dependence of the midpoint potential for plastocyanin and the results indicated that the pK(a) for plastocyanin on PGE was 4.94 compared to that on GCE of 4.66. It is concluded that both the electrode material and the nature of the promoter can influence the position of the redox potentials for proteins measured in vitro. This study extends the range of biogenic promoters used in combination with electrode materials. Thus, we can begin to develop a more comprehensive understanding of electrode-protein interactions and draw conclusions as to metalloprotein function, in vivo. To support these studies, we have sought information as to the nature of the electrode/promoter/protein interaction using scanning tunneling microscopy (STM) to study both the promoter and the plastocyanin protein on a gold surface.

Bis­(di-μ-acetato-aqua{2-[N-ethyl-N-(2-hydroxy-4-methyl­benzyl)­amino­methyl]-1-methyl­benz­imid­az­ole}­nickel)­nickel(II) tetra­hy­drate

The previously unknown title compound, tetra-μ-acexadtato-1:2κ2O;1:2κ2O:O′;xad2:3κ2O;xad2:3κ2O:O′-dixadaqua-1κO,3κO-bisxad(μ-2-{[N-ethyl-N-(2-hyxaddroxy-5-methylbenzyl)xadamxadino]xadmethyl}-1-methyl-1H-benzxadimidxadazxadole)-1κ3N3,N,O:2κO;3κ3N3,N,O:2κO-trixadnickel(II) tetraxadhyxaddrate, [Ni3(C18H22N3O)2(C2H3O2)4(H2O)2]·xad4H2O, (I), is a centrosymmetric linear trinuclear nickel(II) complex, where the Ni atoms are in an octahedral coordination and the ligand heteroatoms act so as to model amino acid residues.

Aqua­bis(4‐morpholinyldi­thio­carbamato)­iron(III) tri­fluoro­methane­sulfonate

In the title compound, [Fe(C5H8NOS2)2(H2O)]CF3SO3, the Fe atom has five-coordinate square-pyramidal stereochemistry, with the Fe atom 0.472u2005(1)u2005A above the plane of four S atoms and a water molxadecule O atom at the apex. The cation has m (Cs) symmetry, with the crystallographic mirror plane passing through the Fe, O and N atoms. One O atom of the anion forms a strong hydrogen bond with the coordinated water molxadecule [O⋯O = 2.642u2005(5)u2005A]. This is the first report of a cationic [Fe(dixadthioxadcarbamate)2L]n+ species.

Aceto­nitrile{N-[2-(4,7-di­methyl-1,4,7-tri­aza­cyclo­non-1-yl)­ethyl]-N-[(1-methyl­benzimidazol-2-yl)­methyl]­methyl­amine}nickel(II) bis­(perchlorate) aceto­nitrile solvate

In the title compound, [Ni(C2H3N)(C20H34N6)](ClO4)2·CH3CN, NiII has approximately octahedral coordination geometry. The ligand donor atoms are five N atoms from the pendant-arm macrocyclic ligand (three from the macrocycle and two from the pendant arm) and one N atom from a coordinated acetoxadnitrile molxadecule.

{2-(4,7-Di­methyl-1,4,7-tri­aza­cyclo­non-1-yl)-N-methyl-N-[(1-methyl­benzimidazol-2-yl)­methyl]­ethyl­amine}­copper(II) bis­(perchlorate)

In the title compound, [Cu(C20H34N6)](ClO4)2, the CuII atom is coordinated to the five ligand donor N atoms of the pendant-arm macrocyclic ligand. The coordination sphere geometry can be described as either distorted square-pyramidal or distorted trigonal-prismatic.