Alvin L. Crumbliss

Chemical aspects of siderophore mediated iron transport

In this mini-review we describe selected aspects of the coordination chemistry relevant to siderophore mediated iron transport and bioavailability. Specific emphasis is placed on a discussion of in vitro kinetic and thermodynamic data that are relevant to elucidating possible in vivo mechanisms for environmental iron acquisition by microbial cells.

Direct electron transfer at horseradish peroxidase—colloidal gold modified electrodes

Abstract The reduction of H2O2 on horseradish peroxidase (HRP) and horseradish peroxidase—gold sol (HRP-Au) modified electrodes has been studied with and without an electron transfer mediator. The amplification effect owing to the enzyme-catalyzed turnover of substrate facilitates our observation that HRP immobilized on colloidal gold and then deposited on a flat electrode surface can be reduced at a convenient rate at 0 V (Ag/AgCl) without an electron transfer mediator. Possible mechanisms and potential applications are discussed.

A xanthine oxidase/colloidal gold enzyme electrode for amperometric biosensor applications

An electrode has been prepared based on xanthine oxidase adsorbed to colloidal gold and evaporated onto the surface of glassy carbon. This electrode responds to xanthine or hypoxanthine in the absence of added mediator by electrochemical oxidation of the enzymatic oxidation product, uric acid, at the electrode surface. The electrode can also be used in the presence of an electron transfer mediator to detect other substrates for xanthine oxidase such as 4-hydroxypyrimidine.

Coordination Chemistry and Redox Processes in Siderophore-Mediated Iron Transport

In this mini-review we present an environmental iron mobility/transport scheme consisting of inter-related controls, whereby the first coordination shell of iron modulates the iron redox potential (E1/2), and the oxidation state of iron controls the chemistry of the first coordination sphere and therefore the immediate chemical environment of the iron. Siderophores (microbially generated iron specific chelators) may be viewed as iron redox mediators. Siderophore chelation of environmental iron in a reduced (Fe(II)) oxidation state results in facile air oxidation of iron due to the negative redox potentials observed for Fe-siderophore complexes. This solubilizes the iron and locks it into a specific coordination environment, thereby preventing hydrolysis and precipitation. The high-spin Fe3+ → Fe+ electron transfer process may be viewed as a switch that controls the thermodynamic stability and kinetic lability of the first coordination shell. Reduction of iron(III)-siderophore complexes to iron(II)-siderophore complexes decreases thermodynamic stability, increases the rate of siderophore ligand exchange, and increases the ease of siderophore donor atom protonation, thus facilitating a rapid turnover of the first coordination shell. Results are presented for iron-siderophore pH and oxidation state dependent speciation studies that are relevant to environmental and microbial iron mobility and transport.

Iron sequestration by small molecules: Thermodynamic and kinetic studies of natural siderophores and synthetic model compounds

Publisher Summary This chapter discusses researches on natural siderophores and synthetic siderophore-like molecules, with an emphasis on results from the laboratories over the past decade in the context of the overall field. These findings are discussed in terms of siderophore structure, the thermodynamics of iron sequestration, the kinetics of iron-exchange reactions in ferri-siderophore systems, and molecular recognition and transport. The chapter also discusses the expanding field of ferri-siderophore chemistry and its application to the design of synthetic iron chelators for the study of siderophore binding and uptake in mimics of biological systems, and in therapeutic applications. The use of iron chelators with high stability and specificity can be linked to the study of iron–siderophore chemistry, as an understanding of the structural and architectural features that microbes use to obtain iron from their environment can be used to design synthetic siderophore-like molecules for chelating iron in biological and engineered systems.

A carrageenan hydrogel stabilized colloidal gold multi-enzyme biosensor electrode utilizing immobilized horseradish peroxidase and cholesterol oxidase/cholesterol esterase to detect cholesterol in serum and whole blood

The preparation of two immobilized enzyme electrodes is described. One electrode contains horseradish peroxidase absorbed to colloidal gold and deposited on a glassy carbon electrode along with cholesterol oxidase entrapped in a carrageenan hydrogel. The second electrode also includes cholesterol esterase entrapped in the carrageenan. The incorporation of ferrocene or ferrocenecarboxylic acid mediator is brought about by either evaporation on the glassy carbon electrode or, in the latter case, entrapment in the carrageenan hydrogel. Amperometric signal generation results from the HRP catalyzed turnover of H2O2, a secondary product of the cholesterol oxidase catalyzed oxidation of cholesterol. Use of these enzyme electrodes makes cholesterol detection possible in human serum, low density lipoprotein, and whole blood.

The influence of the synergistic anion on iron chelation by ferric binding protein, a bacterial transferrin

Although the presence of an exogenous anion is a requirement for tight Fe3+ binding by the bacterial (Neisseria) transferrin nFbp, the identity of the exogenous anion is not specific in vitro. nFbp was reconstituted as a stable iron containing protein by using a number of different exogenous anions [arsenate, citrate, nitrilotriacetate, pyrophosphate, and oxalate (symbolized by X)] in addition to phosphate, predominantly present in the recombinant form of the protein. Spectroscopic characterization of the Fe3+/anion interaction in the reconstituted protein was accomplished by UV-visible and EPR spectroscopies. The affinity of the protein for Fe3+ is anion dependent, as evidenced by the effective Fe3+ binding constants (K′eff) observed, which range from 1 × 1017 M−1 to 4 × 1018 M−1 at pH 6.5 and 20°C. The redox potentials for Fe3+nFbpX/Fe2+nFbpX reduction are also found to depend on the identity of the synergistic anion required for Fe3+ sequestration. Facile exchange of exogenous anions (Fe3+nFbpX + X′ → Fe3+nFbpX′ + X) is established and provides a pathway for environmental modulation of the iron chelation and redox characteristics of nFbp. The affinity of the iron loaded protein for exogenous anion binding at pH 6.5 was found to decrease in the order phosphate > arsenate ∼ pyrophosphate > nitrilotriacetate > citrate ∼ oxalate ≫ carbonate. Anion influence on the iron primary coordination sphere through iron binding and redox potential modulation may have in vivo application as a mechanism for periplasmic control of iron delivery to the cytosol.

Hypothesis: is lung disease after silicate inhalation caused by oxidant generation?

Inhaled silicate dusts may cause lung disease through their surface coordination of iron with subsequent oxidant generation via the Fenton reaction. Pneumoconiosis, irritant bronchitis, focal emphysema, and carcinoma may be produced by oxidants either directly through lipid peroxidation and protein inactivation, or indirectly by oxidant-mediated release of cytokines such as platelet-derived growth factor. The increased incidence of tuberculosis observed among silicate workers could be explained by accumulation of iron complexed by dust particles in the lung and made available to dormant mycobacteria as a virulence factor.

The redox hypothesis in siderophore-mediated iron uptake

The viability of iron(III/II) reduction as the initial step in the in vivo release of iron from its thermodynamically stable siderophore complex is explored.

Comparison of colloidal gold electrode fabrication methods: the preparation of a horseradish peroxidase enzyme electrode

In order to prepare biosensing electrodes which respond to hydrogen peroxide, horseradish peroxidase has been adsorbed to colloidal gold sols and electrodes prepared by deposition of these enzyme-gold sols onto glassy carbon using three methods: evaporation, electrodeposition and electrolyte deposition. In the latter method the enzyme-gold sol is applied to the surface of a glassy carbon disk electrode followed by an equal volume of 2 mM CaCl2. The electrolyte causes the sol to precipitate on the electrode surface, producing an immobilized enzyme electrode. Satisfactory electrodes which gave an electrochemical response to hydrogen peroxide in the presence of the electron transfer mediator ferrocenecarboxylic acid were produced by all three methods. Evaporation of horseradish peroxidase-gold sols produced electrodes with the best reproducibility and the widest linear amperometric response range. These electrodes can also easily be stored in a dry state. Although not as good as evaporation, electrodeposition also produced satisfactory electrodes. Electro-deposition provides the added advantage that it lends itself to the preparation of multi-enzyme/multi-analyte electrodes by the adsorption of different enzymes to separate gold sols, followed by sequential electrodeposition onto discrete areas of a multichannel electrode.