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THERMODYNAMIC AND KINETIC INVESTIGATIONS OF STRUCTURE-FUNCTION RELATIONSHIPS IN FERRIC BINDING PROTEIN, TRANSFERRIN, AND HEMOGLOBIN
- Abstract:
This investigation probes the influences of protein structure-function relationships on iron proteins. Iron transport proteins (transferrins) play a critical role in iron homeostasis in both microbes and man. Possible mechanisms for iron binding by the bacterial transferrin ferric binding protein (FbpA) were investigated using stopped-flow fluorescence spectroscopy on WT and site-directed variants of FbpA. The iron binding site in FbpA is sandwiched between two domains (C-terminal and N-terminal), each of which have been reported as the initial site of interaction. A detailed analysis of the kinetics involved in fluorescence quenching of the binding site tyrosines at the C-terminal domain confirmed that the N-terminal domain interacts with iron in the early stages of iron insertion.
Due to a similar charge and ionic radius, the non-redox active iron analog gallium is commonly used in biochemical assays where the generation of reactive iron species is undesirable. Gallium exhibits cellular toxicity and has been shown to be dependent on FbpA mediated uptake. Supplementation with an exogenous iron source was reported to induce recovery from gallium toxicity. In order to better define FbpA mediated toxicity and iron recovery, the thermodynamic and kinetic consequences of metal mimicry were investigated. While the thermodynamic stability K’eff = 1014 M-1 for GaFbpA indicates a relatively stable assembly, the difference in stability achieved relative to iron is reduced by four orders of magnitude. The stepwise loading of Ga3+ into FbpA was found to be similar to that of iron, but metal exchange with exogenous iron was found to be kinetically facile.
Reactions of hemoglobin and oxygen initiate physiologically adverse oxidative side reactions when cell-free hemoglobins are used as blood substitutes. To clarify the factors governing these reactions, anaerobic oxidation studies were conducted on normal human hemoglobin and modified hemoglobins using spectroelectrochemistry. The reduction potentials of the modified hemoglobins were shifted to higher potentials relative to unmodified hemoglobin indicating a reduced thermodynamic driving force for oxidation to the met (Fe3+) form. The reduced amount of time required for the modified hemoglobins to achieve equilibrium suggested that the modifications altered heme exposure and implied an influence on the kinetics of oxidation.
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