engleski

The Role of Citrate and Phosphate Anions in the Mechanism of Iron(III) Sequestration by Ferric Binding Protein: Kinetic Studies of Holoprotein Formation of Wild Type and Binding Variants of FbpA

The ferric binding protein A (FbpA) plays a central role in the iron acquisition processes of pathogenic Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenza. FbpA functions as an iron shuttle within the periplasmic space of these Gram-negative human pathogens. Iron is picked up by FbpA at the periplasmic aspect of the outer membrane with concomitant acquisition of a synergistic anion. Here we report the kinetics and mechanisms involved with iron(III) loading into iron-free FbpA using iron(III) citrate as an iron source in the presence of excess citrate or phosphate (physiologically available anions) at pH 6.5. In the presence of excess phosphate, iron(III) citrate loads into apo-FbpA in three kinetically distinguishable steps, while in the presence of excess citrate only two steps are discernible. A stable intermediate containing iron(III) citrate bound FbpA is observed in each case. The observation of an additional kinetic step and moderate increase in apparent rate constants suggests an active role for phosphate in the iron insertion process. To further elucidate a mechanism for iron-loading, we report on the sequestration kinetics of iron(III) citrate in the presence of phosphate with six binding site mutant apo-FbpAs, H9E, E57D, E57Q, Q58A, Y195F, and Y196H. Tyrosine mutations drastically alter the kinetics, and reduce iron binding ability. H9E, E57D, and E57Q have near native iron binding behavior, however initial iron binding rates are altered enabling assignment of side chain interactions. Additionally this investigation elaborates on the function of FbpA as a carrier for iron chelates as well as iron salts

FbpA; Ferric binding protein; Fe(Cit)′; the distribution of mono and bis iron(III) citrate complexes at the conditions given (see Experimental Section); WT; wild-type; CTAB; cetyltrimethylammonium bromide; CD; Circular dichroism; LMCT; ligand-to-metal-charge-transfer band

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