Fe3+ Coordination and Redox Properties of a Bacterial Transferrin

Céline H Taboy,Kevin G Vaughan,Timothy A Mietzner,Philip Aisen,Alvin L Crumbliss

doi:10.1074/jbc.m004763200

Céline H Taboy, Kevin G Vaughan + Show 3 more

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https://doi.org/10.1074/jbc.m004763200

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Abstract

The Fe(3+) binding site of recombinant nFbp, a ferric-binding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques. An effective Fe(3+) binding constant in the presence of 350 microm phosphate at pH 6.5 and 25 degrees C was determined as 2.4 x 10(18) m(-1). EPR spectra for the recombinant Fe(3+)nFbp gave g' = 4.3 and 9 signals characteristic of high spin Fe(3+) in a strong ligand field of low (orthorhombic) symmetry. (31)P NMR experiments demonstrated the presence of bound phosphate in the holo form of nFbp and showed that phosphate can be dialyzed away in the absence of Fe(3+) in apo-nFbp. Finally, an uncorrected Fe(3+/2+) redox potential for Fe-nFbp was determined to be -290 mV (NHE) at pH 6.5, 20 degrees C. Whereas our findings show that nFbp and mammalian transferrin have similar Fe(3+) binding constants and EPR spectra, they differ greatly in their redox potentials. This has implications for the mechanism of Fe transport across the periplasmic space of Gram-negative bacteria.

Highlights

The Fe3؉ binding site of recombinant nFbp, a ferricbinding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques
Fe3ϩnFbp reconstituted from the apo protein and freshly prepared Fe(NH4)2(SO4)2 showed a similar spectrum, but addition of bicarbonate to a concentration of 17 mM enhanced the splitting in the spectrum of the reconstituted protein to 5.4 mT, producing a line similar to that given by native Human transferrin (hTf) (Ref. 20, Fig. 1B) and indicating increased axial perturbation in the zero-field tensor
A likely interpretation, is that bicarbonate is capable of fulfilling the anion requirement of Ferric-binding proteins (Fbps) as it is for eukaryotic transferrins

Summary

Introduction

The Fe3؉ binding site of recombinant nFbp, a ferricbinding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques. Whereas our findings show that nFbp and mammalian transferrin have similar Fe3؉ binding constants and EPR spectra, they differ greatly in their redox potentials This has implications for the mechanism of Fe transport across the periplasmic space of Gram-negative bacteria. Ferric-binding proteins (Fbps) are found in Haemophilus influenzae (hFbp), Neisseria gonorrhoeae, and Neisseria meningitidis (nFbp) [3] These bacteria express several different transferrin and lactoferrin receptors, Fbps appear to be the nodal point for transport of iron across the periplasmic space to the cytoplasmic membrane. The inner coordination shell around iron in both nFbp [11] and hFbp [10] show a striking resemblance with the inner coordination sphere of iron in mammalian transferrins These Fbps contain only a single Fe3ϩ binding site composed of four donor groups from protein

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