Spectroscopic and voltammetric characterisation of the bacterioferritin-associated ferredoxin of Escherichia coli

The bacterioferritin-associated ferredoxin (Bfd) of Escherichia coli is a 64-residue polypeptide encoded by the bfd gene located upstream of the gene (bfr) encoding the iron-storage haemoprotein, bacterioferritin. The Bfd sequence resembles those of the similar to 60-residue domains found in NifU proteins (required for metallocluster assembly), nitrite reductases, and Klebsiella pneumoniae nitrate reductase. These related-domains contain four well-conserved cysteine residues, which are thought to function as ligands to a [2Fe-2S] cluster. The Bfd protein was over-produced, purified, and characterised. Bfd was found to be a positively-charged monomer containing two iron atoms and two labile sulphides. Ultraviolet-visible, EPR, variable-temperature magnetic-circular dichroism and resonance Raman spectroscopies, together with cyclic voltogram measurements, revealed the presence of a [2Fe-2S](2+,+) centre (E(12) = -254 mV) having remarkably similar properties to the Fe-S cluster of NifU. Bfd may thus be a 2Fe ferredoxin participating either in release/delivery of iron from/to bacterioferritin (or other iron complexes), or in iron-dependent regulation of bfr expression. (C) 1996 Academic Press, Inc.