Characterization of a flavocytochrome that is induced during the anaerobicrespiration of Fe3+ by Shewanella frigidimarina NCIMB400

A 63.9 kDa periplasmic tetrahaem flavocytochrome c(3), designated Ifc(3), w as found to be expressed in Shewanella frigidimarina NCIMB400 grown anaerob ically with ferric citrate or ferric pyrophosphate as the sole terminal ele ctron acceptor, but not in anaerobic cultures of the bacterium with other r espiratory substrates. Ifc(3) was purified to homogeneity and revealed by b iochemical, spectroscopic and primary structure analyses to contain four lo w-spin bis-His-ligated c(3)-haems, with midpoint reduction potentials of -7 3, -141, -174 and -259 mV. A low-potential flavin was present in the form o f non-covalently bound FAD; the protein possessed a unidirectional fumarate reductase activity. Disruption of the chromosomal gene encoding Ifc(3), if cA, did not lead to a significant change in the rate of Fe3+ reduction in b atch culture. However, during such growth the Ifc(3)-deficient mutant produ ced both a 35 kDa periplasmic c-type cytochrome and a 45 kDa membrane-assoc iated c-type cytochrome at markedly higher levels than did the parent strai n. Nucleotide sequencing data from directly upstream of ifcA indicated the presence of an open reading frame encoding a putative outer-membrane beta-b arrel protein of 324 amino acid residues.