Identification and characterization of a novel cytochrome c(3) from Shewanella frigidimarina that is involved in Fe(III) respiration

Shewanella frigidimarina NCIMB400 is a non-fermenting, facultative anaerobe from the gamma group of proteobacteria, When grown anacrobically this organism produces a wide variety of periplasmic c-type cytochromes, mostly of unknown function, We have purified a small, acidic, low-potential tetrahaem cytochrome with similarities to the cytochromes c(3) from sulphate-reducing bacteria. The N-terminal sequence was used to design PCR primers and the cctA gene encoding cytochrome c(3) was isolated and sequenced. The EPR spectrum of purified cytochrome c(3) indicates that all four haem irons are ligated by two histidine residues, a conclusion supported by the presence of eight histidine residues in the polypeptide sequence, each of which is conserved in a related cytochrome c(3) and in the cytochrome domains of flavocytochromes c(3). All four haems exhibit low midpoint redox potentials that range from -207 to -58 mV at pH 7; these values are not significantly influenced by pH changes. Shewanella cytochrome c(3) consists of a mere 86 amino acid residues with a predicted molecular mass of 11780 Da, including the four attached haem groups. This corresponds closely to the value of 11778 Da estimated by electrospray MS. To examine the function of this novel cytochrome c(3) we constructed a null mutant by gene disruption. S. fi frigidimarina lacking cytochrome c(3) grows well aerobically and its growth rate under anaerobiosis with a variety of electron accepters is indistinguishable from that of the wild-type parent strain, except that respiration with Fe(III) as sole acceptor is severely, although not completely, impaired.