Purification and magneto-optical spectroscopic characterization of cytoplasmic membrane and outer membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400

Two membranous c-type cytochromes from the Fe(III)-respiring bacterium Shew anella frigidimarina NCIMB400, CymA and OmcA, have been purified and charac terized by W-visible, magnetic circular dichroism, and electron paramagneti c resonance spectroscopies. The 20-kDa CymA is a member of the NapC/NirT fa mily of multiheme cytochromes, which are invariably anchored to the cytopla smic membrane of Gram-negative bacteria, and are postulated to mediate elec tron flow between quinols and periplasmic redox proteins. CymA was found to contain four low-spin c-hemes, each with bis-His axial ligation, and midpo int reduction potentials of +10, -108, -136, and -229 mV, The 85-kDa OmcA i s located at the outer membrane of S, frigidimarina NCIMB400, and as such m ight function as a terminal reductase via interaction with insoluble Fe(III ) substrates, This putative role is supported by the finding that the prote in was released into solution upon incubation of harvested intact cells at 25 degrees C, suggesting an attachment to the exterior face of the outer me mbrane, OmcA was revealed by magneto-optical spectrocopies to contain 10 lo w-spin bis-His ligated c-hemes, with the redox titer indicating two sets of near iso-potential components centered at -243 and -324 mV.