Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens

Geobacter sulfurreducens is capable of anaerobic respiration with Fe(III) a s a terminal electron acceptor via a membrane-bound Fe(III) reductase activ ity associated with a large molecular mass cytochrome c. This cytochrome wa s purified by detergent extraction of the membrane fraction. Q-Sepharose io n-exchange chromatography. preparative electrophoresis, and MonoQ ion-excha nge chromatography. Spectrophotometric analysis of the purified cytochrome reveals a c-type haem, with no evidence of haem a. haem b or sirohaem. The cytochrome has an M-r, of 89 000 as determined by denaturing PAGE. and has an isoelectric point of 5.2 as determined by analytical isoelectric focusin g. Dithionite-reduced cytochrome can donate electrons to Fe(III)-nitrilotri acetic acid and synthetic ferrihydrite, thus demonstrating that the cytochr ome has redox and thermodynamic properties required for reduction of Fe(III ). Analysis using cyclic voltammetry confirmed that the reduced cytochrome can catalytically transfer electrons to ferrihydrite, further demonstrating its ability to be an electron transport mediator in anaerobic Fe(III) resp iration. Sequence analysis of a cloned chromosomal DNA fragment revealed a 2307 bp open reading frame (ferA) encoding a 768 amino acid protein corresp onding to the 89 kDa cytochrome. The deduced amino acid sequence (FerA) tra nslated from the open reading frame contained 12 putative haem-binding moti fs., as well as a hydrophobic N-terminal membrane anchor sequence., a lipid -attachment site and an ATP/GTP-binding site. FerA displayed 20% or less id entity with amino acid sequences of other known cytochromes, although it do es share some features with characterized polyhaem cytochromes c.