Isolation, characterization and gene sequence analysis of a membrane-associated 89 kda fe(iii) reducing cytochrome c from geobacter sulfurreducens
Magnuson, Timothy S.
Hodges-Myerson, Allison L.
Maroney, Michael J.
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Geobacter sulfurreducens is capable of anaerobic respiration with Fe(III) as a terminal electron acceptor via a membrane-bound Fe(III) reductase activity associated with a large molecular mass cytochrome c. This cytochrome was purified by detergent extraction of the membrane fraction, Q-Sepharose ion-exchange 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 Mr of 89000 as determined by denaturing PAGE, and has an isoelectric focusing. Dithionite-reduced cytochrome can donate electrons to Fe(III)-nitrilotriacetic acid and synthetic ferrihydrite, thus demonstrating that the cytochrome has redox and thermodynamic properties required for reduction of Fe(III). Analysis using cyclic voltametry 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) respiration. Sequence analysis of a cloned chromosomal DNA fragment revealed a 2307 bp open reading frame (fer A) encoding a 768 amino acid protein corresponding to the 89 kDa cytochrome. The deduced amino acid sequence (FerA) translated from the open reading frame contained 12 putative haem–binding motifs, 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 identity with amino acid sequences of other known cytochromes, although it does share some features with characterized polyhaem cytochromes c.
Magnuson, T.S., N. Isoyama, A.L. Hodges-Myerson, G. Davidson, M.J. Maroney, G.G. Geesey, and D.R. Lovley, "Isolation, Characterization and Gene Sequence Analysis of a Membrane-Associated 89 kDa Fe(III) Reducing Cytochrome c from Geobacter sulfurreducens," Biochem. J., 359:1 (2001).