title: Heterologous expression of hexaheme fragments of a multidomain cytochrome from Geobacter sulfurreducens representing a novel class of cytochromes c.
abstract: Multiheme cytochromes c are of great interest for researchers for a variety of reasons but difficult to obtain in quantities sufficient for the majority of studies. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Three of them represent a new class of multiheme cytochromes characterized by a mixed type of heme coordination and multidomain organization. We cloned and expressed in Escherichia coli three hexaheme fragments corresponding to two-domain fragments of one such protein containing 12 heme binding motifs and believed to consist of four triheme domains. Despite high sequence similarity among the fragments, expression levels varied significantly. Expression was optimized either by host strain variation or by reducing the rate of apoprotein synthesis. All three fragments were purified by cation exchange followed by gel filtration and were shown to contain six covalently attached hemes as confirmed by mass spectrometry. Their visible spectra are typical of c-type cytochromes. One of the fragments was crystallized and its preliminary X-ray structure shows two separate domains.
authors.affiliation: Biosciences Division, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA. londer@anl.gov
authors.forename: Yuri Y
authors.initials: YY
authors.lastname: Londer
authors.forename: P Raj
authors.initials: PR
authors.lastname: Pokkuluri
authors.forename: Jill
authors.initials: J
authors.lastname: Erickson
authors.forename: Valerie
authors.initials: V
authors.lastname: Orshonsky
authors.forename: Marianne
authors.initials: M
authors.lastname: Schiffer
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D045302:Geobacter / Q000201:enzymology* / Q000235:genetics; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010957:Plasmids; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
chemical_list: D003574:Cytochrome c Group; D004269:DNA, Bacterial; D007544:Isopropyl Thiogalactoside; D006418:Heme