Production and preliminary characterization of a recombinant triheme cytochrome c(7) from Geobacter sulfurreducens in Escherichia coli.
Multiheme cytochromes c have been found in a number of sulfate- and metal ion-reducing bacteria. Geobacter sulfurreducens is one of a family of microorganisms that oxidize organic compounds, with Fe(III) oxide as the terminal electron acceptor. A triheme 9.6 kDa cytochrome c(7) from G. sulfurreducens is a part of the metal ion reduction pathway. We cloned the gene for cytochrome c(7) and expressed it in Escherichia coli together with the cytochrome c maturation gene cluster, ccmABCDEFGH, on a separate plasmid. We designed two constructs, with and without an N-terminal His-tag. The untagged version provided a good yield (up to 6 mg/l of aerobic culture) of the fully matured protein, with all three hemes attached, while the N-terminal His-tag appeared to be detrimental for proper heme incorporation. The recombinant protein (untagged) is properly folded, it has the same molecular weight and displays the same absorption spectra, both in reduced and in oxidized forms, as the protein isolated from G. sulfurreducens and it is capable of reducing metal ions in vitro. The shape parameters for the recombinant cytochrome c(7) determined by small angle X-ray scattering are in good agreement with the ones calculated from a homologous cytochrome c(7) of known structure.
Londer
Yuri Y
YY
Biosciences Division, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA
Pokkuluri
P Raj
PR
Tiede
David M
DM
Schiffer
Marianne
M
D003574:Cytochrome c Group / Q000096:biosynthesis / Q000737:chemistry*; D020564:Deltaproteobacteria / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012542:Scattering, Radiation; D014965:X-Rays
D003574:Cytochrome c Group; D011994:Recombinant Proteins; C114913:cytochrome C7