Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA: expression in Escherichia coli confers the ability to reduce soluble Fe(III) chelates.
Shewanella oneidensis MR-1 has the metabolic capacity to grow anaerobically using Fe(III) as a terminal electron acceptor. Growth under these conditions results in the de novo synthesis of a number of periplasmic c-type cytochromes, many of which are multiheme in nature and are thought to be involved in the Fe(III) respiratory process. To begin a biochemical study of these complex cytochromes, the mtrA gene that encodes an approximate 32-kDa periplasmic decaheme cytochrome has been heterologously expressed in Escherichia coli. Co-expression of mtrA with a plasmid that contains cytochrome c maturation genes leads to the assembly of a correctly targeted holoprotein, which covalently binds ten hemes. The recombinant MtrA protein has been characterized by magnetic circular dichroism, which shows that all ten hemes have bis-histidine axial ligation. EPR spectroscopy detected only eight of these hemes, all of which are low spin and provides evidence for a spin-coupled pair of hemes in the oxidized state. Redox titrations of MtrA have been carried out with optical- and EPR-monitored methods, and the hemes are shown to reduce over the potential range -100 to -400 mV. In intact cells of E. coli, MtrA is shown to obtain electrons from the host electron transport chain and pass these onto host oxidoreductases or a range of soluble Fe(III) species. This demonstrates the promiscuous nature of this decaheme cytochrome and its potential to serve as a soluble Fe(III) reductase in intact cells.
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences and School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Katy E
KE
Pitts
Paul S
PS
Dobbin
Francisca
F
Reyes-Ramirez
Andrew J
AJ
Thomson
David J
DJ
Richardson
Harriet E
HE
Seward
D000595:Amino Acid Sequence; D002455:Cell Division; D002614:Chelating Agents / Q000378:metabolism*; D002942:Circular Dichroism; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000378:metabolism*; D013347:Subcellular Fractions; D013696:Temperature; D014466:Ultraviolet Rays
D002614:Chelating Agents; D003580:Cytochromes; D011994:Recombinant Proteins; D006418:Heme; D007501:Iron; D010100:Oxygen