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.
Pitts
Katy E
KE
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences and School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Dobbin
Paul S
PS
Reyes-Ramirez
Francisca
F
Thomson
Andrew J
AJ
Richardson
David J
DJ
Seward
Harriet E
HE
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