Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1.
Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., Delta omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the Delta omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.
Shi
Liang
L
Microbiology Group, Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, MSIN P7-50, Richland, WA 99354, USA. liang.shi@pnl.gov
Chen
Baowei
B
Wang
Zheming
Z
Elias
Dwayne A
DA
Mayer
M Uljana
MU
Gorby
Yuri A
YA
Ni
Shuison
S
Lower
Brian H
BH
Kennedy
David W
DW
Wunschel
David S
DS
Mottaz
Heather M
HM
Marshall
Matthew J
MJ
Hill
Eric A
EA
Beliaev
Alexander S
AS
Zachara
John M
JM
Fredrickson
James K
JK
Squier
Thomas C
TC
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003574:Cytochrome c Group / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004579:Electron Transport; D005290:Ferric Compounds / Q000378:metabolism; D006418:Heme; D005810:Multigene Family; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology*
D001425:Bacterial Outer Membrane Proteins; D003574:Cytochrome c Group; D005290:Ferric Compounds; D011994:Recombinant Proteins; D006418:Heme; C433527:MtrC protein, Shewanella; D010088:Oxidoreductases