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Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1.

Abstract：

: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.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Shi L,Chen B,Wang Z,Elias DA,Mayer MU,Gorby YA,Ni S,Lower BH,Kennedy DW,Wunschel DS,Mottaz HM,Marshall MJ,Hill EA,Beliaev AS,Zachara JM,Fredrickson JK,Squier TC

doi

10.1128/JB.01966-05

subject

Has Abstract

pub_date

2006-07-01 00:00:00

pages

4705-14

issue

eissn

0021-9193

issn

1098-5530

pii

188/13/4705

journal_volume

188

pub_type

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Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1.