Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-Type Cytochromes of Shewanella oneidensis MR-1

Authors

Liang Shi, Pacific Northwest National LaboratoryFollow
Baowei Chen, Pacific Northwest National Laboratory
Zheming Wang, Pacific Northwest National LaboratoryFollow
Dwayne A. Elias, Pacific Northwest National Laboratory
M. Uljana Mayer, Pacific Northwest National Laboratory
Yuri Gorby, Pacific Northwest National Laboratory
Shuison Ni, Pacific Northwest National Laboratory
Brian Lower, Pacific Northwest National Laboratory
David Kennedy, Pacific Northwest National LaboratoryFollow
David S. Wunschel, Pacific Northwest National Laboratory
Heather M. Mottaz, Pacific Northwest National Laboratory
Matthew Marshall, Pacific Northwest National Laboratory
Eric Hill, Pacific Northwest National Laboratory
Alexander Beliaev, Pacific Northwest National LaboratoryFollow
John M. Zachara, Pacific Northwest National LaboratoryFollow
James K. Fredrickson, Pacific Northwest National LaboratoryFollow
Thomas Squier, Pacific Northwest National Laboratory

Date of this Version

2006

Citation

JOURNAL OF BACTERIOLOGY, July 2006, p. 4705–4714 Vol. 188, No. 13; doi:10.1128/JB.01966-05

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., ∆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 ∆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 (K_d < 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.