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.