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Title: 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

SUMMARY Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium that is capable of using insoluble oxidized metals, such as manganese [Mn(III, IV)] and iron [Fe(III)] oxides and oxyhydroxides, as terminal electron acceptors during anaerobic respiration. The ability of S. oneidensis MR-1 to reduce oxidized Mn and/or Fe has previously been linked to OmcA and MtrC: two decaheme c-type cytochromes that are localized to the outer membrane. To investigate how the electron transport proteins OmcA and MtrC are organized, we expressed and purified recombinant OmcA and MtrC from wild type S. oneidensis MR-1 as well as a mutant that lacked OmcA and MtrC (ΔomcA/mtrC). After purification to the nearly electrophoretic homogeneity from the ΔomcA/mtrC mutant, the recombinant OmcA and MtrC exhibited the characteristics of c-type cytochromes, and each of their polypeptides was confirmed to contain 10 hemes. When purified from wild type cells, endogenous MtrC or OmcA was always co-purified with recombinant OmcA or MtrC, respectively. Fluorescence polarization experiment showed that recombinant OmcA bound to the FlAsH-labeled MtrC with a dissociation constant of 7 ×10-7 M. The purified recombinant OmcA or MtrC alone displayed intrinsic ferric reductase activity with NADH used as an electron donor. Ferric reductase specific activity increased bymore » 35 to 41% when nearly equimolar concentrations of OmcA and MtrC were assayed relative to the two proteins assayed independently. These results demonstrate that OmcA and MtrC directly interact with each other to form a stable complex with high ferric reductase activity.« less

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
885441
Report Number(s):
PNNL-SA-45401
Journal ID: ISSN 0021-9193; JOBAAY; 12496; KP1102010; TRN: US200616%%639
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Journal of Bacteriology, 188(13):4705-4714
Additional Journal Information:
Journal Volume: 188; Journal Issue: 13; Journal ID: ISSN 0021-9193
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BINDING ENERGY; CYTOCHROMES; ELECTRONS; FLUORESCENCE; FUNCTIONALS; IRON; MANGANESE; MEMBRANES; MUTANTS; OXIDES; OXIDOREDUCTASES; POLARIZATION; POLYPEPTIDES; PROTEINS; PURIFICATION; RESPIRATION; VALENCE; Environmental Molecular Sciences Laboratory

Citation Formats

Shi, Liang, Chen, Baowei, Wang, Zheming, Elias, Dwayne A, Mayer, M Uljana, Gorby, Yuri A, Ni, Shuisong, Lower, Brian H, Kennedy, David W, Wunschel, David S, Mottaz, Heather M, Marshall, Matthew J, Hill, Eric A, Beliaev, Alex S, Zachara, John M, Fredrickson, Jim K, and Squier, Thomas C. Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-type Cytochromes of Shewanella oneidensis MR-1. United States: N. p., 2006. Web. doi:10.1128/JB.01966-05.
Shi, Liang, Chen, Baowei, Wang, Zheming, Elias, Dwayne A, Mayer, M Uljana, Gorby, Yuri A, Ni, Shuisong, Lower, Brian H, Kennedy, David W, Wunschel, David S, Mottaz, Heather M, Marshall, Matthew J, Hill, Eric A, Beliaev, Alex S, Zachara, John M, Fredrickson, Jim K, & Squier, Thomas C. Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-type Cytochromes of Shewanella oneidensis MR-1. United States. doi:10.1128/JB.01966-05.
Shi, Liang, Chen, Baowei, Wang, Zheming, Elias, Dwayne A, Mayer, M Uljana, Gorby, Yuri A, Ni, Shuisong, Lower, Brian H, Kennedy, David W, Wunschel, David S, Mottaz, Heather M, Marshall, Matthew J, Hill, Eric A, Beliaev, Alex S, Zachara, John M, Fredrickson, Jim K, and Squier, Thomas C. Sat . "Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-type Cytochromes of Shewanella oneidensis MR-1". United States. doi:10.1128/JB.01966-05.
@article{osti_885441,
title = {Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-type Cytochromes of Shewanella oneidensis MR-1},
author = {Shi, Liang and Chen, Baowei and Wang, Zheming and Elias, Dwayne A and Mayer, M Uljana and Gorby, Yuri A and Ni, Shuisong and Lower, Brian H and Kennedy, David W and Wunschel, David S and Mottaz, Heather M and Marshall, Matthew J and Hill, Eric A and Beliaev, Alex S and Zachara, John M and Fredrickson, Jim K and Squier, Thomas C},
abstractNote = {SUMMARY Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium that is capable of using insoluble oxidized metals, such as manganese [Mn(III, IV)] and iron [Fe(III)] oxides and oxyhydroxides, as terminal electron acceptors during anaerobic respiration. The ability of S. oneidensis MR-1 to reduce oxidized Mn and/or Fe has previously been linked to OmcA and MtrC: two decaheme c-type cytochromes that are localized to the outer membrane. To investigate how the electron transport proteins OmcA and MtrC are organized, we expressed and purified recombinant OmcA and MtrC from wild type S. oneidensis MR-1 as well as a mutant that lacked OmcA and MtrC (ΔomcA/mtrC). After purification to the nearly electrophoretic homogeneity from the ΔomcA/mtrC mutant, the recombinant OmcA and MtrC exhibited the characteristics of c-type cytochromes, and each of their polypeptides was confirmed to contain 10 hemes. When purified from wild type cells, endogenous MtrC or OmcA was always co-purified with recombinant OmcA or MtrC, respectively. Fluorescence polarization experiment showed that recombinant OmcA bound to the FlAsH-labeled MtrC with a dissociation constant of 7 ×10-7 M. The purified recombinant OmcA or MtrC alone displayed intrinsic ferric reductase activity with NADH used as an electron donor. Ferric reductase specific activity increased by 35 to 41% when nearly equimolar concentrations of OmcA and MtrC were assayed relative to the two proteins assayed independently. These results demonstrate that OmcA and MtrC directly interact with each other to form a stable complex with high ferric reductase activity.},
doi = {10.1128/JB.01966-05},
journal = {Journal of Bacteriology, 188(13):4705-4714},
issn = {0021-9193},
number = 13,
volume = 188,
place = {United States},
year = {2006},
month = {7}
}