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Characterization of an Expressed Bacterial Mn(II,III)-Oxidizing Multicopper Oxidase Complex and its Mn Oxide Products

EMSL Project ID


Microbial Mn(II) oxidation is an important environmental process that links the biogeochemical cycle of Mn with the cycling of C, N, P, and many trace metals and radionuclides. Microbes that oxidize Mn are known to have activities that enable them to degrade lignin and other recalcitrant organic compounds. The Mn oxide minerals produced by microbes have extraordinary sorptive properties, are highly reactive and are some of the strongest oxidants found in the environment, participating in a wide range of redox reactions with organic and inorganic chemical species and compounds. Our project investigates the mechanism by which a multicopper oxidase (MCO) complex, called “Mnx”, oxidizes Mn(II). We were recently able to express active Mnx in E. coli and have determined that the complex contains an MCO and six accessory proteins. The enzymatic complex is novel because no other known MCOs require accessory proteins and the Mnx complex catalyzes a two electron oxidation of Mn(II) to Mn(IV), whereas other known MCOs that act on metals only catalyze only one-electron oxidations. In this project we propose to use EMSL mass spectrometry and microscopy facilities to help elucidate the structure of the enzyme complex and the mechanism of Mn oxide biomineralization. Specifically, we will examine the role of the accessory proteins in Mn(II) and Mn(III) oxidation, the architecture of the Mn oxidase complex, and the molecular details that lead to the formation of a layered-structure Mn(IV) oxide mineral. The expertise of the staff and the state-of-the-art mass spectrometry facilities provide the capability to analyze native protein with high resolution to observe metal binding and post-translational modifications. The electron microscopy facility has advanced high resolution electron microscopes optimized for imaging and analyses of bacterial-mineral interactions and protein complexes including the analysis of metal content and oxidation states. The Mn(II)-oxidizing complex and the Mn oxides it produces have potential biotechnological applications in environmental remediation (immobilization of metals and radionuclides; degradation of recalcitrant organic compounds), in bioenergy production (e.g., fuel cells, lignin degradation), energy storage (batteries) and as possible catalysts for a wide range of reactions (e.g., water oxidation and hydrogen production).

Project Details

Project type
Large-Scale EMSL Research
Start Date
End Date


Principal Investigator

Bradley Tebo
University of Washington

Team Members

Mowei Zhou
Environmental Molecular Sciences Laboratory

Christine Romano
Oregon Health & Science University

Alexandra Soldatova
University of Washington

Thomas Spiro
University of Washington

Ljiljana Pasa-Tolic
Environmental Molecular Sciences Laboratory

Related Publications

Romano CA, M Zhou, Y Song, VH Wysocki, A Dohnalkova, L Kovarik, L Pasa Tolic, and BM Tebo. 2017. "Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx." Nature Communications 8:746. doi:10.1038/s41467-017-00896-8
Yan J, M Zhou, J Gilbert, JJ Wolff, A Somogyi, RE Pedder, RS Quintyn, L Morrison, ML Easterling, L Pasa Tolic, and VH Wysocki. 2017. "Surface-induced dissociation of protein complexes in a hybrid Fourier transform ion cyclotron resonance mass spectrometer." Analytical Chemistry 89(1):895-901. doi:10.1021/acs.analchem.6b03986
Zhou M, J Yan, CA Romano, BM Tebo, VH Wysocki, and L Pasa Tolic. 2018. "Surface Induced Dissociation coupled with High Resolution Mass Spectrometry Unveils Heterogeneity from Variable Metal Binding and Unexpected Modifications in an Uncharacterized 211 kDa Multicopper Oxidase Protein Complex." Journal of the American Society for Mass Spectrometry. doi:10.1007/s13361-017-1882-x