Visualizing Manganese Biomineralization by the Mnx Protein Complex within the Exosporium of Mn-oxidizing Bacillus species
EMSL Project ID
60773
Abstract
Microbial production of manganese oxide minerals is a globally important biomineralization process that drives the manganese cycle in the environment. This cycle supports life through the manganese catalytic centers of many enzymes and influences elemental biogeochemical cycles, controlling the distribution and bioavailability of many toxic and essential elements. The ability to oxidize dissolved Mn(II) to form solid manganese oxides has been found in a diverse array of bacteria from a variety of geological settings. Among these, manganese-oxidizing Bacillus species represent a unique case, since in this group, dormant spores, not metabolically active cells, catalyze Mn oxidation. Our previous work narrowed down Mn-oxidation activity there to a Mn oxidase located in exosporium, the outermost proteinaceous layer that surrounds the spore, also serving as a deposition site of the manganese oxide product. Little has been known about how microbial Mn(II) oxidation works at the molecular level until Tebo lab succeeded in purifying a recombinant Mn oxidase complex Mnx. Extensive spectroscopic and kinetic studies of Mnx established a mechanism involving dinuclear complexes in successively higher oxidation states. Subsequent cryo electron microscopy structural characterization at EMSL revealed a 3D structure that provides a plausible protein context for this mechanism. However, the steps of biomineralization—nucleation and translocation of the primary enzymatic MnO2 product and its interaction with the protein and the exosporium layer—remains to be established. Exosporium defines the boundary between the spore and its environment, and enzymatic processes there directly influence the spore’s habitat, but our understanding of protein composition and assembly of exosporium is limited.
With this proposal, we aim to visualize molecular-level interactions between the protein and its mineral product as it forms, and to uncover the molecular-level details of exosporium structure and composition, and how manganese oxidation is orchestrated there. Using three selected Bacillus species and isolated Mn oxidase and its structural information, we will utilize the EMSL visual proteomics capabilities to map exosporium architecture and manganese mineralization pathways. By combining cryo-electron microscopy, cryo-electron tomography, and native and cross-linking mass spectrometry, we will focus on five interconnecting questions: (1) How does enzyme scaffold accommodate manganese intermediates and growing MnO2 product? (2) How does the mineral exit the protein? (3) What are the major proteins that organize the exosporium? (4) How is manganese oxidase distributed within the layer? and (5) How does the manganese oxide product propagate within the exosporium?
By visualizing mineral and biological phases together, we will obtain fundamental knowledge about how manganese mineralization is staged by a ubiquitous bacterial enzyme within the biomolecular matrix of exosporium, demonstrating how advanced visual proteomics approaches can be used to solve geochemical and geomicrobiological questions. This mechanistic knowledge will guide future efforts towards new biotechnologies for potential use of Mn-oxidizing spores in production of manganese oxides for use as catalysts in energy storage and photoelectrochemical water splitting systems to generate power in biofuel cells.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2023-10-01
End Date
N/A
Status
Active
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members