CoChat
Sign Up
Cited by 102Metal-Catalyzed Oxygenation MechanismsEnzyme Structure and FunctionGeochemistry and Elemental Analysis

The DOI matches its Crossref record; 66 of the 66 references it cites were found in a registry or an index.

Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx

Open access
Romano, Christine A., Zhou, Mowei, Song, Yang, Wysocki, Vicki H., Dohnalkova, Alice C., Kovarik, Libor, Paša-Tolić, Ljiljana, Tebo, Bradley M.·2017·Nature Communications
DOI 10.1038/s41467-017-00896-8PMC PMC5622069
Open this paper in CoChat — free accountOpen PDF

Abstract

Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase in Bacillus sp. PL-12, Mnx, is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins, MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. Here, we show that native mass spectrometry defines the subunit topology and copper binding of Mnx, while high-resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for understanding Mn biomineralization by such unexplored enzymes.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41467-017-00896-8

Cites

66 of 66 references verified0 citation errors · 0 not checkable

Statistics

Figures

Fig. 1 Native MS of Mnx complex. a Mass spectrum and b ion mobility–mass spectrum of the intact Mnx complex. The major species correspond to the Mnx complex with a molecular weight of 211 kDa. The impurity is a host cell protein complex that carried over during purification (Supplementary Note 2). Identical data are displayed in a, b, with b showing the extra dimension of separation in the vertical axis from the drift time recorded in ion mobility. The IM–MS spectrum has the m/z on the horizontal axis from the mass spectrometry measurement and the drift time measurement from the ion mobility separation in the vertical axis. The ions were separated based on their shape-to-charge ratio in ion mobility and arrived at the mass analyzer for m/z detection at discrete drift times, generating the two-dimensional spectra. The color of the spots indicates the relative abundance as shown by the scale at the bottom. The full peak width at half maximum in a is about 55 m/z, or about 1.6 kDa (±0.8 kDa) in uncertainty in mass determination
Fig. 1 Native MS of Mnx complex. a Mass spectrum and b ion mobility–mass spectrum of the intact Mnx complex. The major species correspond to the Mnx complex with a molecular weight of 211 kDa. The impurity is a host cell protein complex that carried over during purification (Supplementary Note 2). Identical data are displayed in a, b, with b showing the extra dimension of separation in the vertical axis from the drift time recorded in ion mobility. The IM–MS spectrum has the m/z on the horizontal axis from the mass spectrometry measurement and the drift time measurement from the ion mobility separation in the vertical axis. The ions were separated based on their shape-to-charge ratio in ion mobility and arrived at the mass analyzer for m/z detection at discrete drift times, generating the two-dimensional spectra. The color of the spots indicates the relative abundance as shown by the scale at the bottom. The full peak width at half maximum in a is about 55 m/z, or about 1.6 kDa (±0.8 kDa) in uncertainty in mass determinationCC BY · PubMed Central
Fig. 2 Diameters of Mnx and MnxEF particles determined by TEM. Particles were deposited onto a 300 mesh gold TEM grid with ultrathin carbon film on lacey carbon support film and counterstained with nanoW. a Representative image of the Mnx complex. b Representative image of MnxEF particles. Scale bars are 10 nm. Size distribution of c Mnx and d MnxEF particles from TEM measurements. Histograms show the number of particles counted within a specific diameter range (<6.8, between 6.8 and 6.9, and so on) for both Mnx (c) and MnxEF (d)
Fig. 2 Diameters of Mnx and MnxEF particles determined by TEM. Particles were deposited onto a 300 mesh gold TEM grid with ultrathin carbon film on lacey carbon support film and counterstained with nanoW. a Representative image of the Mnx complex. b Representative image of MnxEF particles. Scale bars are 10 nm. Size distribution of c Mnx and d MnxEF particles from TEM measurements. Histograms show the number of particles counted within a specific diameter range (<6.8, between 6.8 and 6.9, and so on) for both Mnx (c) and MnxEF (d)CC BY · PubMed Central
Fig. 3 Dissecting Mnx in the gas phase for characterization of quaternary structure. Ion mobility mass spectra of a the isolated 29+ Mnx complex, b CID at 120 V, c SID at a low collision voltage of 40 V, and d SID at a high-collision voltage of 120 V. The color of the spots represents the relative abundance, with the color scale above the figure. Figure 1a was overlaid in a as the light orange trace to better display the single charge state of Mnx that was isolated. When the Mnx complex was activated in the collision cell pressurized with argon in CID b, monomers of MnxE and MnxF were released (around m/z 2000), leaving behind the stripped complex (Mnx complex losing one MnxE or one MnxF, around m/z 9000). Upon activation of SID at 40 V c, the complex dissociates into MnxEF hexamer (mostly E3F3) and MnxG (highlighted in red parallelograms), suggesting that the Mnx complex is comprised of a MnxEF hexamer attached to a MnxG monomer. At higher activation energy d, more extensive dissociation of the MnxEF hexamer and Mnx complex into smaller subcomplexes can be observed
Fig. 3 Dissecting Mnx in the gas phase for characterization of quaternary structure. Ion mobility mass spectra of a the isolated 29+ Mnx complex, b CID at 120 V, c SID at a low collision voltage of 40 V, and d SID at a high-collision voltage of 120 V. The color of the spots represents the relative abundance, with the color scale above the figure. Figure 1a was overlaid in a as the light orange trace to better display the single charge state of Mnx that was isolated. When the Mnx complex was activated in the collision cell pressurized with argon in CID b, monomers of MnxE and MnxF were released (around m/z 2000), leaving behind the stripped complex (Mnx complex losing one MnxE or one MnxF, around m/z 9000). Upon activation of SID at 40 V c, the complex dissociates into MnxEF hexamer (mostly E3F3) and MnxG (highlighted in red parallelograms), suggesting that the Mnx complex is comprised of a MnxEF hexamer attached to a MnxG monomer. At higher activation energy d, more extensive dissociation of the MnxEF hexamer and Mnx complex into smaller subcomplexes can be observedCC BY · PubMed Central
Fig. 4 Building a structural model of Mnx based on the subunit connectivity determined from native MS experiment. a Determination of the topology of Mnx complex based on observed subcomplexes released in SID. The major species observed for the released MnxEF dimers are E1F1, and for the released trimers are E1F2 (~2/3 of the trimer in the spectrum), and E2F1 (~1/3 of the trimer in the spectrum). The lack of a strong signal for homo-dimers and homo-trimers ruled out some possible configurations for the observed E2F2 and E2F3 subcomplexes, leaving a symmetric hexamer structure being the most reasonable solution. b With the topology proposed based on SID data, computational models of the subunits can be docked to generate possible models of Mnx complex with additional constraints from CCS and surface labeling experiments (Supplementary Methods and Supplementary Fig. 5)
Fig. 4 Building a structural model of Mnx based on the subunit connectivity determined from native MS experiment. a Determination of the topology of Mnx complex based on observed subcomplexes released in SID. The major species observed for the released MnxEF dimers are E1F1, and for the released trimers are E1F2 (~2/3 of the trimer in the spectrum), and E2F1 (~1/3 of the trimer in the spectrum). The lack of a strong signal for homo-dimers and homo-trimers ruled out some possible configurations for the observed E2F2 and E2F3 subcomplexes, leaving a symmetric hexamer structure being the most reasonable solution. b With the topology proposed based on SID data, computational models of the subunits can be docked to generate possible models of Mnx complex with additional constraints from CCS and surface labeling experiments (Supplementary Methods and Supplementary Fig. 5)CC BY · PubMed Central
Fig. 5 Examining metal-binding stoichiometry of in situ dissected MnxE and MnxF subunits. Zoomed-in view of the mass spectra for monomers released from Mnx, MnxF and MnxE, by SID (a, b) or CID (c, d). Peaks corresponding to the apo protein are labeled with open circles; copper bound proteins are labeled with filled circles. Asterisks (*) annotate protein peaks thought to arise from artificial modification during protein expression and purification; the pound sign (#) represents the modified protein bound to copper. MnxE primarily binds one copper with strong affinity. MnxF binds 0–2 copper; the stoichiometry is variable depending on sample conditions, implying a weaker affinity to copper than MnxE. In CID, most of the weakly bound copper on MnxF was lost after the subunits were released from the complex due to protein unfolding. Because minimal unfolding of the subunits occurs in SID, the weakly bound ligands can be preserved (see Supplementary Fig. 6 and Supplementary Note 4 for more details)
Fig. 5 Examining metal-binding stoichiometry of in situ dissected MnxE and MnxF subunits. Zoomed-in view of the mass spectra for monomers released from Mnx, MnxF and MnxE, by SID (a, b) or CID (c, d). Peaks corresponding to the apo protein are labeled with open circles; copper bound proteins are labeled with filled circles. Asterisks (*) annotate protein peaks thought to arise from artificial modification during protein expression and purification; the pound sign (#) represents the modified protein bound to copper. MnxE primarily binds one copper with strong affinity. MnxF binds 0–2 copper; the stoichiometry is variable depending on sample conditions, implying a weaker affinity to copper than MnxE. In CID, most of the weakly bound copper on MnxF was lost after the subunits were released from the complex due to protein unfolding. Because minimal unfolding of the subunits occurs in SID, the weakly bound ligands can be preserved (see Supplementary Fig. 6 and Supplementary Note 4 for more details)CC BY · PubMed Central
Fig. 6 Representative TEM images of nascent Mn oxide nanoparticles produced by Mnx. The reaction proceeded for 30 min at a substrate to enzyme molar ratio of 5000:1. a Agglomerated forms of nanoparticles (crystallites). Individual crystallites are ~1.1–4.2 nm in length, with an average diameter of 2.3 ± 0.7 nm. b Atomic resolution TEM revealed three distinct forms of nanocrystalline material: amorphous clusters consisting of individual Mn atoms (arrows), individual nanocrystals (dashed circle), and agglomerations of crystallites forming lath-like crystals (dashed rectangle—notice the MnOx atomic rows). Scale bars are 2 nm
Fig. 6 Representative TEM images of nascent Mn oxide nanoparticles produced by Mnx. The reaction proceeded for 30 min at a substrate to enzyme molar ratio of 5000:1. a Agglomerated forms of nanoparticles (crystallites). Individual crystallites are ~1.1–4.2 nm in length, with an average diameter of 2.3 ± 0.7 nm. b Atomic resolution TEM revealed three distinct forms of nanocrystalline material: amorphous clusters consisting of individual Mn atoms (arrows), individual nanocrystals (dashed circle), and agglomerations of crystallites forming lath-like crystals (dashed rectangle—notice the MnOx atomic rows). Scale bars are 2 nmCC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 66 references

Research works printed but not linked: 1

  1. Zhou M., et al. Unveiling the heterogeneity of an uncharacterized manganese oxidizing multicopper oxidase using high resolution native mass spectrometry and surface induced dissociation. Proc. 64th Am. Soc. Mass Spectrom. Conf. 279055 (2016).

Cited by

Sorted by citations

Related

See all

Metadata

Authors
Romano, C. A., Zhou, M., Song, Y., Wysocki, V. H., Dohnalkova, A. C., Kovarik, L., Paša-Tolić, L., & Tebo, B. M.
Affiliations
  • Oregon Health & Science University (US)
  • Pacific Northwest National Laboratory (US)
  • The Ohio State University (US)
Corresponding author
  • Romano, Christine A.
  • Zhou, Mowei
  • Tebo, Bradley M.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-09-29
Volume
8
Issue
1
Pages
746-746
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-017-00896-8
PMC
PMC5622069
OpenAlex
W2760615945
Cited by
102
References
66
Citations by year
2026: 11 · 2025: 14 · 2024: 10 · 2023: 12 · 2022: 11 · 2021: 14 · 2020: 11 · 2019: 13 · 2018: 6
FWCI
3.14
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science Foundation (1410688, 1202859)
  • U.S. Department of Energy
  • Bentham-Moxon Trust
  • National Institutes of Health
  • Office of Science
  • National Institute of General Medical Sciences (R01GM113658)
  • Division of Chemistry (CHE-1410688)
  • Biological and Environmental Research
  • Pacific Northwest National Laboratory
Topics
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Physical Sciences › Earth and Planetary Sciences › Geochemistry and Petrology › Geochemistry and Elemental Analysis
Keywords
biogenic manganese oxides, multicopper oxidase, manganese nanoparticles, copper binding, native mass spectrometry, high-resolution electron microscopy
MeSH terms
Bacillus, Bacterial Proteins, Copper, Manganese, Oxides, Oxidoreductases, Mass Spectrometry, Manganese Compounds, Microscopy, Electron, Transmission, Nanoparticles

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review