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Structure of Geobacter cytochrome OmcZ identifies mechanism of nanowire assembly and conductivity

Nature Microbiology volume 8, pages 284–298 (2023)Cite this article

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Abstract

OmcZ nanowires produced by Geobacter species have high electron conductivity (>30 S cm−1). Of 111 cytochromes present in G. sulfurreducens, OmcZ is the only known nanowire-forming cytochrome essential for the formation of high-current-density biofilms that require long-distance (>10 µm) extracellular electron transport. However, the mechanisms underlying OmcZ nanowire assembly and high conductivity are unknown. Here we report a 3.5-Å-resolution cryogenic electron microscopy structure for OmcZ nanowires. Our structure reveals linear and closely stacked haems that may account for conductivity. Surface-exposed haems and charge interactions explain how OmcZ nanowires bind to diverse extracellular electron acceptors and how organization of nanowire network re-arranges in different biochemical environments. In vitro studies explain how G. sulfurreducens employ a serine protease to control the assembly of OmcZ monomers into nanowires. We find that both OmcZ and serine protease are widespread in environmentally important bacteria and archaea, thus establishing a prevalence of nanowire biogenesis across diverse species and environments.

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Fig. 1: Cryo-EM structure of purified OmcZ nanowires.
Fig. 2: Linear haem arrangement and closer stacking in OmcZ than in OmcS.
Fig. 3: OmcZ nanowires show solvent-exposed haems and non-canonical loop that are involved in electron transfer.
Fig. 4: Organization of OmcZ nanowire network changes with biochemical environment.
Fig. 5: A protease-mediated switch regulates OmcZ nanowire assembly.
Fig. 6: omcZ operon homologues are widespread in environmentally important bacteria and archaea.

Data availability

The key relevant datasets generated during and/or analysed during the current study are publicly available. Cryo-EM data were deposited with the Electron Microscopy Data Bank (ID EMD-23481) and with the Protein Data Bank (ID 7LQ5). Source data are provided with this paper.

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Acknowledgements

We thank E. Martz for help with structural analysis, C. Shipps for help with UV–vis spectroscopy of reduced OmcZ nanowires, and T. Pollard, M. Hochstrasser and B. Kazmierczak for helpful suggestions. This research was supported by the NSF CAREER award no. 1749662 (to N.S.M.), the NSF EAGER award no. 2038000 (to N.S.M.), the NSF-ANR award no. 2210473 (to N.S.M. and V.S.B.) and the National Institutes of Health Director’s New Innovator award (1DP2AI138259-01 to N.S.M. and R01GM141192 to K.G.). Research was sponsored by the Defense Advanced Research Project Agency Army Research Office and was accomplished under Cooperative Agreement Number W911NF-18-2-0100 (with N.S.M. and V.S.B.). High-resolution cryo-EM data collection was performed at the Case Western Reserve University Cryo-Electron Microscopy Core facility with the assistance of Kunpeng Li and Sudha Chakrapani.

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Author notes

  1. Yangqi Gu

    Present address: PNAC division, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

Authors and Affiliations

  1. Microbial Sciences Institute, Yale University, West Haven, CT, USA

    Yangqi Gu, Matthew J. Guberman-Pfeffer, Vishok Srikanth, Cong Shen, Yuri Londer, Fadel A. Samatey & Nikhil S. Malvankar

  2. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA

    Yangqi Gu

  3. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Matthew J. Guberman-Pfeffer, Vishok Srikanth, Yuri Londer, Fadel A. Samatey & Nikhil S. Malvankar

  4. Department of Microbiology, Yale University, New Haven, CT, USA

    Cong Shen

  5. Department of Cell Biology, Yale University, New Haven, CT, USA

    Fabian Giska & Kallol Gupta

  6. Nanobiology Institute, Yale University, West Haven, CT, USA

    Fabian Giska & Kallol Gupta

  7. Department of Chemistry, Yale University, New Haven, CT, USA

    Victor S. Batista

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Contributions

Y.G. purified OzpA, OmcZ50 and OmcZ nanowires, collected and analysed negative-stain and cryo-EM data to build the atomic model, performed biochemical analyses, UV–vis, CD spectroscopy, solubility test, nanowire alignment, in vitro digestion experiment of OmcZ nanowires, immune-gold labelling and power spectra analyses. M.J.G.-P. performed molecular dynamics and CD simulations. Y.L. constructed the expression vector for OmcZ50 in E. coli. Y.G. and F.A.S. optimized the culturing condition and purified OmcZ50 from E. coli. V.S. helped with OmcZ nanowire purification. F.A.S. and V.S. helped with model building. C.S. constructed the omcS:pk18 strain for OmcZ purification. F.G. performed the native mass spectrometry experiments and analysed the data under the supervision of K.G. V.S.B. guided computational studies. Y.G. and N.S.M. conceived and designed the project. N.S.M. supervised the work. Y.G. and N.S.M. wrote the paper with input from all the authors.

Corresponding authors

Correspondence to Yangqi Gu or Nikhil S. Malvankar.

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Nature Microbiology thanks Thomas Boesen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Illustration of cryo-EM map quality.

a–g, Density fitting of individual heme cofactors. Low iron density is due to the non-selection of coordinated histidine when rendering the figures at the given threshold of the density. i, Resolution distribution of the cryo-EM map. j, Representative density of sidechains. k, Map to map FSC. l, Map to model FSC. m, Real space correlation coefficient (RSCC) for each residue. n, Heme RSCC.

Source data

Extended Data Fig. 2 omcZ homologues show high sequence similarity.

omcZ homologues show up to 68% sequence similarity. In addition to all heme binding motifs, key residues are also highly conserved which we found to be critical for high conductivity. For example, a consecutive pair of histidines that brings T-stacked hemes closer is also highly conserved in all omcZ homologs. Thus significant sequence identities are present throughout the protein.

Extended Data Fig. 3 Circular Dichroism (CD) spectroscopy shows unique heme arrangements for OmcZ nanowires.

a, CD spectra OmcZ nanowire shows similar secondary structure at pH 10.5 and pH 7 buffer. b, CD spectra of OmcS and OmcZ nanowire hemes. Reduced spectrum show narrowing, and a red-shift of the peaks compared to the oxidized spectrum for c, OmcZ. d, OmcS nanowires as expected. e, The double positive peak feature is unique to the OmcZ nanowires, while the non-filamentous OmcZ30 fails to show a such feature. f, Simulated CD spectra for various sextets of hemes from OmcZ showing that the line shape is independent of six hemes included in the computations. Simulated CD spectra of g, OmcS (green) and h, OmcZ (pink), using only hemes and heme binding motifs show the peak features observed in the experimental data (black). The simulated spectra were uniformly shifted to longer wavelength by 0.06 eV (see methods for details) i, Resolving the contribution of individual hemes to the CD spectra for OmcS and OmcZ reveals that the double peak is a specific feature of hemes in OmcZ nanowires. Evolution of the CD spectra as adjacent hemes in OmcS (green) and OmcZ (pink) were sequentially added to the computations. The labels in each panel indicate which hemes were added for the spectra. The sequence of addition is from left-to-right in both rows.

Source data

Extended Data Fig. 4 OmcZ nanowires reduce diverse electron acceptors.

a, Buffer Control: OmcZ nanowire spectra do not change after 1 hour upon addition of degassed deionized bis-tris buffer (20 mM, pH=7.2) used for all substrate reduction experiments. b, OmcZ nanowires could not be fully reduced by DTT but could be reduced by Sodium dithionite. c, OmcZ cannot reduce riboflavin.

Source data

Extended Data Fig. 5 OmcZ nanowires are robust electronic biomaterials that function under extreme environments.

Negative staining TEM images of OmcZ nanowires treated with following protein denaturants. a, No denaturant. b, 6 M urea, c, 5 M GuHCl, d, Phosphoric acid at low pH (pH = 1.6), and e, boiled in 2% SDS. All scale bars: 100 nm. f, OmcZ nanowires are more stable than OmcS at low pH (pH = 1.6) as evident by retention of its red colour due to hemes. OmcZ nanowires also retained their red colour characteristic of haems under all other denaturants. g, Solution CD spectra and h, UV-vis spectra show the stability of protein and electronic structure of OmcZ nanowires in denaturants.

Source data

Extended Data Fig. 6 In vitro Assembly of OmcZ nanowires from native OmcZ50.

a, TEM image of native G. sulfurreducens OmcZ50 showing lack of nanowire assembly. b, Western immunoblot using OmcZ antibody showing OzpA digesting OmcZ50 into OmcZ30. c, Digested OmcZ30. self-assembles into nanowires in vitro. d, SDS-PAGE gel of purified OzpA showing three major bands: premature protein, mature protein, and the cleaved inhibitor domain (I9). e, Power spectrum of the reconstituted OmcZ nanowires shows the same helical parameters as cell-produced OmcZ nanowires. f, 2D average of reconstituted OmcZ nanowires. g, CD spectra of reconstituted and native OmcZ nanowires is similar. Immuno-gold labeling of reconstituted OmcZ nanowires using h, No-primary antibody, i, antibody for OmcZ30 and j, antibody for OmcZ50. Scale bars, a, 100 nm. c, 50 nm. c, 2 nm. h, 100 nm, i, 50 nm, j, 100 nm.

Source data

Extended Data Fig. 7 Native mass spectrometry of OmcZ50 purified from E. coli shows molecular weight and heme groups consistent with native OmcZ50.

a, Coomassie and Heme staining gel of OmcZ50 purified from E. coli. b, Mass spectrum of OmcZ purified from G. sulfurreducens. Detected mass corresponds to the OmcZ monomer covalently bound to eight hemes. c-d, Mass spectrum of OmcZ50 purified from E. coli. Detected proteins exist as monomer and homodimer. Mass analysis shows that OmcZ50 monomer is bound covalently to primarily c, eight haems but sometimes also to d, six hemes.

Extended Data Fig. 8 OmcZ nanowire assembly could be environmentally controlled.

a, omcZ operon genes with intergenic region in G. sulfurreducens. b, RT-PCR shows that omcZ and ozpA are co-transcribed. Lanes 1: genomic DNA (gDNA), 2: cDNA, 3: cDNA control. c, Predicted RNA structure of the intergenic region of mRNA using ViennaRNA suite. Color code shows the probability of each base or base pair’s placement in the secondary structure.

Extended Data Fig. 9 OmcZ operon is found in diverse microbes.

Phylogenetic tree derived from OmcZ amino acid sequence alignments using homology cut-off score of 100 bit. Light blue circles represent bootstrap values.

Extended Data Table 1 Statistics of OmcZ refinement and heme distances
Full size table

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Fig. 1.

Reporting Summary

Supplementary Video 1

OzpA cleaves OmcZ50 into OmcZ30 that self-assembles into nanowires.

Source data

Source Data Fig. 1

Raw data for size exclusion chromatography.

Source Data Fig. 3

Raw data for iron reduction measurements.

Source Data Extended Data Fig. 1

Raw data for cryo-EM structural analysis.

Source Data Extended Data Fig. 3

Raw data for CD measurements and modelling.

Source Data Extended Data Fig. 4

Raw data for reduction measurements of electron acceptors.

Source Data Extended Data Fig. 5

Raw data for CD and UV–vis measurements.

Source Data Extended Data Fig. 6

Raw data for CD measurements.

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Gu, Y., Guberman-Pfeffer, M.J., Srikanth, V. et al. Structure of Geobacter cytochrome OmcZ identifies mechanism of nanowire assembly and conductivity. Nat Microbiol 8, 284–298 (2023). https://doi.org/10.1038/s41564-022-01315-5

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