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An allosteric redox switch involved in oxygen protection in a CO2 reductase

Nature Chemical Biology volume 20, pages 111–119 (2024)Cite this article

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Abstract

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

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Fig. 1: Enzymatic activity of FdhAB and variants.
Fig. 2: EPR spectra of WV species in WT FdhAB enzyme, C872A and M405A variants.
Fig. 3: Structural changes induced by the allosteric cleavage of the C845–C872 bond.
Fig. 4: Effect of oxygen on catalysis by WT FdhAB and C872A variant.
Fig. 5: Reversibility of the FdhAB redox switch.

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Data availability

The data that support the findings of this study are available within the main text and its Supplementary Information file. The atomic coordinates and structure factors for the D. vulgaris H C872A variant structures have been deposited in the PDB under accession codes 8CM4, 8CM5, 8CM6 and 8CM7. Source data are provided with this paper.

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Acknowledgements

This work was financially supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through fellowship nos. SFRH/BD/116515/2016 (A.R.O.), DFA/BD/7897/2020 (R.R.M.) and COVID/BD/151766/2021 (A.R.O.), grant no. PTDC/BII-BBF/2050/2020 (I.A.C.P. and M.J.R.) and R&D units MOSTMICRO-ITQB (grant nos. UIDB/04612/2020 and UIDP/04612/2020) (I.A.C.P.) and UCIBIO (grant nos. UIDP/04378/2020 and UIDB/04378/2020) (M.J.R.), and Associated Laboratories LS4FUTURE (grant no. LA/P/0087/2020) (I.A.C.P.) and i4HB (grant no. LA/P/0140/2020) (M.J.R.). The European Union’s Horizon 2020 research and innovation program (grant no. 810856) is also acknowledged (I.A.C.P.). This work was also funded by the French national research agency (ANR – MOLYERE project, grant no. 16-CE-29-0010-01) (B.G.), and supported by the computing facilities of the Centre Régional de Compétences en Modélisation Moléculaire de Marseille. We thank the excellent technical assistance of João Carita from ITQB NOVA on microbial cell growth. We are also grateful to the EPR-MRS facilities of the Aix-Marseille University EPR centre and acknowledge the support of the European research infrastructure MOSBRI (grant no. 101004806) (B.G.) and the French research infrastructure INFRANALYTICS (FR2054) (B.G.). We also acknowledge the ESRF and ALBA Synchrotron for provision of synchrotron radiation facilities, and we thank the staff of the ESRF and EMBL Grenoble and ALBA for assistance and support in using beamlines ID23-1, ID30A-3, ID30B and XALOC.

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Author notes

  1. These authors contributed equally: Ana Rita Oliveira, Cristiano Mota.

Authors and Affiliations

  1. Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal

    Ana Rita Oliveira, Rita Rebelo Manuel, Neide Pedrosa & Inês A. Cardoso Pereira

  2. Associate Laboratory i4HB – Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal

    Cristiano Mota, Guilherme Vilela-Alves, Kateryna Klymanska & Maria João Romão

  3. UCIBIO, Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal

    Cristiano Mota, Guilherme Vilela-Alves, Kateryna Klymanska & Maria João Romão

  4. Laboratory of Bioenergetics and Protein Engineering, Aix Marseille University, CNRS, BIP, Marseille, France

    Vincent Fourmond, Christophe Léger & Bruno Guigliarelli

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  1. Ana Rita Oliveira
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Contributions

I.A.C.P. and A.R.O. conceived and designed biochemical experiments and sequence analysis. A.R.O. performed molecular biology experiments, protein purification, biochemical characterization, enzymatic assays, thermal shift assays, sequence analysis, EPR and electrochemical studies of WT, C845A and C872A variants and figure preparation. R.R.M. produced and characterized the M405A variant and contributed to CO2 reduction assays and figure preparation. C.M., M.J.R. and G.V.-A. designed the crystallography experiments and analyzed the crystal structures. C.M. and G.V.-A. crystallized the proteins, solved and refined all structures. G.V.-A. prepared all figures with crystal structures. K.K. helped with crystallization assays and first stages of refinement of the 8CM4 and 8CM5 structures. V.F. and C.L. designed electrochemical experiments, performed by V.F. and A.R.O. B.G. designed and analyzed EPR experiments, performed by B.G. and A.R.O. I.A.C.P., R.R.M. and N.P. designed in vivo experiments, performed by N.P. and R.R.M. I.A.C.P. and M.J.R. supervised and funded the project. A.R.O., I.A.C.P., M.J.R., C.M. and G.V.-A. wrote the manuscript with inputs from coauthors V.F., C.L. and B.G. All authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Maria João Romão or Inês A. Cardoso Pereira.

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Nature Chemical Biology thanks Russ Hille 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 Steady-state formate oxidation (A, C, and E) and CO2 reduction kinetics (B, D and F).

Formate oxidation (A, C, and E) and CO2 reduction (B, D and F). A and B – As-isolated WT FdhAB assays without DTT and with DTT activation, respectively. C and D – C872A variant anaerobically isolated (no DTT activation). E and F – M405A variant anaerobically isolated (no DTT activation). The concentration of enzyme used in the assays was 14 nM for A, E and F, and 0.9-2 nM for B, C and D. Data are presented as mean values ± s.d. (n = 2 or 3 assay technical replicates). Lines represent the direct fitting of Michaelis-Menten equation to experimental data.

Source data

Extended Data Fig. 2 EPR spectra of WV species in WT FdhAB enzyme and in C872A and M405A variants.

Experimental spectra are in black, and simulations are shown below in red. a) resting WT sample poised at −468 mV by reduction with dithionite; b) DTT activated WT sample poised at −395 mV by reduction with formate; c) C872A sample poised at −443 mV by reduction with formate; d) C872A sample poised at −469 mV by reduction with dithionite; e) M405A reduced by dithionite. EPR conditions: temperature 80 K; microwave power 40 mW at 9.479 GHz; modulation amplitude 1 mT at 100 kHz. Simulated spectra result from the superimposition of WVF and WVD signals with parameters given in Table S2 with the following WVD /WVF ratio: a) 90/10; b) 5/95; c) 10/90; d) 80/20. For the sake of clarity, the radical signal at g=2.00 arising from mediators in redox titrations was not simulated.

Extended Data Fig. 3 FdhAB W active site.

FdhAB W active site in aerobic C872A_ox crystal structure (a, b, c) and C872A_anox anaerobic one (d). The results for three different labile group refinement strategies of the labile ligand, for the C872A-O2 model, are presented: (a) refinement with an oxygen replacing the sulfido ligand; (b) refinement with a sulfido ligand with an occupancy of 1; and (c) refinement with a sulfido ligand refined with an occupancy of 0.556. In (d) the W center for the C872Aanoxic-CHNH2O is shown. The pterin domains of both MGD cofactors and the sidechain of U192 are represented as light blue or blue sticks, respectively, for C872A-O2 and C872Aanoxic-CHNH2O, the W ion as a blue sphere and the sulfido ligand by a yellow stick. The 2Fo-Fc electron density map for each refinement is shown as a magenta mesh, at 1σ, and the Fo-Fc map is represented as a green mesh, for positive density, and as a red mesh, for negative density, both at 3σ.

Extended Data Fig. 4 Comparison of the active site in WT and C872A_anox structures.

Superposition of FdhAB WT (gray) and C872A_anox structures (blue) showing the new H193 conformation and respective hydrogen bonding network. The W ion is represented as a violet sphere and its two bound MGDs are shown as sticks. Distances are in Å.

Extended Data Fig. 5 A formamide molecule is present near the active site (C872A_anox structure) in the formate channel.

R441 and T450 are hydrogen bonding the formamide ligand. The W ion is represented as a violet sphere and its two bound MGDs are shown as sticks. The 2Fo-Fc electron density map is shown as a magenta mesh, at 1σ. Distances are in Å.

Extended Data Fig. 6 EPR spectrum of WV in reduced M405A variant.

a) M405A sample as-isolated anaerobically; b) after reduction with dithionite; c) after reduction with formate; d) formate-reduced sample after washing-off formate. EPR conditions: temperature 80 K; microwave power 40 mW; modulation amplitude 1 mT at 100 kHz.

Source data

Extended Data Fig. 7 Active site environment of the M405A variant.

Superposition of WT (grey) and M405A variant (yellow) is shown. Black arrows highlight the conformational changes. The W ion is represented as a violet sphere and its two bound MGDs are shown as sticks.

Extended Data Fig. 8 Rate of O2 inactivation.

Solid lines show the derivative of the logarithm of the current for a– DTT-activated WT FdhAB, b– Anaerobically purified WT FdhAB, c- C845A and d- C872A variants, after injection of 30 µM of O2. Dashed lines are the representative fits of the kinetic model in equation [3] (Supplementary Information).

Source data

Supplementary information

Supplementary Information

Supplementary Data 1–3, Figs. 1–3, Tables 1–7 and references.

Reporting Summary

Supplementary Data 1

Source Data for Supplementary Fig. 2.

Supplementary Data 2

Source Data for Supplementary Fig. 3.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1.

Source Data Fig. 2

Source data for Fig. 2.

Source Data Fig. 4

Statistical source data for Fig. 4.

Source Data Fig. 5

Statistical source data for Fig. 5.

Source Data Table 1

Statistical source data for Table 1.

Source Data Extended Data Fig. 1

Statistical source data for Extended Data Fig. 1.

Source Data Extended Data Fig. 6

Source data for Extended Data Fig. 6.

Source Data Extended Data Fig. 8

Source data for Extended Data Fig. 8.

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Oliveira, A.R., Mota, C., Vilela-Alves, G. et al. An allosteric redox switch involved in oxygen protection in a CO2 reductase. Nat Chem Biol 20, 111–119 (2024). https://doi.org/10.1038/s41589-023-01484-2

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