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Stepwise assembly of the active site of [NiFe]-hydrogenase

Nature Chemical Biology volume 19, pages 498–506 (2023)Cite this article

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Abstract

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2e− and 2H+ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN− molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

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Fig. 1: Model of the sequential assembly mechanism of the NiFe(CN)2(CO) center in [NiFe]-hydrogenase.
Fig. 2: Purification and IR spectroscopic characterization of the HoxG maturation intermediates.
Fig. 3: Mössbauer spectra of the 57Fe-labeled HoxG maturation intermediates.
Fig. 4: NRVS of 57Fe-labeled HoxG maturation intermediates.
Fig. 5: XAS analysis of mHoxG and preHoxG.

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Data availability

The authors declare that the data supporting the findings of this study are available within the article and the Supplementary Information. Raw XAS data were obtained at the SSRL synchrotron. Raw NRVS data were generated at the synchrotron facilities Petra III and SPring-8, and are available in their processed form upon request. Additional reagents will be made available by the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

G.C., S.H., O.L., I.Z. and P.H. are grateful to the Einstein Foundation Berlin (grant number EVF-2016-277) for funding. This work was also supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through the PP 1927 ‘Iron Sulfur for Life’ (project no. DE1877/1-1 (S.D.), 311062227 (O.L., I.Z.)) and the cluster of excellence ‘UniSysCat’ under Germany’s Excellence Strategy-EXC2008-390540038. The authors are indebted for EU financial support (Article 38.1.2, GA) within the European Union’s Horizon 2020 research and innovation program under grant agreement no. 810856. C.v.S. and S.D. thank the Max-Planck Society for funding. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. C.v.S. and S.D. gratefully acknowledge M. Latimer for his support and assistance during XAS measurements at beamline 9-3. NRVS data collection was supported by the proposal at BL09XU [2019A1201] at SPring-8. We acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at Petra III and we thank A. Jafari for assistance in using P01. Beamtime was allocated for proposal I-20200452. J.-P. Oudsen is gratefully acknowledged for assistance in the NRVS data acquisition. We thank S. P. Cramer for fruitful discussions on NRVS data and S. Leimkühler for ICP-OES measurements. We are grateful to J. Fritsch for performing preliminary experiments on the HoxG intermediates, and to A. C. Schulz for HypCD sample preparations.

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  1. These authors contributed equally: Giorgio Caserta, Sven Hartmann.

Authors and Affiliations

  1. Institut für Chemie, Technische Universität Berlin, Berlin, Germany

    Giorgio Caserta, Sven Hartmann, Chara Karafoulidi-Retsou, Christian Lorent, Janna Schoknecht, Peter Hildebrandt, Ingo Zebger, Stefan Frielingsdorf & Oliver Lenz

  2. Department of Inorganic Spectroscopy, Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr, Germany

    Casey Van Stappen & Serena DeBeer

  3. Department of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany

    Stefan Yelin, Matthias Keck & Christian Limberg

  4. Deutsches Elektronen-Synchrotron, Hamburg, Germany

    Ilya Sergueev

  5. Japan Synchrotron Radiation Research Institute (JASRI), Hyogo, Japan

    Yoshitaka Yoda

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Contributions

G.C., S.H., S.F. and O.L. conceived and designed experiments. G.C., S.H., S.F. and J.S. conducted molecular biology experiments. S.H. and G.C. performed sample preparations and biochemical assays. G.C. performed sample preparation for synchrotron measurements. G.C., S.H., C.K.-R. and I.Z. performed and analyzed IR spectroscopic experiments. C. Lorent performed and analyzed EPR measurements. M.K., S.Y. and C. Limberg performed and analyzed Mössbauer experiments. G.C., Y.Y. and I.S. acquired and analyzed NRVS data. C.v.S. and S.D. performed and analyzed EXAFS experiments. G.C., S.H., O.L, S.F., I.Z. and P.H. analyzed the data. G.C., S.H., S.F. and O.L. wrote the manuscript with input from all co-authors. All authors have given approval to the final version of the manuscript.

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Caserta, G., Hartmann, S., Van Stappen, C. et al. Stepwise assembly of the active site of [NiFe]-hydrogenase. Nat Chem Biol 19, 498–506 (2023). https://doi.org/10.1038/s41589-022-01226-w

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