ArticlePloS one2012
Site saturation mutagenesis demonstrates a central role for cysteine 298 as proton donor to the catalytic site in CaHydA [FeFe]-hydrogenase.
Article in PloS one, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 23 papers.
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Who cites it
23 citing papers in PubMed, 65 citations in OpenAlex.
- Probing the Influence of the Protein Scaffold on H-Cluster Reactivity via Gain-of-Function Studies─Improved HJournal of the American Chemical Society · 2025Article
- Probing Substrate Transport Effects on Enzymatic Hydrogen Catalysis: An Alternative Proton Transfer Pathway in Putatively Sensory [FeFe] Hydrogenase.ACS catalysis · 2023Article
- Redox tuning of the H-cluster by second coordination sphere amino acids in the sensory [FeFe] hydrogenase fromChemical science · 2023Article
- Binding of exogenous cyanide reveals new active-site states in [FeFe] hydrogenases.Chemical science · 2023Article
- Editorial: Hydrogenase: structure, function, maturation, and application.Frontiers in microbiology · 2023Article
- Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase.Chemical reviews · 2022Review
- Fast Proton Transport in FeFe Hydrogenase via a Flexible Channel and a Proton Hole Mechanism.The journal of physical chemistry. B · 2022Article
- The Contribution of Proton-Donor pKa on Reactivity Profiles of [FeFe]-hydrogenases.Frontiers in microbiology · 2022Article
- Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site.Dalton transactions (Cambridge, England : 2003) · 2021Article
- A safety cap protects hydrogenase from oxygen attack.Nature communications · 2021Article
- Generating dihydrogen by tethering an [FeFe]hydrogenase via a molecular wire to the APhotosynthesis research · 2020Article
- How [FeFe]-Hydrogenase Facilitates Bidirectional Proton Transfer.Journal of the American Chemical Society · 2019Article
- Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases.Nature communications · 2018Article
- Terminal Hydride Species in [FeFe]-Hydrogenases Are Vibrationally Coupled to the Active Site Environment.Angewandte Chemie (International ed. in English) · 2018Article
- From protein engineering to artificial enzymes - biological and biomimetic approaches towards sustainable hydrogen production.Sustainable energy & fuels · 2018Review
- Influence of the [4Fe-4S] cluster coordinating cysteines on active site maturation and catalytic properties ofChemical science · 2017Article
- Reaction Coordinate Leading to HJournal of the American Chemical Society · 2017Article
- Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases.Nature communications · 2017Article
- Mechanism of ONature chemistry · 2017Article
- Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution.Chemical science · 2016Article
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
[FeFe]-hydrogenases reversibly catalyse molecular hydrogen evolution by reduction of two protons. Proton supply to the catalytic site (H-cluster) is essential for enzymatic activity. Cysteine 298 is a highly conserved residue in all [FeFe]-hydrogenases; moreover C298 is structurally very close to the H-cluster and it is important for hydrogenase activity. Here, the function of C298 in catalysis was investigated in detail by means of site saturation mutagenesis, simultaneously studying the effect of C298 replacement with all other 19 amino acids and selecting for mutants with high retained activity. We demonstrated that efficient enzymatic turnover was maintained only when C298 was replaced by aspartic acid, despite the structural diversity between the two residues. Purified CaHydA C298D does not show any significant structural difference in terms of secondary structure and iron incorporation, demonstrating that the mutation does not affect the overall protein fold. C298D retains the hydrogen evolution activity with a decrease of k(cat) only by 2-fold at pH 8.0 and it caused a shift of the optimum pH from 8.0 to 7.0. Moreover, the oxygen inactivation rate was not affected demonstrating that the mutation does not influence O(2) diffusion to the active site or its reactivity with the H-cluster. Our results clearly demonstrate that, in order to maintain the catalytic efficiency and the high turnover number typical of [FeFe] hydrogenases, the highly conserved C298 can be replaced only by another ionisable residue with similar steric hindrance, giving evidence of its involvement in the catalytic function of [FeFe]-hydrogenases in agreement with an essential role in proton transfer to the active site.
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