Transport limited adsorption experiments give a new lower estimate of the turnover frequency of Escherichia coli hydrogenase 1

Aldinio-Colbachini, Anna; Fasano, Andrea; Guendon, Chloé; Bailly, Aurore; Wozniak, Jeremy; Baffert, Carole; Kpebe, Arlette; Léger, Christophe; Brugna, Myriam; Fourmond, Vincent

doi:10.1016/j.bbadva.2023.100090

Cited by 1 publication

(2 citation statements)

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“…The enzyme E. coli Hyd 1 and the double mutant C19G/C120G were produced as described in our previous work , (SI sections S4–S6), from plasmids (pET24a) in the E. coli FTD147 (DE3) and the E. coli FTD147 (DE3) Δ recA strains carrying chromosomal in-frame deletions of the genes encoding the large subunits of hydrogenase-1, -2, and -3. − The recombinant Hyd 1 hydrogenase was produced as a dimeric, soluble form, consisting of only a large subunit (L1, or HyaB) and a small subunit (S1, or HyaA) or L1S1. The C-terminal membrane-anchored hydrophobic helix of S1 was replaced with a Streptag II.…”

Section: Resultsmentioning

confidence: 99%

“…The film of enzymes were made by polishing the surface of an homemade pyrolytic graphite edge electrode (3 mm diameter) and drop-casting 0.5–1 μL of protein solution (between 0.5 and 10 μM). For WT Hyd 1 and C19G/C120G Hyd 1, the films were also made by slow mass transport limited adsorption from the electrochemical cell solution, as described in ref . All of the experiments were performed in a buffer containing MES, CHES, HEPES, TAPS, Na acetate (each 5 mM), and NaCl (0.1 M).…”

Section: Methodsmentioning

confidence: 99%

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A Chimeric NiFe Hydrogenase Heterodimer to Assess the Role of the Electron Transfer Chain in Tuning the Enzyme’s Catalytic Bias and Oxygen Tolerance

Fasano,

Guendon,

Jacq-Bailly

et al. 2023

J. Am. Chem. Soc.

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The observation that some homologous enzymes have the same active site but very different catalytic properties demonstrates the importance of long-range effects in enzyme catalysis, but these effects are often difficult to rationalize. The NiFe hydrogenases 1 and 2 (Hyd 1 and Hyd 2) from E. coli both consist of a large catalytic subunit that embeds the same dinuclear active site and a small electron-transfer subunit with a chain of three FeS clusters. Hyd 1 is mostly active in H 2 oxidation and resistant to inhibitors, whereas Hyd 2 also catalyzes H 2 production and is strongly inhibited by O 2 and CO. Based on structural and site-directed mutagenesis data, it is currently believed that the catalytic bias and tolerance to O 2 of Hyd 1 are defined by the distal and proximal FeS clusters, respectively. To test these hypotheses, we produced and characterized a hybrid enzyme made of the catalytic subunit of Hyd 1 and the electron transfer subunit of Hyd 2. We conclude that catalytic bias and sensitivity to CO are set by the catalytic subunit rather than by the electron transfer chain. We confirm the importance of the proximal cluster in making the enzyme Hyd 1 resist long-term exposure to O 2 , but we show that other structural determinants, in both subunits, contribute to O 2 tolerance. A similar strategy based on the design of chimeric heterodimers could be used in the future to elucidate various structure− function relationships in hydrogenases and other multimeric metalloenzymes and to engineer useful hydrogenases that combine the desirable properties of distinct, homologous enzymes.

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