A De Novo‐Designed Artificial Metallopeptide Hydrogenase: Insights into Photochemical Processes and the Role of Protonated Cys

Parambath, Sreya Malayam; Williams, Ashley E.; Hunt, Leigh Anna; Selvan, Dhanashree; Hammer, Nathan I.; Chakraborty, Saumen

doi:10.1002/cssc.202100122

Cited by 10 publications

(25 citation statements)

References 56 publications

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“…We believe this process leads to more dimer production upon Ni II addition to the apo mixture. This is consistent with our previous observation that the Ni II (Cys) 4 coordination is also stabilized when Ni II is added to the 2SCC peptide that is designed to exist as a dimer under similar experimental conditions [3c] . To test how the oligomerization behavior is affected by the introduction of Cys residues, we prepared the Cys‐free peptide (4SCC‐Cys: Table 1) where the 9th and 12th layer contained Leu and Ile, respectively, instead of Cys.…”

Section: Resultssupporting

confidence: 83%

Photocatalytic Hydrogen Evolution by a De Novo Designed Metalloprotein that Undergoes Ni‐Mediated Oligomerization Shift

Prasad

Hunt

Pall

et al. 2023

Chemistry A European J

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De novo metalloprotein design involves the construction of proteins guided by specific repeat patterns of polar and apolar residues, which, upon self-assembly, provide a suitable environment to bind metals and produce artificial metalloenzymes. While a wide range of functionalities have been realized in de novo designed metalloproteins, the functional repertoire of such constructs towards alternative energy-relevant catalysis is currently limited. Here we show the application of de novo approach to design a functional H 2 evolving protein. The design involved the assembly of an amphiphilic peptide featuring cysteines at tandem a/d sites of each helix. Intriguingly, upon Ni II addition, the oligomers shift from a major trimeric assembly to a mix of dimers and trimers. The metalloprotein produced H 2 photocatalytically with a bell-shape pH dependence, having a maximum activity at pH 5.5. Transient absorption spectroscopy is used to determine the timescales of electron transfer as a function of pH. Selective outer sphere mutations are made to probe how the local environment tunes activity. A preferential enhancement of activity is observed via steric modulation above the Ni II site, towards the N-termini, compared to below the Ni II site towards the C-termini.

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Section: Resultssupporting

confidence: 83%

Photocatalytic Hydrogen Evolution by a De Novo Designed Metalloprotein that Undergoes Ni‐Mediated Oligomerization Shift

Prasad

Hunt

Pall

et al. 2023

Chemistry A European J

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“…Our results not only provide novel insights into the sophisticated mechanism of H 2 activation in [NiFe]-hydrogenases, but also deciphered the oftenoverlooked contribution of the protein scaffold to fine-tune proton and electron dynamics, which is of general importance for structure/function studies of (metallo)enzymes operating with effective proton/electron transfer mechanisms. In this context, our results also have implications for the design of effective synthetic complexes and artificial metallopeptides (see, e.g., ref61 ) featuring hydrogenaselike reactivity.…”

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confidence: 65%

Structural Determinants of the Catalytic Nia-L Intermediate of [NiFe]-Hydrogenase

Waffo

Lorent

Katz

et al. 2023

Preprint

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[NiFe]-hydrogenases catalyze the reversible cleavage of H2 into two protons and two electrons at the inorganic heterobimetallic NiFe center of the enzyme. Their catalytic cycle involves at least four intermediates, some of which are still under debate. While the core reaction, including H2/H- binding, takes place at the inorganic cofactor, a major challenge lies in identifying those amino acid residues that contribute to the reactivity and how they stabilize (short-lived) intermediate states. Using cryogenic infrared and electron paramagnetic resonance spectroscopy on the regulatory [NiFe]-hydrogenase from Cupriavidus necator, a model enzyme for the analysis of catalytic intermediates, we deciphered the structural basis of the hitherto elusive Nia-L intermediates. We unveiled the protonation states of a proton-accepting glutamate and a Ni-bound cysteine residue in the Nia-L1, Nia-L2, and the hydride-binding Nia-C intermediates, as well as previously unknown conformational changes of amino acid residues in proximity of the bimetallic active site. As such, this study unravels the complexity of the Nia-L intermediate and reveals the importance of the protein scaffold in fine-tuning proton and electron dynamics in [NiFe]-hydrogenase.

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“…[7] A particular focus is to expand the functional diversity of these constructs into previously unexplored areas, [8] including energy relevant catalysis. [7,9] As complex enzyme structures are made up of peptides, which are subsequently synthesized from amino acid building blocks, de novo metalloenzyme design approach provides a handle to explore how primary sequence determines the folding and function of complex metalloenzymes. [10] In addition, the simpler de novo proteins help elucidate the catalytic and mechanistic underpinning of complex native enzymes.…”

Section: Introductionmentioning

confidence: 99%

Abiotic Reactivity of De Novo Designed Artificial Copper Peptides (ArCuPs): The Case of C-H Activation

Prakash

Mitra

Murphy

et al. 2022

Preprint

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We report a series of de novo designed Artificial Cu Peptides (ArCuPs) that oxidize and peroxygenate C-H bonds of model abiotic substrates via electrochemically generated Cu-oxygen species using H2O2 as the terminal oxidant, akin to native Cu enzymes. Detailed assessment of kinetic parameters established the catalytic nature of the ArCuPs. Selective alteration of outer sphere steric at the d layers above and below the Cu site allows facilitated access of substrates, where a more pronounced effect on catalysis is observed when space is created at the d layer below the Cu site via Ile to Ala mutation producing a kcat of 6.2 s-1, TONmax of 14800 and catalytic proficiency (kcat/KM/kuncat) of 340 M-1 for the oxidation of benzyl alcohol. Independent spectroscopic studied revealed that the rate of formation of the Cu-oxygen species and the spectroscopic feature of the most active variant is distinct compared to the other ArCuPs. Systematic alteration of outer sphere hydrophobicity led to a correlated tuning of the T2 Cu site redox potentials by ~80 mV. The enhanced activity of the ArCuP variant is attributed to a combination of steric effect that allows easy access of substrates, the nature of Cu-oxygen species, and stability of this construct compared to others, where Ile to Ala mutation unexpectedly leads to a higher thermostability which is further augmented by Cu binding.

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A De Novo‐Designed Artificial Metallopeptide Hydrogenase: Insights into Photochemical Processes and the Role of Protonated Cys

Cited by 10 publications

References 56 publications

Photocatalytic Hydrogen Evolution by a De Novo Designed Metalloprotein that Undergoes Ni‐Mediated Oligomerization Shift

Photocatalytic Hydrogen Evolution by a De Novo Designed Metalloprotein that Undergoes Ni‐Mediated Oligomerization Shift

Structural Determinants of the Catalytic Nia-L Intermediate of [NiFe]-Hydrogenase

Abiotic Reactivity of De Novo Designed Artificial Copper Peptides (ArCuPs): The Case of C-H Activation

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