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Nickel-substituted rubredoxin (NiRd) from Desulfovibrio desulfuricans has previously been shown to act as both a structural and functional mimic of the [NiFe] hydrogenase. However, improvements both in turnover frequency (TOF) and overpotential are needed to rival the native [NiFe] hydrogenase enzymes. Characterization of a library of NiRd mutants with variations in the secondary coordination sphere suggested protein dynamics played a substantial role in modulating activity. In this work, rubredoxin scaffolds were selected from diverse organisms to study the effects of distal sequence variation on catalytic activity. It was found that though electrochemical catalytic activity was only slightly impacted across the series, the Rd sequence from a psychrophilic organism exhibited substantially higher levels of solution-phase hydrogen production. Additionally, Eyring analyses suggest that catalytic activation properties relate to the growth temperature of the parent organism, implying that the general correlation between parent organism environment and catalytic activity often seen in naturally occurring enzymes may also be observed in artificial enzymes. Selecting protein scaffolds from hosts that inhabit diverse environments, particularly low temperature environments, represents an alternative approach for engineering artificial metalloenzymes.
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Design of a minimal di-nickel hydrogenase peptide
Ancestral metabolic processes involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13–amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe] hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase, two ancient, extant proteins central to metabolism. These experimental results demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth.
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- Award ID(s):
- 2025200
- PAR ID:
- 10493981
- Editor(s):
- Thorp, Holden
- Publisher / Repository:
- Science Advances
- Date Published:
- Journal Name:
- Science Advances
- Edition / Version:
- 1
- Volume:
- 9
- Issue:
- 10
- ISSN:
- 2375-2548
- Subject(s) / Keyword(s):
- Enzyme Hydrogenase nickel acetyl-CoA synthase nickelback
- Format(s):
- Medium: X Other: pdf
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1126/sciadv.abq1990
