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Designed surface residue substitutions in [NiFe] hydrogenase that improve electron transfer characteristics.

Yonemoto IT, Smith HO, Weyman PD - Int J Mol Sci (2015)

Bottom Line: Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons.While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor.Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme.

View Article: PubMed Central - PubMed

Affiliation: J. Craig Venter Institute, Synthetic Biology and Bioenergy Group, 4120 Capricorn Lane, La Jolla, CA 92037, USA. isaac.yonemoto@gmail.com.

ABSTRACT
Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii "Deep Ecotype" [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons necessary for hydrogen production. Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons. Thus, we engineered a hydrogenase-ferredoxin fusion that also contained several other modifications. In addition to the C-terminal ferredoxin fusion, we truncated the C-terminus of the hydrogenase small subunit, identified as the available terminus closer to the electron transfer region. We also neutralized an anionic patch surrounding the interface Fe-S cluster to improve transfer kinetics with the negatively charged ferredoxin. Initial screening showed the enzyme tolerated both truncation and charge neutralization on the small subunit ferredoxin-binding face. While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor. Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme.

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Methyl viologen-mediated (A) and methyl viologen-free (dithionite only) (B) in vitro hydrogen production assay from extracts of E. coli over-expressing the G1 hydrogenase and Δ15 and Δ22 truncations of the C-terminal tail.
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ijms-16-02020-f003: Methyl viologen-mediated (A) and methyl viologen-free (dithionite only) (B) in vitro hydrogen production assay from extracts of E. coli over-expressing the G1 hydrogenase and Δ15 and Δ22 truncations of the C-terminal tail.

Mentions: Two designs for C-terminal truncation were tested. Based on homology to the newly released structure of E. coli hydrogenase-1 [20], a Δ15 construct was built to trim the A. macleodii hydrogenase small subunit C-terminus to the equivalent point where the membrane-embedded alpha-helix of the E. coli hydrogenase-1 begins. A more aggressive Δ22 construct was built to trim the C-terminal tail to the point where sequence conservation begins, and the polypeptide chain is exposed to solvent in multiple structures [20]. Neither truncation had an appreciable effect on the hydrogenase activity (Figure 3), so the Δ22 truncation strategy was employed moving forward.


Designed surface residue substitutions in [NiFe] hydrogenase that improve electron transfer characteristics.

Yonemoto IT, Smith HO, Weyman PD - Int J Mol Sci (2015)

Methyl viologen-mediated (A) and methyl viologen-free (dithionite only) (B) in vitro hydrogen production assay from extracts of E. coli over-expressing the G1 hydrogenase and Δ15 and Δ22 truncations of the C-terminal tail.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307346&req=5

ijms-16-02020-f003: Methyl viologen-mediated (A) and methyl viologen-free (dithionite only) (B) in vitro hydrogen production assay from extracts of E. coli over-expressing the G1 hydrogenase and Δ15 and Δ22 truncations of the C-terminal tail.
Mentions: Two designs for C-terminal truncation were tested. Based on homology to the newly released structure of E. coli hydrogenase-1 [20], a Δ15 construct was built to trim the A. macleodii hydrogenase small subunit C-terminus to the equivalent point where the membrane-embedded alpha-helix of the E. coli hydrogenase-1 begins. A more aggressive Δ22 construct was built to trim the C-terminal tail to the point where sequence conservation begins, and the polypeptide chain is exposed to solvent in multiple structures [20]. Neither truncation had an appreciable effect on the hydrogenase activity (Figure 3), so the Δ22 truncation strategy was employed moving forward.

Bottom Line: Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons.While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor.Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme.

View Article: PubMed Central - PubMed

Affiliation: J. Craig Venter Institute, Synthetic Biology and Bioenergy Group, 4120 Capricorn Lane, La Jolla, CA 92037, USA. isaac.yonemoto@gmail.com.

ABSTRACT
Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii "Deep Ecotype" [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons necessary for hydrogen production. Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons. Thus, we engineered a hydrogenase-ferredoxin fusion that also contained several other modifications. In addition to the C-terminal ferredoxin fusion, we truncated the C-terminus of the hydrogenase small subunit, identified as the available terminus closer to the electron transfer region. We also neutralized an anionic patch surrounding the interface Fe-S cluster to improve transfer kinetics with the negatively charged ferredoxin. Initial screening showed the enzyme tolerated both truncation and charge neutralization on the small subunit ferredoxin-binding face. While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor. Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme.

Show MeSH
Related in: MedlinePlus