Limits...
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.

Show MeSH

Related in: MedlinePlus

Methyl viologen-mediated in vitro hydrogen production assay (A) from tandem immobilized metal affinity chromatography (IMAC)/streptactin-purified G1 and G2 hydrogenases, and their respective ferredoxin fusions. In vitro H2 production of the same hydrogenases in the methyl viologen-free assay (dithionite only) (B) and expressed as the ratio of methyl viologen-free activity to activity in assays containing methyl viologen (C), plotted on a log scale over an 100-fold range to compare fold-changes. Sypro-ruby stained gel (D) and Anti-HynL western blot (E) of the same constructs from the same protein preparations presented in (A,B).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4307346&req=5

ijms-16-02020-f004: Methyl viologen-mediated in vitro hydrogen production assay (A) from tandem immobilized metal affinity chromatography (IMAC)/streptactin-purified G1 and G2 hydrogenases, and their respective ferredoxin fusions. In vitro H2 production of the same hydrogenases in the methyl viologen-free assay (dithionite only) (B) and expressed as the ratio of methyl viologen-free activity to activity in assays containing methyl viologen (C), plotted on a log scale over an 100-fold range to compare fold-changes. Sypro-ruby stained gel (D) and Anti-HynL western blot (E) of the same constructs from the same protein preparations presented in (A,B).

Mentions: Also as predicted, the ferredoxin fusions exhibited approximately 2-fold improved methyl viologen-free activity compared to the unfused hydrogenase, suggesting enhanced electron transfer to the enzyme as a result of both modifications (Figure 4). This increased activity attributable to the fusion is not explained by differences in protein abundance as determined by Western blot (Figure 4).


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 in vitro hydrogen production assay (A) from tandem immobilized metal affinity chromatography (IMAC)/streptactin-purified G1 and G2 hydrogenases, and their respective ferredoxin fusions. In vitro H2 production of the same hydrogenases in the methyl viologen-free assay (dithionite only) (B) and expressed as the ratio of methyl viologen-free activity to activity in assays containing methyl viologen (C), plotted on a log scale over an 100-fold range to compare fold-changes. Sypro-ruby stained gel (D) and Anti-HynL western blot (E) of the same constructs from the same protein preparations presented in (A,B).
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-02020-f004: Methyl viologen-mediated in vitro hydrogen production assay (A) from tandem immobilized metal affinity chromatography (IMAC)/streptactin-purified G1 and G2 hydrogenases, and their respective ferredoxin fusions. In vitro H2 production of the same hydrogenases in the methyl viologen-free assay (dithionite only) (B) and expressed as the ratio of methyl viologen-free activity to activity in assays containing methyl viologen (C), plotted on a log scale over an 100-fold range to compare fold-changes. Sypro-ruby stained gel (D) and Anti-HynL western blot (E) of the same constructs from the same protein preparations presented in (A,B).
Mentions: Also as predicted, the ferredoxin fusions exhibited approximately 2-fold improved methyl viologen-free activity compared to the unfused hydrogenase, suggesting enhanced electron transfer to the enzyme as a result of both modifications (Figure 4). This increased activity attributable to the fusion is not explained by differences in protein abundance as determined by Western blot (Figure 4).

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