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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.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.

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 WT hydrogenase, G1 hydrogenase, and progressive substitutions to G2 hydrogenase (see Table 1 for sequence identities). Activities are plotted on a log scale over a 10-fold and 100-fold ranges, respectively to compare fold improvements.
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ijms-16-02020-f002: Methyl viologen-mediated (A) and methyl viologen-free (dithionite only) (B) in vitro hydrogen production assay from extracts of E. coli over-expressing the WT hydrogenase, G1 hydrogenase, and progressive substitutions to G2 hydrogenase (see Table 1 for sequence identities). Activities are plotted on a log scale over a 10-fold and 100-fold ranges, respectively to compare fold improvements.

Mentions: A more modest series of surface residue modifications was pursued (see Table 1). Proceeding from the G1 enzyme, the double substitution E261Q, E268Q (“I1”) was constructed, followed by further substitution of E227Q (“I2”). Finally, the residue D231 was substituted as a histidine (D231H) following the substitution in a hydrogenase from Azoarcus sp. BH72 (YP_935309). While no appreciable difference could be measured between the variants with an in vitro hydrogen evolution assay using methyl viologen (Figure 2A), omission of the positively charged methyl viologen in favor of direct electron transfer from the anionic reducing agent dithionite (or possibly other soluble redox transfer species present in crude lysates) resulted in a dramatic 5-fold improvement in hydrogen evolution for the fully substituted 2nd generation “G2” enzyme compared to the G1 enzyme (Figure 2B).


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 WT hydrogenase, G1 hydrogenase, and progressive substitutions to G2 hydrogenase (see Table 1 for sequence identities). Activities are plotted on a log scale over a 10-fold and 100-fold ranges, respectively to compare fold improvements.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-02020-f002: Methyl viologen-mediated (A) and methyl viologen-free (dithionite only) (B) in vitro hydrogen production assay from extracts of E. coli over-expressing the WT hydrogenase, G1 hydrogenase, and progressive substitutions to G2 hydrogenase (see Table 1 for sequence identities). Activities are plotted on a log scale over a 10-fold and 100-fold ranges, respectively to compare fold improvements.
Mentions: A more modest series of surface residue modifications was pursued (see Table 1). Proceeding from the G1 enzyme, the double substitution E261Q, E268Q (“I1”) was constructed, followed by further substitution of E227Q (“I2”). Finally, the residue D231 was substituted as a histidine (D231H) following the substitution in a hydrogenase from Azoarcus sp. BH72 (YP_935309). While no appreciable difference could be measured between the variants with an in vitro hydrogen evolution assay using methyl viologen (Figure 2A), omission of the positively charged methyl viologen in favor of direct electron transfer from the anionic reducing agent dithionite (or possibly other soluble redox transfer species present in crude lysates) resulted in a dramatic 5-fold improvement in hydrogen evolution for the fully substituted 2nd generation “G2” enzyme compared to the G1 enzyme (Figure 2B).

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.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.

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