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Open AccessArticle

Designed Surface Residue Substitutions in [NiFe] Hydrogenase that Improve Electron Transfer Characteristics

J. Craig Venter Institute, Synthetic Biology and Bioenergy Group, 4120 Capricorn Lane, La Jolla, CA 92037, USA
Author to whom correspondence should be addressed.
Academic Editor: Patrick Hallenbeck
Int. J. Mol. Sci. 2015, 16(1), 2020-2033;
Received: 11 December 2014 / Accepted: 12 January 2015 / Published: 16 January 2015
(This article belongs to the Special Issue Photosynthesis and Biological Hydrogen Production)
PDF [1578 KB, uploaded 16 January 2015]


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. View Full-Text
Keywords: hydrogenase; ferredoxin; Alteromonas macleodii hydrogenase; ferredoxin; Alteromonas macleodii

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Yonemoto, I.T.; Smith, H.O.; Weyman, P.D. Designed Surface Residue Substitutions in [NiFe] Hydrogenase that Improve Electron Transfer Characteristics. Int. J. Mol. Sci. 2015, 16, 2020-2033.

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