Insights in enzyme modification for renewable solar hydrogen

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Insights in enzyme modification for renewable solar hydrogen

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Insights in enzyme modification for renewable solar hydrogen

Isaac T Yonemoto, Philip Weyman J. Craig Venter Institute

A Little Bit of Basic Biology

Conventional Biofuels are Steppy

Intercept Electrons at the Source

GOS identified the Alteromonas "Uptake" Hydrogenase

[NiFe] Hydrogenase Features a Complex Organometallic Active Site

Our Studies Focus on the Fe-S Clusters Carrying Electrons to the Active Site

Recap on Fe-S Clusters

Initial Modifications to Hydrogenase Small Subunit

Altermonas Hydrogenase Oxidizes Hydrogen

Can Altermonas Hydrogenase Reduce Proton?

Previous Literature Suggested Amino Acid Substitutions

Species Substitution Oxidation Effect Reduction Effect D. fructosovorans Medial Pro to Cys -38% +60%[1] D. fructosovorans Distal His to Cys -98.7% -53%[2]
  • [1]Rousset, et al. PNAS 1998
  • [2]Dementin, et al. JACS 2006

Crude Thought Model Suggests a Mechanism

Hydrogen is produced using a simple in vitro assay

Neither Homologous Substitution Improved Activity

Combining Substitutions Improved Activity

How Else Can we Play with Electron Transport?

Can Tweaking the Midpoint Potentials Help?

Literature Precedent for "Strange" Fe-S Clusters

Species Location Substitution M. barkeri Proximal 1 Cys to Asp G. metallireducens Proximal 2 Cys to Asp N. punctiforme Proximal 2 Cys to Asn N. punctiforme Distal 1 His to Gln [NiFeSe] hydrogenases Medial 2 Pro to Cys
  • [1]Raleiras, et al. JBC 2013

We Screened 48 Substitutions of Fe-S ligating Cysteines

"Native-like" Substitutions are All Functional

Aspartic Acid Substitutions are Generally Well-Tolerated

...And Some of them Outperform the Wild-Type Enzyme

Conclusions and Future Directions

  • We can make Alteromonas hydrogenase better
  • We have a library of 48 variants with varying activity
  • We're working towards understanding why the enzyme has a bias
  • Hydrogenase may be a good platform for putting Marcus Theory to rigorous test

Acknowledgement

  • Philip Weyman
  • Hamilton O Smith
  • Pin-Ching Maness
  • Ben Clarkson

Funding

Department of Energy Fuel Cells Office DE-FG36-05GO15027