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]

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

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