Characterization and Extension of a Bio-hybrid Photosystem I-hydrogenase Nanoconstruct Device For Hydrogen Generation

Open Access
Author:
Applegate, Amanda Marie
Graduate Program:
Chemistry
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
December 15, 2014
Committee Members:
  • John H Golbeck, Thesis Advisor
Keywords:
  • Photosystem I
  • hydrogenase
  • hydrogen
  • solar fuels
Abstract:
As a result of the ever increasing demanded for fossil fuels, there is a drive to develop robust strategies for the conversion of solar energy into a storable alternative fuel such as dihydrogen (H2). Previously, a hydrogen generation strategy was developed and optimized that couples Photosystem I (PSI), which harvests and stores solar energy, with an [FeFe]-Hydrogenase (H2ase) enzyme catalyst for direct generation of H2 from sunlight. The two proteins are connected at a short distance using a dithiol molecular wire and generate H2 at a rate of 2200 µmol H2¬•mg Chl-1•h-1, under optimized conditions. Described here is the characterization and extension of the PSI—molecular wire—[FeFe]-H2ase (PSIC13G—H2aseC97G) nanoconstruct device to expand this technology further for improved solar H2 generation. These PSIC13G—H2aseC97G nanoconstructs are prepared through the self-assembly of iron-sulfur coordination bonds between the dithiol molecular wire and the low potential [4Fe-4S] clusters found in both proteins. Quantum yield measurements of the light-induced H2 generation were conducted to determine the efficiency or number of molecules of H2 produced per photon of light absorbed under 400-700 nm illumination. Sample composition was assayed using analytical methods to provide insight into the number of species generated in each self-assembled sample and how to further improve upon this technology. Sample preparation techniques were employed to exclude species such as PSI—molecular wire—PSI dimers from PSIC13G—H2aseC97G nanoconstructs chromatographically. Replacement of the [FeFe]-¬H2ase with a more oxygen-tolerant [NiFe]-H2ase catalyst was explored improve the oxygen stability of this technology. The preliminary in vitro characterization of PSI timers isolated from a strain of PSI containing the genes for the PsaCC13G/C33S mutations in vivo was also performed.