Expanding the Versatility of the PS I-HydA Nanoconstruct
Open Access
- Author:
- Yeung, Simon
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 02, 2013
- Committee Members:
- Donald Ashley Bryant, Thesis Advisor/Co-Advisor
John H Golbeck, Thesis Advisor/Co-Advisor
John Michael Regan, Thesis Advisor/Co-Advisor - Keywords:
- photosystem
hydrogenase
HydA
N75
crosslinker
BIDBE
DTME
molecular wire
nanoconstruct - Abstract:
- In 2010, our group devised a photosystem I (PS I)—[FeFe] hydrogenase (CaHydA) nanoconstruct capable of light-driven H2 evolution with a rate of 30.3 ± 0.8 μmol of H2 (mg of Chl)-1 h-1. PS I from Synechococcus PCC 7002 and HydA from Clostridium acetobutylicum ATCC 824 were tethered by a molecular wire, 1,6-hexanedithiol. The molecular wire links PS I and CaHydA through the FB and Fe/S clusters in the enzymes, respectively, while eliminating diffusion as a factor for electron transfer between the two enzymes. Both clusters are [4Fe-4S] clusters missing one of the cysteine ligands generated by site-directed mutagenesis, leaving an open coordination site for the molecular wire to rescue the clusters chemically. Upon illumination, an electron is transferred along the electron transport chain (ETC) of PS I to CaHydA. Electrons ultimately reach the active site of CaHydA, the H-cluster, which catalyzes proton reduction to form molecular hydrogen, H2 (g). This success has sparked interest in the application of PS I, especially the extraction of low-potential electrons from the FB cluster. Firstly, attempts were made to optimize the production of CaHydA in Shewanella oneidensis MR-1. Secondly, this study explored an alternative method serving to connect PS I with a redox-active protein reporter by using sulfhydryl crosslinkers. This protein reporter (i.e., N75) is the N-terminal domain of CaHydA containing a single prosthetic group, a [2Fe-2S] cluster. Therefore, N75 can demonstrate the possibility of directing an electron from PS I to an external protein redox cofactor that is not a [4Fe-4S] cluster. These studies expand the versatility of the nanoconstruct platform by extending its connectivity to allow studies of other protein cofactors of interest.