PHOTOSYSTEM I AS A LIGHT INDUCED ELECTRON PUMP FOR DIRECT ENZYME CATALYSIS
Open Access
- Author:
- Walters, Karim Andre
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 10, 2016
- Committee Members:
- Dr. John Golbeck, Dissertation Advisor/Co-Advisor
Dr. John Golbeck, Committee Chair/Co-Chair
Dr. Squire Booker, Committee Member
Dr. Christine Keating, Committee Member
Dr. Wayne Curtis, Outside Member - Keywords:
- Photosynthesis
Ferredoxin
Sulfite Reductase - Abstract:
- Molecular wire technology developed in our lab utilizes an aliphatic or aromatic hydrocarbon terminated by thiols to tether the [4Fe-4S] cluster FB of Photosystem I (PS I) to the [4Fe-4S] cluster of a [Fe-Fe] hydrogenase or an inorganic stabilized thiolate platinum nanoparticle (Pt-NP) to create a light induced hydrogen-producing complex. The work described in this dissertation is an extension of this molecular wire technology and involves designing a modified dicluster ferredoxin from Clostridium pasteurianum to tether PS I to catalytic enzymes containing surface exposed [4Fe-4S] clusters or Pt-NP. The presence of two externally facing cysteine residues should permit the dicluster ferredoxin to take the place of the molecular wire, allowing it to function in vivo. To provide free thiolate groups in close proximity to each [4Fe-4S] cluster, which can act as external rescue ligands to an open coordination site, cysteine double mutants of the dicluster ferredoxin from C. pasteurianum have been successfully cloned and expressed in Escherichia coli. The first studies were accomplished using Pt-NP as the test bed. The PS I/dicluster ferredoxin/ Pt nanoparticle complex was able to produce hydrogen when illuminated with visible light. This is the first instance in which a 2[4Fe-4S] bacterial ferredoxin was used to tether two redox-active species, replacing the need for a dithiol molecular wire. To test the ability of PS I/dicluster ferredoxin to take part in multi-electron chemistry, ferredoxin sulfite reductase (FdSiR) was chosen as a redox partner. FdSiR has several interesting features that make it an excellent candidate for conducting these studies: (i) it contains a unique active site known, as siroheme, an iron tetrahydroporphyrin of the isobacteriochlorin type (adjacent pyrrole rings reduced) with eight carboxylate side chains, and (ii) it has the ability to perform six-electron reduction of sulfite to sulfide or nitrite to ammonia although with a much higher Km. A FdSiRWT and the FdSiRC491G variant from Synechococcus elongatus PCC 7942 has been successfully cloned and expressed in E. coli. Both FdSiRWT and FdSiRC491G variant were shown to be enzymatically active. This is the first reported expression of a FdSiRC491G variant in which one cysteine ligand to the [4Fe-4S] cluster has been altered while maintaining enzymatic activity. Initial attempts to tether the FdSiRC491G variant to PS I using the dithiol molecular wire 1,6-hexanedithiol and 1,8-octane dithiol were inconclusive.