Dissertations, Theses, and Capstone Projects

Date of Degree


Document Type


Degree Name





Ronald L Koder

Committee Members

Rein Ulijn

Marilyn Gunner

Raymond Tu

Hiroshi Matsui

Subject Categories

Analytical Chemistry | Biochemistry | Biological and Chemical Physics | Biophysics | Molecular Biology | Organic Chemistry


Photosynthesis, Protein engineering, de novo proteins, viologens, Charge separation


To create an efficient de novo photosynthetic protein it is important to create long lived charge separated states. Achieving stable charge separation leads to an increase in the efficiency of the photosynthetic reaction which in turn leads to higher yields of end products, such as biofuels, electrical charge, or synthetic chemicals. In an attempt to create charge separated states in de novo proteins we hypothesized that we could engineer the free energy gaps in the proteins from excited primary donor (PD) to acceptor (A), and A back to ground state PD such that the forward electron transfer (ET) would be close to the Marcus parabola maximum to maximize the forward ET rate. At the same time, the reverse reaction would be overdriven such that it lies in the Marcus inverted region where ET is slow. This would have the effect of creating a charge separated state that lasts long enough for the electron to move away from the electron hole to further electron acceptors creating a truly charge separated state. Our approach was to design proteins that incorporate a synthetic amino acid able to attach covalently to a set of different viologens using Copper assisted alkyne-azide cycloaddition (CuAAC) click chemistry. The proteins incorporate zinc porphyrins as the primary electron donor, and an iron porphyrin as a placeholder cofactor or final electron acceptor. The proteins constructs were investigated using steady-state fluorescence quenching for investigating the rate of ET, TCSPC for fluorescence lifetime and transient absorption measurements at the picosecond timescale. v Protein constructs with viologens had their redox potentials determined through Dutton solution potentiometry and it was found that covalent attachment has little effect compared to free viologen potential. We have determined that it is feasible to click viologens onto the proteins, and we show that the ET obeys Marcus kinetics. We also see evidence of ET in transient absorption measurements made on proteins where the viologen forms a bridging position between the PD and the ultimate iron porphyrin electron acceptor. These findings introduce a novel range of artificial redox co-factors as well as providing evidence that it is possible to use the Marcus inverted region to achieve charge separation in de novo proteins.