Applications of Electroabsorption Spectroscopy to Enzymology and Molecular Electronics

Abstract

Absorption spectra of molecules encode structural details of the local electrostatic environment via coupling of the local electric field with charge redistribution through the molecule upon optical excitation. Electroabsorption spectroscopy, also known as Stark spectroscopy, provides a simple method for determining dependence of electronic transition energies on the local field by measuring and interpreting perturbations to the absorption spectrum due to a well-characterized external electric field. In this dissertation, electroabsorption spectroscopy has been utilized, first, to estimate the second-harmonic generation cross section of the intracellular signaling metabolite lumichrome, second, to measure optical band gap tuning of the molecular electronic component perylene diimide by an external electric field, and, third, to exploit optical transition energies of the ubiquitous enzyme cofactor flavin to measure flavoenzyme active site electric fields. Experimental measurements are complemented by time-dependent density functional theory calculations to interpret experimental results and localize charge redistribution within the molecular frame.Lumichrome is a photodegradation product and catabolite of flavin cofactors that additionally serves as a signaling molecule for plants and bacteria. As a mediator of bacterial quorum sensing, lumichrome is transported across bacterial membranes. While transiting bacterial membranes, the average orientation of the trafficked lumichrome may be sufficiently anisotropic to allow detection by second-harmonic generation spectroscopy, the magnitude of which is a function of charge redistribution upon optical excitation and may be estimated by Stark spectroscopy. Using Stark spectroscopy we have estimated lumichrome’s second harmonic generation cross-section to be approximately 80% that of flavin adenine dinucleotide, which has previously successfully been used as a second-harmonic generation probe. Perylene diimide is an n-type organic semiconductor that is widely used in organic photovoltaics, light emitting diodes, and other molecular opto-electronics. Electric fields within electronic devices utilizing monomeric and / or aggregated perylene diimide alter the band structure of the chromophore, possibly providing a tunable parameter for the improvement of these devices. Stark spectroscopy has been used to measure tuning of the optical bandgap of monomeric perylene diimide by an external electric field. The centrosymmetry of the molecule causes the first optical excitation's difference permanent dipole moment to approach zero. The change in polarizability following excitation is also modest, with the trace of the difference polarizability equal to 42 cubic angstroms and the component of the difference polarizability along the transition dipole moment equal to 29 cubic angstroms. Electric fields within molecular electronics are on the order of 1 MV/cm. Such an electric field would redshift the first optical excitation energy of monomeric perylene diimide by 16.25 wavenumbers, which is less than 0.1% of the transition energy (18500 wavenumbers). Flavin adenine dinucleotide and flavin mononucleotide are ubiquitous enzymatic cofactors mediating catalytic transfer of one or two electrons. Flavin cofactors are ubiquitous because of their versatile and tunable reactivity. For example, the two-electron reduction potential of flavin can be continuously tuned through a range of over 400 mV by differing interactions with flavoenzyme active site residues. We have utilized Stark spectroscopy to measure charge redistribution upon optical excitation for the first two excited states of oxidized flavin in the aprotic, non-polar solvent toluene, with and without a tridentate hydrogen-bonding ligand. In comparing this data with previously reported measurements, we conclude that charge redistribution is independent of solvent polarity. As such, flavin’s optical transition energies may be used to indirectly measure flavoenzyme active site electric fields directly from the perspective of the electrostatically-tuned flavin cofactor, enhancing our understanding of flavin-dependent biochemistry.