Molecular mechanism of light activation in the blue-light photoreceptor BLUF

Photoreceptor proteins are molecular sensors that translate photon energy into biological information. The BLUF (Blue Light using FAD) protein is such a sensor that switches between its dark and light states by means of photoinduced proton-coupled electron transfer (PCET). In this thesis, I present the first detailed and systematic computational study of photoinduced PCET in BLUF using state-of-the-art electronic-structure methods. The photoactivation in BLUF results in the tautomerization and rotation of a conserved glutamine side chain. The computed potential-energy landscapes presented in this thesis reveal the energies of glutamine rotamers and tautomers and serve as a basis to identify the structure of the glutamine side chain in the functional dark and light states of BLUF. To map the pathway connecting the dark and light states on the excited-state potential-energy surface, I established a computational procedure employing multi-configurational multi-reference electronic-structure methods, and built and characterized quantum-mechanical cluster and hybrid quantum-mechanical/molecular-mechanical models. After establishing and benchmarking the computational protocol, I computed several PCET photoreaction pathways. The energy profiles obtained serve as a basis to answer, for the first time, questions related to how PCET is realized in photoactivation, photostability, and redox tuning in BLUF

Role of the Molecular Environment in Flavoprotein Color and Redox Tuning: QM Cluster versus QM/MM Modeling

We investigate the origin of the excitation energy shifts induced by the apoprotein in the active site of the bacterial photoreceptor BLUF (<u>B</u>lue <u>L</u>ight sensor <u>U</u>sing <u>F</u>lavin adenine dinucleotide). In order to compute the vertical excitation energies of three low-lying electronic states, including two π–π* states of flavin (S₁ and S₂) and a π–π* tyrosine-flavin electron-transfer state (ET), with respect to the energy of the closed-shell ground state (S₀), we prepared alternative quantum mechanical (QM) cluster and quantum mechanics/molecular mechanics (QM/MM) models. We found that the excitation energies computed with both types of models correlate with the magnitude of the charge transfer character of the excitation. Accordingly, we conclude that the small charge transfer character of the light absorbing S₀–S₁ transition and the substantial charge transfer character of the nonabsorbing but redox active S₀–ET transition explain the small color changes but substantial redox tuning in BLUF and also in other flavoproteins. Further analysis showed that redox tuning is governed by the electrostatic interaction in the QM/MM model and transfer of charge between the active site and its environment in the QM cluster. Moreover, the wave function polarization of the QM subsystem by the MM subsystem influences the magnitude of the charge transfer, resulting in the QM/MM and QM excitation energies that are not entirely consistent

Toward a Scalable Synthesis and Process for EMA401 Part III: Using an Engineered Phenylalanine Ammonia Lyase Enzyme to Synthesize a Non-natural Phenylalanine Derivative

A process using engineered phenylalanine ammonia lyase (PAL) enzymes was developed as part of an alternative route to a key intermediate of olodanrigan (EMA401). In the first part of the manuscript, the detailed results from a screening for the optimal reaction conditions are presented, followed by the discussion of several work-up strategies investigated. In the PAL catalyzed reaction, 70–80% conversion of a cinnamic acid derivative to the corresponding phenylalanine derivative could be achieved. The phenylalanine derivative was subsequently telescoped to a Pictet-Spengler reaction with formaldehyde and the corresponding tetrahydroisoquinoline derivative was isolated in 60–70% yield with >99.9:0.1 er. Based on our screenings, carbonate/carbamate buffered ammonia at 9–10 M NH3 concentration and pH 9.5–10.5 were found as the optimal conditions. Enzyme loadings down to 2.5wt% (E:S 1:40 w/w) could be achieved and substrate concentrations between 3–9 v/w (1.17–0.39 M) were found to be compatible with the reaction conditions. A temperature gradient was applied in the final process: a pre-equilibrium was established at 45 °C, before making use of the temperature-dependence of the entropy term with subsequent cooling to 20 °C and achieving maximum conversion. This temperature gradient also allowed balancing enzyme stability (low at 45 °C, high at 20 °C) with activity (high at 45 °C, low at 20 °C) in order to achieve optimal conversion (low at 45 °C, high at 20 °C). From the various work-up operations investigated, a sequence consisting of denaturation of the enzyme, followed by NH3/CO2 removal by distillation, acidification and telescoping to the subsequent Pictet-Spengler cyclization was our preferred approach. The process presented in this study is a more sustainable, shorter and more cost effective alternative to the previous process

Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon

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