Using genetic code expansion to investigate electron transfer and protein electrostatics in the photosynthetic reaction center
Keywords: Chemistry.

The photosynthetic reaction center (RC) has been intensely studied for decades as a model system for understanding ultrafast electron transfer and protein design. The RC is a pseudo-C2 symmetric, ten-cofactor, integral membrane protein which catalyzes the initial electron transfers in photosynthesis which enable solar energy capture. Within the RC, bacteriochlorophyll, bacteriopheophytin, and quinone cofactors are arranged into two symmetric branches. Despite this symmetry, RCs catalyze electron transfer down only the A-branch of chromophores (96%> ) with picosecond efficiency. The structural origins of this unidirectional electron transfer have been a longstanding question and we use genetic code expansion in the RC to investigate electron transfer. This included site-specifically genetically encoding noncanonical amino acids in the form of tyrosine analogs at a key position (M210) and as vibrational probes of electron transfer and protein electrostatics. RCs, as photosynthetic membrane protein complexes, must be produced in the organism R. sphaeroides; genetic code expansion tools, however, are developed often to work only in E. coli or other model organisms. To incorporate noncanonical amino acids in RCs, I adapted tools from molecular biology evolved to be used in E. coli such that they functioned in R. sphaeroides. I found initial genetic expansion systems tested were toxic and noncompatible with R. sphaeroides. By moving to a more generally orthogonal system derived from an archaeal Pyrrolysyl-tRNA synthetase and changing transcriptional regulation of genes used in genetic code expansion (aminoacyl tRNA synthetase and tRNA), I enabled noncanonical amino acid incorporation in RCs in R. sphaeroides. Based on prior theoretical work, I varied the electronic properties of a key tyrosine in the RC via its replacement with noncanonical amino acid tyrosine analogs. I verified noncanonical amino acid incorporation with mass spectrometry and X-ray crystallography. Furthermore, with X-ray crystal structures of a variety of RC variants, I demonstrate tyrosine substitution with tyrosine analogs is minimally perturbative, causing no significant structural perturbation of surrounding cofactors or changes in other amino acids surrounding electron donors and acceptors. Using ultrafast kinetic analysis, I show the electron transfer mechanism was primarily affected, where tyrosine analogs caused increasingly larger fractions of the sample to perform a slower 1-step electron transfer which bypassed BA (P* --> P+HA-- ) as opposed to the more typical, faster 2-step electron transfer which occurs in wild-type (P* --> P+BA-- --> P+HA--). This correlated with electron transfer intermediate energetics I characterized using resonance Stark spectroscopy, where RC variants which destabilized the P+BA more had increasingly larger fractions of sample where P* decays though 1-step electron transfer. In the most destabilized RC variant, 3-nitrotyrosine at site M210, the signal associated with the P+BA-- formation at 1030 nm was dramatically reduced. This matched the ~110 meV increase in free energy in NO2Y RCs and indicated that the interaction of tyrosine at M210 with BA tunes the electron transfer mechanism to be a faster two-step electron transfer. Systems exist for incorporating noncanonical amino acids that can act as vibrational probes of electron transfer and protein electrostatics. One common vibrational probe is the nitrile, whose frequency has been shown to be sensitive to electric field changes through the vibrational Stark effect (VSE). Since electron transfer intermediates would be expected to exert an electric field, nitrile infrared (IR) frequency changes serve as ideal reporters of electron transfer. Though changes in RC chromophore absorbance are typically used to probe electron transfer, chromophores are known to be electronically coupled and to have spectral overlap; nitrile absorbance occurs in a much more background-free mid-IR region and are not electronically coupled to RC chromophores. I take a system made to genetically encode o-cyanophenylalanine (oCNF) in E. coli and adapt it to function in R. sphaeroides. By replacing native phenylalanine (F) sites about the A-branch of chromophores with oCNF, I incorporate multiple vibrational probes to study electron transfer in the RC. I verify oCNF incorporation with mass spectrometry and characterize oCNF-containing RC structures with X-ray crystallography. When standard kinetic measurements of electron transfer are obtained for an oCNF-containing RC variant (L97oCNF) using chromophore visible absorbance changes (visible pump -- visible probe), we find that nitrile vibrational probes to not appear to significantly effect wild-type electron transfer kinetics and that the nitrile probe is a true spectator of electron transfer. Experiments (visible pump -- IR probe) are currently in progress to characterize nitrile absorption changes during electron transfer with collaborators. Though the nitrile vibrational frequency is governed by the VSE, it is often complicated by hydrogen bonding interactions which impede electric field characterization. While calibrating the oCNF nitrile frequency-field response to monitor RC electron transfer, I discovered a new VSE which correlates electric field with integrated nitrile peak intensity. This calibration functions without complications (i.e. stays linear) in both aprotic and protic environment. In E. coli, I genetically encode oCNF at a range of positions in photoactive yellow protein (PYP), a model protein system, to site-specifically encode nitrile vibrational probes. I characterize high-resolution X-ray crystal structures of nitrile containing PYP variants and demonstrate that electric fields obtained using absolute peak intensities correlate with the electrostatics expected based on PYP crystal structures. In sum, I was able to develop a tool to help characterize the role of protein electrostatics in catalyzing electron transfer in RCs and what is more, discovered a technique which allows us to measure electrostatics in proteins and in any system where nitriles can be incorporated