Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism

Abstract

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a ∼4-ps and a ∼20-ps population to produce the charge-separated state P+HA− in all variants. Global analysis indicates that in the ∼4-ps population, P+HA− forms through a two-step process, P*→ P+BA−→ P+HA−, while in the ∼20-ps population, it forms via a one-step P* → P+HA− superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from ∼25 to ∼45% among variants, while in WT, this percentage is ∼15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P+BA− intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an ∼110-meV increase in the free energy of P+BA− along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P+BA– formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.

High Yield of B-Side Electron Transfer at 77 K in the Photosynthetic Reaction Center Protein from Rhodobacter sphaeroides

“…Representative time profiles for P* stimulated emission decay are shown in Figures S38–S41. As is well-known, fits of the P* decay for WT RCs require two exponentials. − ,− A common interpretation is that the faster (∼3 ps), dominant (80–90%) component reflects the first step of P* → P + B A – → P + H A – two-step ET (the second step taking <1 ps) and the slower (∼10 ps), smaller amplitude component reflects P* → P + H A – one-step superexchange ET (Figure C). The same assignments can be made for the dual-exponential fits for WT VW here (Table ).…”

“…This static picture averages over underlying complexity. We recently presented a view of this complexity that attempts to unify perspectives that have been presented in the literature ,,− ,, concerning multiexponential P* decays . In short, the protein scaffolding provides a fluctuating environment for the embedded cofactors.…”

Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme

“…(A) GFP (green) and Dronpa2 (gold) protein environments suppress excited-state isomerization of the chromophore to different degrees compared to that in vacuum (gray), rendering GFP less photoisomerizable than Dronpa2 (Figure D). (B) Y­(M210)F mutant (purple) of Rhodobacter sphaeroides photosynthetic reaction center reveals that tyrosine at M210, which stabilizes the first intermediate, is in part responsible for the unidirectional excited-state electron transfer of wild type (orange). , (C) Wild-type Fe­(II)/2-oxoglutarate (2OG)-dependent halogenases (orange) chlorinate their substrates, but their intrinsic hydroxylating power can be unleashed upon mutation (purple). ,, The default (blue) and the side pathways (red for all and green for panel A) are shown on the right and left for each panel, respectively. Energies are not drawn to scale.…”

“…It is the ability to prepare subtle mutants or variants that facilitates deeper mechanistic studies and energetic probing, in combination with rigorous characterizations, such as spectroscopic, kinetic, functional, and structural analyses and theoretical modeling. Subtle electrostatic/electronic and steric modulation to critical structural elements and/or physical properties for protein functions, including but not limited to cation−π interactions, 67 π−π interactions, 23 hydrogen bonds, 68,69 dipole−π interactions, 63 pK a , 70 nucleophilicity, 71 electronic distribution, 22 redox potential, 72 active-site electric field, 73 and isomerization efficiency, 74 can then be quantified by a suitable parameter that is derived from the physical chemistry or physical organic 43 tradition. The explored sequence space can accordingly be encoded with such parameter to interrogate its importance on energetics during catalysis, providing insights into the design principles of the large classes of enzymes adopting polar or radical mechanisms.…”

Protein Tyrosine Nitration in Plant Nitric Oxide Signaling

“…Site-specific nitration of calmodulin at its two Tyr residues using genetic code expansion technology allowed assessing the effects of these alterations on calcium binding by calmodulin, and on the subsequent binding and activation of human endothelial NOS (eNOS; Porter et al, 2020 ). By substituting a key Tyr residue by nitroTyr using genetic code expansion technologies, the primary electron transfer in the reaction centers of the photosynthetic bacteria Rhodobacter sphaeroides has been studied and further engineered ( Weaver et al, 2021 ). This genetically encoded synthesis of proteins containing 3-nitroTyr residues opens a potentially valuable strategy to assess the functional relevance of site-specific nitration on diverse target proteins, but to date no such application has been reported for any plant protein.…”