Deborah K. Hanson

In all photosynthetic reaction centers (RC), transmembrane electron-transfer (ET) cofactor pathways are coordinated by homologous peptides and arranged with approximate C ₂ symmetry, providing two possible paths for charge separation (designated A and B). In type II RCs—Photosystem II and the purple bacterial RCs—only the A branch is active for ET. A variant bacterial RC containing nine amino acid substitutions that result in high-yield ET along the normally nonfunctional B branch was designed. Structural and theoretical studies were combined to understand factors that control unique ET reactions in the complex. Serial femtosecond crystallography performed at an X-ray Free Electron Laser afforded the room temperature X-ray-damage-free crystal structure of the variant RC, revealing interactions between the substituted amino acids and the ET cofactors. The positions of the bacteriochlorin cofactors on the symmetry-related pathways are unaffected, underscoring that the substitutions alter the energetics of ET, leading to the functional changes. In the binding pockets for the terminal electron acceptor quinones, alterations in the positions of neighboring amino acids and water molecules are coordinated with the change of the substituted side chains. Modifications deactivated A-branch ET and activated transmembrane charge separation along the B branch. Electrostatic calculations based on the structure reveal how differences in stabilization of charge-separated states by the protein environment underlie the change in direction of electron flow in this variant RC and provide insights into mechanisms by which B-branch charge separation across the photosynthetic membrane can be achieved in high yield.

Photosynthetic reaction centers carry out a series of electron transfer reactions that convert the light energy into reduced products with chemical energy. However, only the A branch is active despite the structural symmetry.

The primary electron transfer (ET) processes at 295 and 77 K are compared for the Rhodobacter sphaeroides reaction center (RC) pigment-protein complex from 13 mutants including a wild-type control. The engineered RCs bear mutations in the L and M polypeptides that largely inhibit ET from the excited state P* of the primary electron donor (P, a bacteriochlorophyll dimer) to the normally photoactive A-side cofactors and enhance ET to the C₂-symmetry related, and normally photoinactive, B-side cofactors. P* decay is multiexponential at both temperatures and modeled as arising from subpopulations that differ in contributions of two-step ET (e.g., P* → P⁺B_B^- → P⁺H_B^-), one-step superexchange ET (e.g., P* → P⁺H_B^-), and P* → ground state. [H_B and B_B are monomeric bacteriopheophytin and bacteriochlorophyll, respectively.] The relative abundances of the subpopulations and the inherent rate constants of the P* decay routes vary with temperature. Regardless, ET to produce P⁺H_B^- is generally faster at 77 K than at 295 K by about a factor of 2. A key finding is that the yield of P⁺H_B^-, which ranges from ∼5% to ∼90% among the mutant RCs, is essentially the same at 77 K as at 295 K in each case. Overall, the results show that ET from P* to the B-side cofactors in these mutants does not require thermal activation and involves combinations of ET mechanisms analogous to those operative on the A side in the native RC.

All possible natural amino acids have been substituted for the native LeuL185 positioned near the B-side bacteriopheophytin (H_B) in the bacterial reaction center (RC) from Rhodobacter sphaeroides. Additional mutations that enhance electron transfer to the normally inactive B-side cofactors are present. Approximately half of the isolated RCs with Glu at L185 contain a magnesium chlorin (C_B) in place of H_B. The chlorin is not the common BChl a oxidation product 3-desvinyl-3-acetyl chlorophyll a with a C–C bond in ring D and a C═C bond in ring B but has properties consistent with reversal of these bond orders, giving 17,18-didehydro BChl a. In such RCs, charge-separated state P⁺C_B^– forms in ∼5% yield. The other half of the GluL185-containing RCs have a bacteriochlorophyll a (BChl a) denoted β_B in place of H_B. Residues His, Asp, Asn, and Gln at L185 yield RCs with ≥85% β_B in the H_B site, while most other amino acids result in RCs that retain H_B (≥95%). To the best of our knowledge, neither bacterial RCs that harbor five BChl a molecules and one chlorophyll analogue nor those with six BChl a molecules have been reported previously. The finding that altering the local environment within a cofactor binding site of a transmembrane complex leads to in situ generation of a photoactive chlorin with an unusual ring oxidation pattern suggests new strategies for amino acid control over pigment type at specific sites in photosynthetic proteins.

Significance: Photosynthetic organisms use pigment–protein complexes called reaction centers (RCs)—effectively nature’s solar cells—to convert the energy of sunlight into charge-separated species that power life processes. Whether from plants, algae, or photosynthetic bacteria, RCs feature 2 quasi-mirror-image arrangements of protein and pigment cofactors. In RCs from purple photosynthetic bacteria, photon-induced electron transfer (ET) reduces the A-branch bacteriopheophytin (BPh) with near-unity quantum yield, while ET to the symmetry-related B-branch BPh is suppressed. Amino acid changes made here in the Rhodobacter sphaeroides RC nearly completely reverse this design. Specifically, we report RCs wherein ET to the B-branch BPh occurs with 90% yield. We draw insight regarding architectural and mechanistic foundations of the primary photon-driven charge-separation events of photosynthesis.

In bacterial reaction centers (RCs), photon-induced initial charge separation uses an A-side bacteriochlorophyll (BChl, B_A) and bacteriopheophytin (BPh, H_A), while the near-mirror image B-side B_B and H_B cofactors are inactive. Two new sets of Rhodobacter capsulatus RC mutants were designed, both bearing substitution of all amino acids for the native histidine M180 (M-polypeptide residue 180) ligand to the core Mg ion of B_B. Residues are identified that largely result in retention of a BChl in the B_B site (Asp, Ser, Pro, Gln, Asn, Gly, Cys, Lys, and Thr), ones that largely harbor the Mg-free BPh in the B_B site (Leu and Ile), and ones for which isolated RCs are comprised of a substantial mixture of these two RC types (Ala, Glu, Val, Met and, in one set, Arg). No protein was isolated when M180 is Trp, Tyr, Phe, or (in one set) Arg. These findings are corroborated by ground state spectra, pigment extractions, ultrafast transient absorption studies, and the yields of B-side transmembrane charge separation. The changes in coordination chemistries did not reveal an RC with sufficiently precise poising of the redox properties of the B_B-site cofactor to result in a high yield of B-side electron transfer to H_B. Insights are gleaned into the amino acid properties that support BChl in the B_B site and into the widely observed multi-exponential decay of the excited state of the primary electron donor. The results also have direct implications for tuning free energies of the charge-separated intermediates in RCs and mimetic systems.

Seemingly redundant parallel pathways for electron transfer (ET), composed of identical sets of cofactors, are a cornerstone feature of photosynthetic reaction centers (RCs) involved in light-energy conversion. In native bacterial RCs, both A and B branches house one bacteriochlorophyll (BChl) and one bacteriopheophytin (BPh), but the A branch is used exclusively. Described herein are the results obtained for two Rhodobacter capsulatus RCs with an unnaturally high degree of cofactor asymmetry, two BPh on the RC’s B side and two BChl on the A side. These pigment changes derive, respectively, from the His(M180)Leu mutation [a BPh (Φ_B) replaces the B-side BChl (B_B)], and the Leu(M212)His mutation [a BChl (β_A) replaces the A-side BPh (H_A)]. Additionally, Tyr(M208)Phe was employed to disfavor ET to the A branch; in one mutant, Val(M131)Glu creates a hydrogen bond to H_B to enhance ET to H_B. In both Φ_B mutants, the decay kinetics of the excited primary ET donor (P*) resolve three populations with lifetimes of ∼9 ps (50–60%), ∼40 ps (10–20%), and ∼200 ps (20–30%), with P⁺Φ_B^– formed predominantly from the 9 ps fraction. The 50–60% yield of P⁺Φ_B^– is the highest yet observed for a Φ_B-containing RC. The results provide insight into factors needed for efficient multistep ET.