Rational design of photosynthetic reaction center protein maquettes.

New technologies for efficient solar-to-fuel energy conversion will help facilitate a global shift from dependence on fossil fuels to renewable energy. Nature uses photosynthetic reaction centers to convert photon energy into a cascade of electron-transfer reactions that eventually produce chemical fuel. The design of new reaction centers de novo deepens our understanding of photosynthetic charge separation and may one day allow production of biofuels with higher thermodynamic efficiency than natural photosystems. Recently, we described the multi-step electron-transfer activity of a designed reaction center maquette protein (the RC maquette), which can assemble metal ions, tyrosine, a Zn tetrapyrrole, and heme into an electron-transport chain. Here, we detail our modular strategy for rational protein design and show that the intended RC maquette design agrees with crystal structures in various states of assembly. A flexible, dynamic apo-state collapses by design into a more ordered holo-state upon cofactor binding. Crystal structures illustrate the structural transitions upon binding of different cofactors. Spectroscopic assays demonstrate that the RC maquette binds various electron donors, pigments, and electron acceptors with high affinity. We close with a critique of the present RC maquette design and use electron-tunneling theory to envision a path toward a designed RC with a substantially higher thermodynamic efficiency than natural photosystems.

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution.

In the structure of photosynthetic reaction center (RC) of the purple bacterium Cereibacter sphaeroides the highly conserved amino acid residue Ile-M206 is located near the bacteriochlorophyll dimer P, which is the primary electron donor, and the monomeric bacteriochlorophyll BA, which is the nearest electron acceptor. Since Ile-M206 is close to the C2-acetyl group of bacteriochlorophyll PB, the hydroxyl group of Tyr-M210, and to the C9-keto group of bacteriochlorophyll BA, as well as to the water molecule near the latter group, this site can be used for introducing mutations in order to study mechanisms of primary photochemical processes in the RC. Previously it was shown that the Ile→Glu substitution at the M204 position (analog of M206 in the RC of C. sphaeroides) in the RC of the closely related purple non-sulfur bacterium Rhodobacter capsulatus significantly affected kinetics of the P+HA- state formation, whereas the M204 Ile→Gln substitution led to the loss of BChl BA molecule from the complex structure. In the present work, it is shown that the single I(M206)Q or double I(M206)Q + F(M208)A amino acid substitutions in the RC of C. sphaeroides do not change the pigment composition and do not markedly influence redox potential of the primary electron donor. However, substitution of Ile M206 by Gln affected positions and amplitudes of the absorption bands of bacteriochlorophylls, increased lifetime of the primary electron donor P* excited state from 3.1 ps to 22 ps, and decreased quantum yield of the P+QA- state formation to 60%. These data suggest significant changes in the pigment-protein interactions in the vicinity of the primary electron donor P and the nearest electron acceptor BA. A considerable decrease was also noticed in the resistance of the mutant RC to thermal denaturation, which was more pronounced in the RC with the double substitution I(M206)Q + F(M208)A. This was likely associated with the disruption of the dense packing of the protein near bacteriochlorophylls PB and BA. Possible reasons for different effects of identical mutations on the properties of two highly homologous RCs from closely related purple non-sulfur bacteria are discussed.

The primary donor of far-red photosystem II: ChlD1 or PD2?

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210-1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ±â€¯4 cm-1 electrochromic shift due to formation of the semiquinone anion, QA-. Modelling indicates that no other FRL pigment is located among the 6 central reaction center chlorins: PD1, PD2 ChlD1, ChlD2, PheoD1 and PheoD2. Two of these chlorins, ChlD1 and PD2, are located at a distance and orientation relative to QA- so as to account for the observed electrochromic shift. Previously, ChlD1 was taken as the most likely candidate for the primary donor based on spectroscopy, sequence analysis and mechanistic arguments. Here, a more detailed comparison of the spectroscopic data with exciton modelling of the electrochromic pattern indicates that PD2 is at least as likely as ChlD1 to be responsible for the 726 nm absorption. The correspondence in sign and magnitude of the CD observed at 726 nm with that predicted from modelling favors PD2 as the primary donor. The pros and cons of PD2 vs ChlD1 as the location of the FRL-primary donor are discussed.