Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides

Abstract

T he biological role of bacterial reaction center (RC) membrane proteins is to convert light energy into chemical free energy. The sequential absorption of two photons by the system results into the production of the doubly reduced and doubly protonated form of the ultimate electron acceptor of the complex, a ubiquinone (Q B ). The formed Q B H 2 molecule then delivers its reducing power to the cytochrome bc1 complex, resulting in the release of protons on the periplasmic side of the membrane. The resulting transmembrane proton gradient drives ATP synthesis through the ATP synthase. The reduction of Q B coupled to the uptake of protons from the bulk is an important step shared by many systems involved either in photosynthesis or in respiratory chains (1).The three-dimensional structure of the reaction center from the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is known at atomic resolution (2-4). Three subunits with a total molecular weight of about 100 kDa compose these RCs. The transmembrane L and M subunits carry the nine pigments and cofactors: four bacteriochlorophylls, two bacteriopheophytins, two ubiquinones 10, and one non-heme iron atom. The third subunit, H, caps the reaction center on the cytoplasmic side of the membrane. The initial photochemical event induced by the absorption of a photon is the creation of the singlet excited state of a dimer of bacteriochlorophylls (P3P*), which constitutes the primary electron donor. P* is a strong reducing species that initiates the electron transfer reaction through the protein. In about 200 ps, the charge separation occurs between P and the first quinone electron acceptor, Q A , situated on the cytoplasmic side of the complex. The electron is then transferred from Q A Ϫ to a secondary quinone Q B within 10-100 s (5-7). Both Q A and Q B are deeply buried within the reaction center protein. The role of the protein in stabilizing the redox species is essential to ensure high forward electron transfer rates and to prevent charge recombinations to occur. Although chemically identical, Q A and Q B behave differently. Q A , bound to the M subunit in a relatively hydrophobic pocket, functions as one electron acceptor and is never protonated. At variance, Q B , bound to the L subunit, is surrounded by charged and polar residues and behaves as a two-electron gate, accepting sequentially two electrons from Q A and two protons from the cytoplasm. In chromatophores, the semiquinone Q B Ϫ can bind a proton below pH 6.8 (8). However, in isolated RCs, the semiquinones are not directly protonated but induce the shift of the pKas of ionizable interacting residues, which results in substoichiometric proton uptake by the protein (9, 10). The proton uptake may occur through a number of water molecules and protonatable amino acid residues situated between Q B and the cytoplasmic surface. Of main interest is to identify the dynamical and structural role of the protein that contributes to the stability of the Q A Ϫ and Q B Ϫ states and to the energetic and functiona...

pH Modulates the Quinone Position in the Photosynthetic Reaction Center from Rhodobacter sphaeroides in the Neutral and Charge Separated States

“…The computed proton uptake data in Figure 6 are similar to the experimental values, 25,26 although the shape of the experimental curve is not reproduced exactly. Between pH 4 and 8, Asp L213 and the ubiquinone (see below) almost exclusively account for the shape of the proton uptake curve.…”

“…In RCs isolated in detergent, the formation of Q B¯l eads to substoichiometric proton uptake in the pH range from 5 to 10. [25][26][27][28] At variance, it has been reported that in chromatophores from Rb. capsulatus and Rb.…”

Effect of Binding of Cd2+ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways

“…We have recently reported the stoichiometries of proton uptake by the PQ A -and PQ B -states in the L209PY and L209PF mutants (25). We have shown that the replacement of L209Pro by aromatic residues is likely to affect the functioning of protonation groups lying between Q B and the water phase and which role is to provide protons to the close environment of Q B (i.e., to the ultimate proton donors to Q B (L212Glu and L213Asp)).…”

Evidence for Delocalized Anticooperative Flash Induced Proton Binding as Revealed by Mutants at the M266His Iron Ligand in Bacterial Reaction Centers

“…The scheme represents the reaction center structure from R. sphaeroides in the quinone region (from (53)). The involvement in the cluster of, at least, L212Glu, L213Asp, L209Pro, M17Asp, L210Asp, M266His, and L234Glu has previously been shown (2,25,30,33,71,85). M249Ala is also mentioned here since it has been shown that when Tyr replaces Ala, its side chain allows it to connect to the interactive network of water molecules and polar residues that spans the region between Q A and Q B on the cytoplasmic side of the complex (29).…”