ENDOR Spectroscopy Reveals Light Induced Movement of the H-Bond from Ser-L223 upon Forming the Semiquinone (Q_B^-•) in Reaction Centers fromRhodobacter sphaeroides

Abstract

Proton ENDOR spectroscopy was used to monitor local conformational changes in bacterial reaction centers (RC) associated with the electron transfer reaction DQ B → D +• Q B −• using mutant RCs capable of photo-reducing Q B at cryogenic temperatures. The charge separated state D +• Q B −• was studied in mutant RCs formed by either (i) illuminating at low temperature (77K) a sample frozen in the dark (ground state protein conformation) or (ii) illuminating at room temperature prior to and during the freezing (charge separated state protein conformation). The charge recombination rates from the two states differed greatly (>10 6 fold) as shown previously, indicating a structural change (Paddock et al (2006) Biochemistry 45, 14032 -14042). ENDOR spectra of Q B −• from both samples (35 GHz, 77K) showed three nearly identical sets of hyperfine couplings due to exchangeable protons that were similar to those for Q B −• in native RCs indicating that in all RCs, Q B −• was located at the proximal position near the metal site. In contrast, one set of H-bond couplings was observed only in the sample frozen under illumination in which the protein can relax prior to freezing. This H-bond was assigned to an interaction between the Ser-L223 hydroxyl and Q B −• based on its absence in Ser L223 → Ala mutant RCs. The Ser-L223 hydroxyl H-bond was also observed in the native RCs frozen under illumination. Thus, part of the protein relaxation in response to light induced charge separation involves the formation of an H-bond between the OH group of Ser-L223 and the anionic semiquinone Q B −• . This proton movement serves to stabilize the charge separated state and facilitate proton transfer to reduced Q B . KeywordsB-branch; bacterial reaction center; quinone movement; bacterial photosynthesis; electron transfer; nanoswitch Conformational changes in protein structure associated with electron transfer reactions play an important role in stabilizing the resultant charge separated state and determining the rates of electron transfer (1-3). In photosynthetic bacteria, reaction centers (RCs) perform the initial photochemical reactions in photosynthesis (see e.g. 4,5) resulting in the light induced reduction of a bound quinone, Q B . The reduction of Q B clearly demonstrates the influence of conformational dynamics on electron transfer. The overall reaction is shown by the solid curves in Figure 1. The first step is the initial photochemistry, a rapid (τ = 10 -10 s) light induced electron transfer from a electron donor species D (a bacteriochlorophyll dimer) along the A-branch (one of two pseudo-symmetric pathways) through a series of acceptor molecules bacteriochlorphyll, B A , bacteriopheophytin, H A , to a tightly bound quinone, Q A ( Figure 1). This is followed by a *To whom correspondence should be addressed. Phone: (858) 534-2504, Fax: (858) , is coupled to protein dynamics as shown by several experimental observations (for a review see 9). First, the rate of reaction is dependent on protein structure as illustrated by the ef...

Hydrogen Bonding and Spin Density Distribution in the QBSemiquinone of Bacterial Reaction Centers and Comparison with the QASite

“…In the proximal position, Q B is clearly hydrogen bonded through the C 4 carbonyl to N δ of His-L190 (an Fe-ligand), but a second H-bond to the C 1 carbonyl is variously indicated from the backbone NH groups of Ile-L224 and/or Gly-L225 as potential donors (Figure 1). 7–9 The hydroxyl from Ser-L223 is also thought to form a hydrogen bond with either the quinone C 1 carbonyl - especially in the semiquinone state - or Asp-L213 2,3,6,10. An additional H-bond from the peptide NH of Thr-L226 to one methoxy group was inferred from a recent structure 9.…”

“…This, however, can be addressed through the application of pulsed EPR methods,3,11 and the Q A - and Q B -sites have been amongst the most thoroughly explored by EPR techniques 10–16. To observe the SQ signals directly by these techniques it is necessary to replace the native high spin Fe 2+ with Zn 2+ , which does not perturb the function of the acceptor quinones 11,12…”

Mechanism of proton-coupled quinone reduction in Photosystem II

“…When Asp-L213 is protonated (via the same proton pathway), reorientation of the Ser-L223 hydroxyl group occurs and an H-bond forms between Ser-L223 and O QB(distal) (17)(18)(19)(20). Protonation of Q B…”

Redox Potential Tuning through Differential Quinone Binding in the Photosynthetic Reaction Center ofRhodobacter sphaeroides

“…We do not detect any significant interaction between SerL223 and the quinone within our simulations. SerL223 has been suggested by mutagenesis and EPR experiments (72–74) to be involved in proton transfer from solution to the quinone. When AspL213 is deprotonated in our simulations, SerL223 interacts with AspL213, a state that has previously been shown computationally to be favored for deprotonated AspL213 (73, 75, 76).…”