Hydrogen Bonding between the Q_BSite Ubisemiquinone and Ser-L223 in the Bacterial Reaction Center: A Combined Spectroscopic and Computational Perspective

Abstract

In the QB site of the Rba. sphaeroides photosynthetic reaction centre the donation of a hydrogen bond from the hydroxyl group of Ser-L223 to the ubisemiquinone formed after the first flash is debatable. In this study we use a combination of spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations to comprehensively explore this topic. We show that ENDOR, ESEEM and HYSCORE spectroscopic differences between the mutant L223SA and the wild type sample (WT) are negligible, indicating only minor perturbations in the ubisemiquinone spin density for the mutant sample. Qualitatively this suggests that a strong hydrogen bond does not exist in the WT between the Ser-L223 hydroxyl group and the semiquinone O1 atom, as removal of this hydrogen bond in the mutant should cause a significant redistribution of spin density in the semiquinone. We show quantitatively, using QM/MM calculations, that a WT model in which the Ser-L223 hydroxyl group is rotated to prevent hydrogen bond formation with the O1 atom of the semiquinone predicts negligible change for the L223SA mutant. This, together with the better agreement between key QM/MM calculated and experimental hyperfine couplings for the non-hydrogen bonded model, leads us to conclude that no strong hydrogen bond is formed between the Ser-L223 hydroxyl group and the semiquinone O1 atom after the first flash. The implications of this finding for quinone reduction in photosynthetic reaction centres are discussed.

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

“…The orientation of SerL223 has been shown to depend on several factors. Some crystallographic structures suggest that SerL223 interacts via a hydrogen bond to the quinone (19, 20, 66) whereas electrostatics calculations suggest that deprotonated AspL213 might be the significant partner to SerL223 (73, 75, 76), consistent with what we observe in our deprotonated simulations. In our additional simulation of UbiQ in the Q A and Q B sites with protonated AspL213, SerL223 shifts from interacting with AspL213 to ArgL217 (Table S5).…”

“…3MeO-Q lacks this 2-methoxy oxygen, and therefore exhibits weaker binding to the Q B site than either UbiQ or 2MeO-Q, consistent with its observed non-functionality as an electron acceptor in Q B (12), the weak binding shown through TI calculations and spontaneous unbinding in equilibrium, its poor competition for the Q B site, and lack of any observable ${\mathrm{Q}}_{\mathrm{A}}^{-}{\mathrm{Q}}_{\mathrm{B}}^{-}$ biradical signal in the CW EPR spectrum upon borohydride reduction at pH 10.5. In addition, we determined that SerL223 does not form direct interactions with the neutral quinone either with or without the protonation of AspL213, suggesting that its role as the proton donor in the formation of quinol is mediated by the formation of the semiquinone or biradical, as has been postulated previously (75). These mechanistic details may not be unique to the RC, and are a clear example of the detailed interactions that lie at the heart of protein redox chemistry.…”

“…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). The most significant O1 interactions are formed with IleL224 across all three quinones simulated (Table 3 and Figs.…”

Conformational Differences between the Methoxy Groups of QA and QB Site Ubisemiquinones in Bacterial Reaction Centers: A Key Role for Methoxy Group Orientation in Modulating Ubiquinone Redox Potential

“…The shape of the ridge is described by the general equation ν α =(Qν β 2 + G) 1/2 , where Q and G are coefficients that are functions of a , T and ν C . 39,40 This lineshape transforms to a straight line segment in the coordinates (ν α ) 2 vs . (ν β ) 2 .…”

Defining a direction: Electron transfer and catalysis in Escherichia coli complex II enzymes

“…Even an attempt to identify H-bond donors around the [2Fe-2S] cluster using comprehensive 2D ESEEM spectroscopy was not able to unambiguously assign hydrogen bonded nitrogens because of the complex character of the orientation-selected spectra and dependence of the simulated spectra from a large number of parameters [65]. A more promising approach applied recently to iron-sulfur clusters [66, 67] and semiquinones [68, 69] is based on selective 15 N isotope labeling of residues of interest using a set of auxotrophs in a commonly used E. coli expression strain C43(DE3), a derivative of E. coli BL21(DE3) [66, 67]. The nitrogen H-bond donors carry unpaired spin density producing an isotropic hyperfine coupling and were identified unambiguously using 15 N selective labeling approach in conjunction with 2D ESEEM.…”