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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.

ABSTRACT: Ubiquinone is an almost universal, membrane-associated redox mediator. Its ability to accept either one or two electrons allows it to function in critical roles in biological electron transport. The redox properties of ubiquinone in vivo are determined by its environment in the binding sites of proteins and by the dihedral angle of each methoxy group relative to the ring plane. This is an attribute unique to ubiquinone among natural quinones and could account for its widespread function with many different redox complexes. In this work, we use the photosynthetic reaction center as a model system for understanding the role of methoxy conformations in determining the redox potential of the ubiquinone/semiquinone couple. Despite the abundance of X-ray crystal structures for the reaction center, quinone site resolution has thus far been too low to provide a reliable measure of the methoxy dihedral angles of the primary and secondary quinones, QA and QB. We performed 2D ESEEM (HYSCORE) on isolated reaction centers with ubiquinones (13)C-labeled at the headgroup methyl and methoxy substituents, and have measured the (13)C isotropic and anisotropic components of the hyperfine tensors. Hyperfine couplings were compared to those derived by DFT calculations as a function of methoxy torsional angle allowing estimation of the methoxy dihedral angles for the semiquinones in the QA and QB sites. Based on this analysis, the orientation of the 2-methoxy groups are distinct in the two sites, with QB more out of plane by 20-25°. This corresponds to an ?50 meV larger electron affinity for the QB quinone, indicating a substantial contribution to the experimental difference in redox potentials (60-75 mV) of the two quinones. The methods developed here can be readily extended to ubiquinone-binding sites in other protein complexes.

SUBMITTER: Taguchi AT

PROVIDER: S-EPMC3800232 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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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.

Taguchi Alexander T AT O'Malley Patrick J PJ Wraight Colin A CA Dikanov Sergei A SA

Biochemistry 20130624 27

Ubiquinone is an almost universal, membrane-associated redox mediator. Its ability to accept either one or two electrons allows it to function in critical roles in biological electron transport. The redox properties of ubiquinone in vivo are determined by its environment in the binding sites of proteins and by the dihedral angle of each methoxy group relative to the ring plane. This is an attribute unique to ubiquinone among natural quinones and could account for its widespread function with man ...[more]

PMID: 23745576

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Project description:The role of protein dynamics in the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), was studied at room temperature in isolated reaction centers (RC) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in trehalose water systems of different trehalose/water ratios. The effects of dehydration on the reaction kinetics were examined by analyzing charge recombination after different regimes of RC photoexcitation (single laser pulse, double flash, and continuous light) as well as by monitoring flash-induced electrochromic effects in the near infrared spectral region. Independent approaches show that dehydration of RC-containing matrices causes reversible, inhomogeneous inhibition of Q(A)(-)-to-Q(B) electron transfer, involving two subpopulations of RCs. In one of these populations (i.e., active), the electron transfer to Q(B) is slowed but still successfully competing with P(+)Q(A)(-) recombination, even in the driest samples; in the other (i.e., inactive), electron transfer to Q(B) after a laser pulse is hindered, inasmuch as only recombination of the P(+)Q(A)(-) state is observed. Small residual water variations ( approximately 7 wt %) modulate fully the relative fraction of the two populations, with the active one decreasing to zero in the driest samples. Analysis of charge recombination after continuous illumination indicates that, in the inactive subpopulation, the conformational changes that rate-limit electron transfer can be slowed by >4 orders of magnitude. The reported effects are consistent with conformational gating of the reaction and demonstrate that the conformational dynamics controlling electron transfer to Q(B) is strongly enslaved to the structure and dynamics of the surrounding medium. Comparing the effects of dehydration on P(+)Q(A)(-)-->PQ(A) recombination and Q(A)(-)Q(B)-->Q(A)Q(B)(-) electron transfer suggests that conformational changes gating the latter process are distinct from those stabilizing the primary charge-separated state.

Project description:In the photosynthetic reaction center from Rhodobacter sphaeroides, the primary (Q(A)) and secondary (Q(B)) electron acceptors are both ubiquinone-10, but with very different properties and functions. To investigate the protein environment that imparts these functional differences, we have applied X-band HYSCORE, a 2D pulsed EPR technique, to characterize the exchangeable protons around the semiquinone (SQ) in the Q(A) and Q(B) sites, using samples of (15)N-labeled reaction centers, with the native high spin Fe(2+) exchanged for diamagnetic Zn(2+), prepared in (1)H(2)O and (2)H(2)O solvent. The powder HYSCORE method is first validated against the orientation-selected Q-band ENDOR study of the Q(A) SQ by Flores et al. (Biophys. J.2007, 92, 671-682), with good agreement for two exchangeable protons with anisotropic hyperfine tensor components, T, both in the range 4.6-5.4 MHz. HYSCORE was then applied to the Q(B) SQ where we found proton lines corresponding to T ? 5.2, 3.7 MHz and T ? 1.9 MHz. Density functional-based quantum mechanics/molecular mechanics (QM/MM) calculations, employing a model of the Q(B) site, were used to assign the observed couplings to specific hydrogen bonding interactions with the Q(B) SQ. These calculations allow us to assign the T = 5.2 MHz proton to the His-L190 N(?)H···O(4) (carbonyl) hydrogen bonding interaction. The T = 3.7 MHz spectral feature most likely results from hydrogen bonding interactions of O1 (carbonyl) with both Gly-L225 peptide NH and Ser-L223 hydroxyl OH, which possess calculated couplings very close to this value. The smaller 1.9 MHz coupling is assigned to a weakly bound peptide NH proton of Ile-L224. The calculations performed with this structural model of the Q(B) site show less asymmetric distribution of unpaired spin density over the SQ than seen for the Q(A) site, consistent with available experimental data for (13)C and (17)O carbonyl hyperfine couplings. The implications of these interactions for Q(B) function and comparisons with the Q(A) site are discussed.

Dataset Information

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.

Publications

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.

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