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Modeling binding kinetics at the Q(A) site in bacterial reaction centers.

ABSTRACT: Bacterial reaction centers (RCs) catalyze a series of electron-transfer reactions reducing a neutral quinone to a bound, anionic semiquinone. The dissociation constants and association rates of 13 tailless neutral and anionic benzo- and naphthoquinones for the Q(A) site were measured and compared. The K(d) values for these quinones range from 0.08 to 90 microM. For the eight neutral quinones, including duroquinone (DQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ(0)), the quinone concentration and solvent viscosity dependence of the association rate indicate a second-order rate-determining step. The association rate constants (k(on)) range from 10(5) to 10(7) M(-)(1) s(-)(1). Association and dissociation rate constants were determined at pH values above the hydroxyl pK(a) for five hydroxyl naphthoquinones. These negatively charged compounds are competitive inhibitors for the Q(A) site. While the neutral quinones reach equilibrium in milliseconds, anionic hydroxyl quinones with similar K(d) values take minutes to bind or dissociate. These slow rates are independent of ionic strength, solvent viscosity, and quinone concentration, indicating a first-order rate-limiting step. The anionic semiquinone, formed by forward electron transfer at the Q(A) site, also dissociates slowly. It is not possible to measure the association rate of the unstable semiquinone. However, as the protein creates kinetic barriers for binding and releasing anionic hydroxyl quinones without greatly increasing the affinity relative to neutral quinones, it is suggested that the Q(A) site may do the same for anionic semiquinone. Thus, the slow semiquinone dissociation may not indicate significant thermodynamic stabilization of the reduced species in the Q(A) site.

SUBMITTER: Madeo J

PROVIDER: S-EPMC2727067 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Modeling binding kinetics at the Q(A) site in bacterial reaction centers.

Madeo Jennifer J Gunner M R MR

Biochemistry 20050801 33

Bacterial reaction centers (RCs) catalyze a series of electron-transfer reactions reducing a neutral quinone to a bound, anionic semiquinone. The dissociation constants and association rates of 13 tailless neutral and anionic benzo- and naphthoquinones for the Q(A) site were measured and compared. The K(d) values for these quinones range from 0.08 to 90 microM. For the eight neutral quinones, including duroquinone (DQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ(0)), the quinone concentratio ...[more]

PMID: 16101283

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Project description:Previously we have shown that ONIOM type (QM/MM) calculations can be used to simulate isotope edited FTIR difference spectra for neutral ubiquinone in the QA binding site in Rhodobacter sphaeroides photosynthetic reaction centers. Here we considerably extend upon this previous work by calculating isotope edited FTIR difference spectra for reaction centers with a variety of unlabeled and (18)O labeled foreign quinones incorporated into the QA binding site. Isotope edited spectra were calculated for reaction centers with 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone (MQ0), 2,3,5,6-tetramethyl-1, 4-benzoquinone (duroquinone, DQ), and 2,3-dimethyl-l,4-naphthoquinone (DMNQ) incorporated, and compared to corresponding experimental spectra. The calculated and experimental spectra agree well, further demonstrating the utility and applicability of our ONIOM approach for calculating the vibrational properties of pigments in protein binding sites. The normal modes that contribute to the bands in the calculated spectra, their composition, frequency, and intensity, and how these quantities are modified upon (18)O labeling, are presented. This computed information leads to a new and more detailed understanding/interpretation of the experimental FTIR difference spectra. Hydrogen bonding to the carbonyl groups of the incorporated quinones is shown to be relatively weak. It is also shown that there is some asymmetry in hydrogen bonding, accounting for 10-13 cm(-1) separation in the frequencies of the carbonyl vibrational modes of the incorporated quinones. The extent of asymmetry in H-bonding could only be established by considering the spectra for various types of quinones incorporated into the QA binding site. The quinones listed above are "tail-less." Spectra were also calculated for reaction centers with corresponding "tail" containing quinones incorporated, and it is found that replacement of the quinone methyl group by a phytyl or prenyl chain does not alter ONIOM calculated spectra.

Project description:Reaction centers (RCs) are integral membrane proteins that undergo a series of electron transfer reactions during the process of photosynthesis. In the Q(A) site of RCs from Rhodobacter sphaeroides, ubiquinone-10 is reduced, by a single electron transfer, to its semiquinone. The neutral quinone and anionic semiquinone have similar affinities, which is required for correct in situ reaction thermodynamics. A previous study showed that despite similar affinities, anionic quinones associate and dissociate from the Q(A) site at rates ≈10(4) times slower than neutral quinones indicating that anionic quinones encounter larger binding barriers (Madeo, J.; Gunner, M. R. Modeling binding kinetics at the Q(A) site in bacterial reaction centers. Biochemistry 2005, 44, 10994-11004). The present study investigates these barriers computationally, using steered molecular dynamics (SMD) to model the unbinding of neutral ground state ubiquinone (UQ) and its reduced anionic semiquinone (SQ(-)) from the Q(A) site. In agreement with experiment, the SMD unbinding barrier for SQ(-) is larger than for UQ. Multi Conformational Continuum Electrostatics (MCCE), used here to calculate the binding energy, shows that SQ(-) and UQ have comparable affinities. In the Q(A) site, there are stronger binding interactions for SQ(-) compared to UQ, especially electrostatic attraction to a bound non-heme Fe(2+). These interactions compensate for the higher SQ(-) desolvation penalty, allowing both redox states to have similar affinities. These additional interactions also increase the dissociation barrier for SQ(-) relative to UQ. Thus, the slower SQ(-) dissociation rate is a direct physical consequence of the additional binding interactions required to achieve a Q(A) site affinity similar to that of UQ. By a similar mechanism, the slower association rate is caused by stronger interactions between SQ(-) and the polar solvent. Thus, stronger interactions for both the unbound and bound states of charged and highly polar ligands can slow their binding kinetics without a conformational gate. Implications of this for other systems are discussed.

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

Modeling binding kinetics at the Q(A) site in bacterial reaction centers.

Publications

Modeling binding kinetics at the Q(A) site in bacterial reaction centers.

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