Proton uptake associated with the reduction of the primary quinone Q(A) influences the binding site of the secondary quinone Q(B) in Rhodopseudomonas viridis photosynthetic reaction centers

Previously, two binding sites for the secondary quinone Q(B) in the photosynthetic reaction center (RC) from Rhodopseudomonas viridis were identified by X-ray crystallography, a 'proximal' binding site close to the non-heme iron, and a 'distal' site, displaced by 4.2 A along the path of the isoprenoid tail [C.R.D. Lancaster and H. Michel, Structure 5 (1997) 1339-1359]. The quinone ring planes in the two sites differ by roughly a 180 degrees rotation around the isoprenoid tail. Here we present molecular dynamics simulations, which support the theory of a spontaneous transfer of Q(B) between the distal site and the proximal site. In contrast to earlier computational studies on RCs, the molecular dynamics simulations of Q(B) migration resulted in a proximal Q(B) binding pattern identical to that of the crystallographic findings. Also, we demonstrate that the preference towards the proximal Q(B) location is not necessarily attributed to reduction of Q(B) to the semiquinone, but already to the preceding reduction of the primary quinone Q(A) and resulting protonation changes in the protein. Energy mapping of the Q(B) binding pocket indicates that the quinone ring rotation required for completion of the transfer between the two sites is improbable at the distal or proximal binding sites due to high potential barriers, but may be possible at a newly identified position near the distal binding site.     

Exploring the energy profile of the Q(A)(-) to Q(B) electron transfer reaction in bacterial photosynthetic reaction centers: pH dependence of the conformational gating step

Both large- and small-scale conformational changes are needed as proteins carry out reactions. However, little is known about the identity, energy of, and barriers between functional substates on protein reaction coordinates. In isolated bacterial photosynthetic reaction centers, the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), is rate limited by conformational changes at low pH and by proton binding at high pH. The kinetics and thermodynamics of this reaction were determined between 200 and 300 K from pH 6 to pH 10.5. A model with two substates of the reactant, P(+)Q(A)(-)Q(B), one protonated (state A) and one unprotonated (alpha), and one state of the product, P(+)Q(A)Q(B)(-) (B), was able to simulate the dependence of the rate on temperature and pH fairly well. The equilibrium between the three states were measured in situ at each temperature. Proton binding (alpha to A transition) has a favorable DeltaH and unfavorable DeltaS as does the conformational changes required for electron transfer at low pH (A to B). The pK for the A to alpha transition is 9.7 at room temperature, consistent with previous measurements, and equivalent to 13.5 at 200 K. The activation barriers were determined for each transition. Both the alpha to A and the A to B transitions are limited primarily by the activation enthalpy with modest DeltaS.     

An isotope-edited FTIR investigation of the role of Ser-L223 in binding quinone (QB) and semiquinone (QB-) in the reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (Q(B)) site. Several nearby residues are important for both binding and redox chemistry involved in the light-induced conversion from Q(B) to quinol Q(B)H(2). Ser-L223 is one of the functionally important residues located near Q(B). To obtain information on the interaction between Ser-L223 and Q(B) and Q(B)(-), isotope-edited Q(B)(-)/Q(B) FTIR difference spectra were measured in a mutant RC in which Ser-L223 is replaced with Ala and compared to the native RC. The isotope-edited IR fingerprint spectra for the C=O [see text] and C=C [see text] modes of Q(B) (Q(B)(-)) in the mutant are essentially the same as those of the native RC. These findings indicate that highly equivalent interactions of Q(B) and Q(B)(-) with the protein occur in both native and mutant RCs. The simplest explanation of these results is that Ser-L223 is not hydrogen bonded to Q(B) or Q(B)(-) but presumably forms a hydrogen bond to a nearby acid group, preferentially Asp-L213. The rotation of the Ser OH proton from Asp-L213 to Q(B)(-) is expected to be an important step in the proton transfer to the reduced quinone. In addition, the reduced quinone remains firmly bound, indicating that other distinct hydrogen bonds are more important for stabilizing Q(B)(-). Implications on the design features of the Q(B) binding site are discussed.     

Kinetics of electron transfer from Q(a) to Q(b) in photosystem II

The oxidation kinetics of the reduced photosystem II electron acceptor Q(A)(-) was investigated by measurement of the chlorophyll fluorescence yield transients on illumination of dark-adapted spinach chloroplasts by a series of saturating flashes. Q(A)(-) oxidation depends on the occupancy of the "Q(B) binding site", where this reaction reduces plastoquinone to plastoquinol in two successive photoreactions. The intermediate, one-electron-reduced plastosemiquinone anion Q(B)(-) remains tightly bound, and its reduction by Q(A)(-) may proceed with simple first-order kinetics. The next photoreaction, in contrast, may find the Q(B) binding site occupied by a plastoquinone, a plastoquinol, or neither of the two, resulting in heterogeneous Q(A)(-) oxidation kinetics. The assumption of monophasic Q(B)(-) reduction kinetics is shown to allow unambiguous decomposition of the observed multiphasic Q(A)(-) oxidation. At pH 6.5 the time constant for Q(A)(-) oxidation was found to be 0.2-0.4 ms with Q(B) in the site, 0.6-0.8 ms with Q(B)(-) in the site, 2-3 ms when the site is empty and Q(B) has to bind first, and of the order of 0.1 s if the site is temporarily blocked by the presence of Q(B)H(2) or other low-affinity inhibitors such as carbonyl cyanide m-chlorophenylhydrazone (CCCP). Effects of pH and H(2)O/D(2)O exchange were found to be remarkably nonspecific. No influence of the S-states could be demonstrated.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ(-)B and free energy and kinetic relations between Q(-)AQB and QAQ(-)B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: (Formula: see text). We found that the charge recombination pathway of D+QAQ(-)B proceeds via the intermediate state D+Q(-)AQB, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product kAD-times the fraction of reaction centers in the Q-AQB state) with the observed recombination rate, kobsD+----D. The kinetic measurements were used to obtain the pH dependence (6.1 smaller than or equal to pH smaller than or equal to 11.7) of the free energy difference between the states Q(-)AQB and QAQ(-)B. At low pH (less than 9) QAQ(-)B is stabilized relative to Q(-)AQB by 67 meV, whereas at high pH Q(-)AQB is energetically favored. Both Q(-)A and Q(-)B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q(-)B provides the driving force for the forward electron transfer.     

Conformation-driven and semiquinone-gated proton-pump mechanism in the NADH-ubiquinone oxidoreductase (complex I)

A novel mechanism for proton/electron transfer is proposed for NADH-quinone oxidoreductase (complex I) based on the following findings: (1) EPR signals of the protein-bound fast-relaxing semiquinone anion radicals (abbreviated as Q(Nf)-) are observable only in the presence of proton-transmembrane electrochemical potential; (2) Iron-sulfur cluster N2 and Q(Nf)- are directly spin-coupled; and (3) The projection of the interspin vector extends only 5A along the membrane normal [Yano, T., Dunham, W.R. and Ohnishi, T. (2005) Biochemistry, 44, 1744-1754]. We propose that the proton pump is operated by redox-driven conformational changes of the quinone binding protein. In the input state, semiquinone is reduced to quinol, acquiring two protons from the N (matrix) side of the mitochondrial inner membrane and an electron from the low potential (NADH) side of the respiratory chain. A conformational change brings the protons into position for release at the P (inter-membrane space) side of the membrane via a proton-well. Concomitantly, an electron is donated to the quinone pool at the high potential side of the coupling site. The system then returns to the original state to repeat the cycle. This hypothesis provides a useful frame work for further investigation of the mechanism of proton translocation in complex I.     

-deltaG(AB) and pH dependence of the electron transfer from P(+)Q(A)(-)Q(B) toP(+)Q(A)Q(B)(-) in Rhodobacter sphaeroides reaction centers

The electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) can occur in two phases with a well-characterized 100 micros component (tau(2)) and a faster process occurring in less than 10 micros (tau(1)). The fast reaction is clearly seen when the native ubiquinone-10 at Q(A) is replaced with naphthoquinones. The dependence of tau(1) on the free-energy difference between the P(+)Q(A)(-)Q(B) and P(+)Q(A)Q(B)(-) states (-) and on the pH was measured using naphthoquinones with different electrochemical midpoint potentials as Q(A) in Rhodobacter sphaeroides reaction centers (RCs) and in RCs where - is changed by mutation of M265 in the Q(A) site from Ile to Thr (M265IT). Q(B) was ubiquinone (UQ(B)) in all cases. Electron transfer was measured by using the absorption differences of the naphthosemiquinone at Q(A) and the ubisemiquinone at Q(B) between 390 and 500 nm. As - was changed from -90 to -250 meV tau(1) decreased from 29 to 0.2 micros. The free-energy dependence of tau(1) provides a reorganization energy of 850 +/- 100 meV for the electron transfer from Q(A)(-) to Q(B). The slower reaction at tau(2) is free-energy independent, so processes other than electron transfer determine the observed rate. The fraction of the reaction at tau(1) increases with increasing driving force and is 100% of the reaction when - is approximately 100 meV more favorable than in the native RCs with ubiquinone as Q(A). The fast phase, tau(1), is pH independent from pH 6 to 11 while tau(2) slows above pH 9. As the Q(A) isoprene tail length is increased from 2 to 10 isoprene units the fraction at tau(1) decreases. However, tau(1), tau(2), and the fraction of the reaction in each phase are independent of the tail length of UQ(B).     

Exploring the energy landscape for Q(A)(-) to Q(B) electron transfer in bacterial photosynthetic reaction centers: effect of substrate position and tail length on the conformational gating step

The ability to initiate reactions with a flash of light and to monitor reactions over a wide temperature range allows detailed analysis of reaction mechanisms in photosynthetic reaction centers (RCs) of purple bacteria. In this protein, the electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) is rate-limited by conformational changes rather than electron tunneling. Q(B) movement from a distal to a proximal site has been proposed to be the rate-limiting change. The importance of quinone motion was examined by shortening the Q(B) tail from 50 to 5 carbons. No change in rate was found from 100 to 300 K. The temperature dependence of the rate was also measured in three L209 proline mutants. Under conditions where Q(B) is in the distal site in wild-type RCs, it is trapped in the proximal site in the Tyr L209 mutant [Kuglstatter, A., et al. (2001) Biochemistry 40, 4253-4260]. The electron transfer slows at low temperature for all three mutants as it does in wild-type protein, indicating that conformational changes still limit the reaction rate. Thus, Q(B) movement is unlikely to be the sole, rate-limiting conformational gating step. The temperature dependence of the reaction in the L209 mutants differs somewhat from wild-type RCs. Entropy-enthalpy compensation reduces the difference in rates and free energy changes at room temperature.     

Quinone (QB) reduction by B-branch electron transfer in mutant bacterial reaction centers from Rhodobacter sphaeroides: quantum efficiency and X-ray structure

The photosynthetic reaction center (RC) from purple bacteria converts light into chemical energy. Although the RC shows two nearly structurally symmetric branches, A and B, light-induced electron transfer in the native RC occurs almost exclusively along the A-branch to a primary quinone electron acceptor Q(A). Subsequent electron and proton transfer to a mobile quinone molecule Q(B) converts it to a quinol, Q(B)H(2). We report the construction and characterization of a series of mutants in Rhodobacter sphaeroides designed to reduce Q(B) via the B-branch. The quantum efficiency to Q(B) via the B-branch Phi(B) ranged from 0.4% in an RC containing the single mutation Ala-M260 --> Trp to 5% in a quintuple mutant which includes in addition three mutations to inhibit transfer along the A-branch (Gly-M203 --> Asp, Tyr-M210 --> Phe, Leu-M214 --> His) and one to promote transfer along the B-branch (Phe-L181 --> Tyr). Comparing the value of 0.4% for Phi(B) obtained in the AW(M260) mutant, which lacks Q(A), to the 100% quantum efficiency for Phi(A) along the A-branch in the native RC, we obtain a ratio for A-branch to B-branch electron transfer of 250:1. We determined the structure of the most effective (quintuple) mutant RC at 2.25 A (R-factor = 19.6%). The Q(A) site did not contain a quinone but was occupied by the side chain of Trp-M260 and a Cl(-). In this structure a nonfunctional quinone was found to occupy a new site near M258 and M268. The implications of this work to trap intermediate states are discussed.     

Quinone (Q(B)) binding site and protein stuctural changes in photosynthetic reaction center mutants at Pro-L209 revealed by vibrational spectroscopy

The effect of substituting Pro-L209 with Tyr, Phe, Glu, and Thr in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides was investigated by monitoring the light-induced FTIR absorption changes associated with the photoreduction of the secondary quinone Q(B). Pro-L209 is close to a chain of ordered water molecules connecting Q(B) to the bulk phase. In wild-type RCs, two distinct main Q(B) binding sites (distal and proximal to the non-heme iron) have been described in the literature. The X-ray structures of the mutant RCs Pro-L209 --> Tyr, Pro-L209 --> Phe, and Pro-L209 --> Glu have revealed that Q(B) occupies a proximal, intermediate, and distal position, respectively [Kuglstatter, A., Ermler, U., Michel, H., Baciou, L., and Fritzsch, G. (2001) Biochemistry 40, 4253-4260]. FTIR absorption changes associated with the reduction of Q(B) in Pro-L209 --> Phe RCs reconstituted with (13)C-labeled ubiquinone show a highly specific IR fingerprint for the C=O and C=C modes of Q(B) upon selective labeling at C(1) or C(4). This IR fingerprint is similar to those of wild-type RCs and the Pro-L209 --> Tyr mutant [Breton, J., Boullais, C., Mioskowski, C., Sebban, P., Baciou, L., and Nabedryk, E. (2002) Biochemistry 41, 12921-12927], demonstrating that equivalent interactions occur between neutral Q(B) and the protein in wild-type and mutant RCs. It is concluded that in all RCs, neutral Q(B) in its functional state occupies a unique binding site which is favored to be the proximal site. This result contrasts with the multiple Q(B) binding sites found in crystal structures. With respect to wild-type RCs, the largest FTIR spectral changes upon Q(B)(-) formation are observed for the Phe-L209 and Tyr-L209 mutants which undergo similar protein structural changes and perturbations of the semiquinone modes. Smaller changes are observed for the Glu-L209 mutant, while the vibrational properties of the Thr-L209 mutant are essentially the same as those for native RCs.     

Cumulant analysis of charge recombination kinetics in bacterial reaction centers reconstituted into lipid vesicles

The kinetics of charge recombination between the primary photoxidized donor (P(+)) and the secondary reduced quinone acceptor (Q(B)(-)) have been studied in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides incorporated into lecithin vesicles containing large ubiquinone pools over the temperature range 275 K = (50 +/- 15) nm). Following these premises, we describe the kinetics of P(+)Q(B)(-) recombination with a truncated cumulant expansion and relate it to P(Q) and to the free energy changes for Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer (DeltaG(AB)(o)) and for quinone binding (DeltaG(bind)(o)) at Q(B). The model accounts well for the temperature and quinone dependence of the charge recombination kinetics, yielding DeltaG(AB)(o) = -7.67 +/- 0.05 kJ mol(-1) and DeltaG(bind)(o) = -14.6 +/- 0.6 kJ mol(-1) at 298 K.     

Proton and electron transfer in bacterial reaction centers

The bacterial reaction center couples light-induced electron transfer to proton pumping across the membrane by reactions of a quinone molecule Q(B) that binds two electrons and two protons at the active site. This article reviews recent experimental work on the mechanism of the proton-coupled electron transfer and the pathways for proton transfer to the Q(B) site. The mechanism of the first electron transfer, k((1))(AB), Q(-)(A)Q(B)-->Q(A)Q(-)(B), was shown to be rate limited by conformational gating. The mechanism of the second electron transfer, k((2))(AB), was shown to involve rapid reversible proton transfer to the semiquinone followed by rate-limiting electron transfer, H(+)+Q(-)(A)Q(-)(B) ifQ(-)(A)Q(B)H-->Q(A)(Q(B)H)(-). The pathways for transfer of the first and second protons were elucidated by high-resolution X-ray crystallography as well as kinetic studies showing changes in the rate of proton transfer due to site directed mutations and metal ion binding.     

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

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.     

Characterization of mutations in crucial residues around the Q(o) binding site of the cytochrome bc complex from Paracoccus denitrificans

The protonation state of residues around the Q(o) binding site of the cytochrome bc(1) complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site-directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme b(L) edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild-type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Q(o) site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P.denitrificans as well. The protonated residue E295 is proposed to change the hydrogen-bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X-ray structures.     

Ubiquinone (coenzyme Q10) binding sites: low dielectric constant of the gate prevents the escape of the semiquinone

The photosynthetic reaction center (RC) from purple bacteria is frequently used as a model for the interaction of ubiquinones (coenzyme Q) with membrane proteins. Single-turnover flash activation of RC leads to formation of the semiquinone (SQ) of the secondary acceptor quinone after odd flashes and quinol after even flashes. The ubiquinol escapes the binding site in 1 ms, while the SQ does not leave the binding site for at least 5 min. Observed difference between these times suggests a large energetic barrier for the SQ. However, high apparent dielectric constant in the vicinity of the quinone ring (>or=25) results in a relatively small electrostatic energy of SQ stabilization. To resolve this apparent contradiction I suggest that a significant part of the kinetic stabilization of the SQ is achieved by the special topology of the binding site in which quinone can exit the binding site only by moving its headgroup toward the center of the membrane. The large energetic penalty of transferring the charged headgroup to the membrane dielectric can explain the observed kinetic stability of the SQ.     

Effect of metal binding on electrogenic proton transfer associated with reduction of the secondary electron acceptor (QB) in Rhodobacter sphaeroides chromatophores

The influence of metal ion (Cd(2+), Zn(2+), Ni(2+)) binding on the electrogenic phases of proton transfer connected with reduction of quinone Q(B) in chromatophores from Rhodobacter sphaeroides was studied by time-resolved electric potential changes. In the presence of metals, the electrogenic transients associated with proton transfer on first and second flash at pH 8 were found to be slower by factors of 3-6. This is essentially the same effect of metal binding that was observed on optical transients in isolated reaction centers (RC), where the metal ion was shown to inhibit proton transfer [Paddock, M. L., Graige, M. S., Feher, G., and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188]. The effect of metal binding on the kinetics in chromatophores is, therefore, similarly attributed to inhibition of proton uptake, which becomes rate-limiting. A striking observation was an increase in the amplitude of the electrogenic proton-uptake phase after the first flash with bound metal ion. We attribute this to a loss of internal proton rearrangement, requiring that the protons that stabilize Q(B)(-) come from solution. In mutant RCs, in which His-H126 and His-H128 are replaced with Ala, the apparent binding of Cd(2+) and Ni(2+) was decreased, showing that the binding site of these metal ions is the same as found in RC crystals [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. Therefore, the unique proton entry point near His-H126, His-H128, and Asp-M17 that was identified in isolated RCs is also the entry point in chromatophores.     

X-ray structure analyses of photosynthetic reaction center variants from Rhodobacter sphaeroides: structural changes induced by point mutations at position L209 modulate electron and proton transfer

The structures of the reaction center variants Pro L209 --> Tyr, Pro L209 --> Phe, and Pro L209 --> Glu from the photosynthetic purple bacterium Rhodobacter sphaeroides have been determined by X-ray crystallography to 2.6-2.8 A resolution. These variants were constructed to interrupt a chain of tightly bound water molecules that was assumed to facilitate proton transfer from the cytoplasm to the secondary quinone Q(B) [Baciou, L., and Michel, H. (1995) Biochemistry 34, 7967-7972]. However, the amino acid exchanges Pro L209 --> Tyr and Pro L209 --> Phe do not interrupt the water chain. Both aromatic side chains are oriented away from this water chain and interact with three surrounding polar side chains (Asp L213, Thr L226, and Glu H173) which are displaced by up to 2.6 A. The conformational changes induced by the bulky aromatic rings of Tyr L209 and Phe L209 lead to unexpected displacements of Q(B) compared to the wild-type protein. In the structure of the Pro L209 --> Tyr variant, Q(B) is shifted by approximately 4 A and is now located at a position similar to that reported for the wild-type reaction center after illumination [Stowell, M. H. B., et al. (1997) Science 276, 812-816]. In the Pro L209 --> Phe variant, the electron density map reveals an intermediate Q(B) position between the binding sites of the wild-type protein in the dark and the Pro L209 --> Tyr protein. In the Pro L209 --> Glu reaction center, the carboxylic side chain of Glu L209 is located within the water chain, and the binding site of Q(B) remains unchanged compared to the wild-type structure.     

Specific triazine resistance in bacterial reaction centers induced by a single mutation in the QA protein pocket

We report here the first example of a reaction center mutant from Rhodobacter sphaeroides, where a single mutation (M266His --> Leu) taking place in the primary quinone protein pocket confers selective resistance to triazine-type inhibitors (terbutryn, ametryn, and atrazine), which bind in the secondary quinone protein pocket, at about 13 A from the mutation site. The M266His --> Leu mutation involves one of the iron atom ligands. Interestingly, neither the secondary quinone nor the highly specific inhibitor stigmatellin binding affinities are affected by the mutation. It is noticeable that in the M266His --> Ala mutant a nativelike behavior in observed. We suggest that the long side chain of Leu in position M266 may lack space to accommodate in the Q(A) pocket therefore transferring its hindrance to the Q(B) pocket. This may occur via the structural feature formed by the Q(A)-M219His-Fe-L190His-inhibitor (or Q(B)) connection, pushing L189Leu and/or L229Ile in closer contact to the triazine molecules, therefore decreasing their bindings. This opens the possibility to finely tune, in reaction center proteins, the affinity for herbicides by designing mutations distant from their binding sites.