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.     

Electrogenic proton transfer in Rhodobacter sphaeroides reaction centers: effect of coenzyme Q(10) substitution by decylubiquinone in the Q(B) binding site.

An electrometric technique was used to investigate the effect of coenzyme Q(10) (UQ), substitution by decylubiquinone (dQ) at the Q(B) binding site of reaction centers (UQ-RC and dQ-RC, respectively) on the electrogenic proton transfer kinetics upon Q(B) reduction in Rhodobacter sphaeroides chromatophores. Unlike dQ-RC, the kinetics of the second flash-induced proton uptake in UQ-RC clearly deviated from the mono-exponential one. The activation energy (about 30 kJ/mol) and the pH profile of the kinetics in dQ-RC were similar to those in UQ-RC, with the power law approximation used in the latter case. The interpretation of the data presumed the quinone translocation between the two binding positions within the Q(B) site. It is proposed that the native isoprenyl side chain (in contrast to decyl chain) favors the equilibrium binding of neutral quinone at the redox-active 'proximal' position, but causes a higher barrier for the hydroquinone movement from 'proximal' to 'distal' position.     

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.     

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.     

-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).     

Tunnel dynamics of quinone derivatives and its coupling to protein conformational rearrangements in respiratory complex I

Respiratory complex I in mitochondria and bacteria catalyzes the transfer of electrons from NADH to quinone (Q). The free energy available from the reaction is used to pump protons and to establish a membrane proton electrochemical gradient, which drives ATP synthesis. Even though several high-resolution structures of complex I have been resolved, how Q reduction is linked with proton pumping, remains unknown. Here, microsecond long molecular dynamics (MD) simulations were performed on Yarrowia lipolytica complex I structures where Q molecules have been resolved in the ~30 Å long Q tunnel. MD simulations of several different redox/protonation states of Q reveal the coupling between the Q dynamics and the restructuring of conserved loops and ion pairs. Oxidized quinone stabilizes towards the N2 FeS cluster, a binding mode not previously described in Yarrowia lipolytica complex I structures. On the other hand, reduced (and protonated) species tend to diffuse towards the Q binding sites closer to the tunnel entrance. Mechanistic and physiological relevance of these results are discussed.     

Energetics of quinone-dependent electron and proton transfers in Rhodobacter sphaeroides photosynthetic reaction centers

Proteins bind redox cofactors, modifying their electrochemistry and affinity by specific interactions of the binding site with each cofactor redox state. Photosynthetic reaction centers from Rhodobacter sphaeroides have three ubiquinone-binding sites, Q(A), and proximal and distal Q(B) sites. Ubiquinones, which can be doubly reduced and bind 2 protons, have 9 redox states. However, only Q and Q(-) are seen in the Q(A) site and Q, Q(-), and QH(2) in the proximal Q(B) site. The distal Q(B) function is uncertain. Multiple conformation continuum electrostatics (MCCE) was used to compare the ubiquinone electrochemical midpoints (E(m)) and pK(a) values at these three sites. At pH 7, the Q(A)/Q(A)(-) E(m) is -40 mV and proximal Q(B)/Q(B)(-) -10 mV in agreement with the experimental values (assuming a solution ubiquinone E(m) of -145 mV). Q(B) reduction requires changes in nearby residue protonation and SerL223 reorientation. The distal Q(B)/Q(B)(-) E(m) is a much more unfavorable -260 mV. Q(A) and proximal Q(B) sites generally stabilize species with a -1 charge, while the distal Q(B) site prefers binding neutral species. In each site, the dianion is destabilized because favorable interactions with the residues and backbone increase with charge (q), while the unfavorable loss of solvation energy increases with q(2). Therefore, proton binding before a second reduction, forming QH and then QH(-), is always preferred to forming the dianion (Q(-)(2)). The final product QH(2) is higher in energy at the proximal Q(B) site than in solution; therefore, it binds poorly, favoring release. In contrast, QH(2) binds more tightly than Q at the distal Q(B) site.     

Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.     

Coupling of quinone dynamics to proton pumping in respiratory complex I

Respiratory complex I (NADH:quinone oxidoreductase) plays a central role in generating the proton electrochemical gradient in mitochondrial and bacterial membranes, which is needed to generate ATP. Several high-resolution structures of complex I have been determined, revealing its intricate architecture and complementing the biochemical and biophysical studies. However, the molecular mechanism of long-range coupling between ubiquinone (Q) reduction and proton pumping is not known. Computer simulations have been applied to decipher the dynamics of Q molecule in the ~30 Å long Q tunnel. In this short report, we discuss the binding and dynamics of Q at computationally predicted Q binding sites, many of which are supported by structural data on complex I. We suggest that the binding of Q at these sites is coupled to proton pumping by means of conformational rearrangements in the conserved loops of core subunits.     

Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides reconstituted with anthraquinone as primary quinone Q(A)

In the photosynthetic reaction centre (RC) from the purple bacterium Rhodobacter sphaeroides, the primary quinone, a ubiquinone-10 (Q(A)), has been substituted by anthraquinone. Three-dimensional crystals have been grown from the modified RC and its structure has been determined by X-ray crystallography to 2.4 A resolution. The bindings of the head-group from ubiquinone-10 and of the anthraquinone ring are very similar. In particular, both rings are parallel to each other and the hydrogen bonds connecting the native ubiquinone-10 molecule to AlaM260 and HisM219 are conserved in the anthraquinone containing RC. The space of the phytyl tail missing in the anthraquinone exchanged RC is occupied by the alkyl chain of a detergent molecule. Other structural changes of the Q(A)-binding site are within the limit of resolution. Our structural data bring strong credit to the very large amount of spectroscopic data previously achieved in anthraquinone-replaced RCs and which have participated in the determination of the energetics of the quinone system in bacterial RCs.     

A tightly bound quinone functions in the ubiquinone reaction sites of quinoprotein alcohol dehydrogenase of an acetic acid bacterium, Gluconobacter suboxydans

Quinoprotein alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound enzyme that functions as the primary dehydrogenase in the ethanol oxidase respiratory chain. It consists of three subunits and has a pyrroloquinoline quinone (PQQ) in the active site and four heme c moieties as electron transfer mediators. Of these, three heme c sites and a further site have been found to be involved in ubiquinone (Q) reduction and ubiquinol (QH2) oxidation respectively (Matsushita et al., Biochim. Biophys. Acta, 1409, 154-164 (1999)). In this study, it was found that ADH solubilized and purified with dodecyl maltoside, but not with Triton X-100, had a tightly bound Q, and thus two different ADHs, one having the tightly bound Q (Q-bound ADH) and Q-free ADH, could be obtained. The Q-binding sites of both the ADHs were characterized using specific inhibitors, a substituted phenol PC16 (a Q analog inhibitor) and antimycin A. Based on the inhibition kinetics of Q2 reductase and ubiquinol-2 (Q2H2) oxidase activities, it was suggested that there are one and two PC16-binding sites in Q-bound ADH and Q-free ADH respectively. On the other hand, with antimycin A, only one binding site was found for Q2 reductase and Q2H2 oxidase activities, irrespective of the presence of bound Q. These results suggest that ADH has a high-affinity Q binding site (QH) besides low-affinity Q reduction and QH2 oxidation sites, and that the bound Q in the QH site is involved in the electron transfer between heme c moieties and bulk Q or QH2 in the low-affinity sites.     

Absence of large-scale displacement of quinone QB in bacterial photosynthetic reaction centers

Photosynthesis transforms light into chemical energy by coupling electron transfer to proton uptake at the quinone Q(B). The possibility of initiating this process with a brief pulse of light and the known X-ray structure makes the photosynthetic bacterial reaction center a paradigm for studying coupled electron-proton transfer in biology. It has been established that electron transfer from the primary quinone Q(A) to Q(B) is gated by a protein conformational change. On the basis of a dramatic difference in the location of Q(B) in structures derived from crystals cooled to 90 K either under illumination or in the dark, a functional model for the gating mechanism was proposed whereby neutral Q(B) moves 4.5 A before receiving the electron from Q(A)(-) [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816]. Isotope-edited FTIR difference spectroscopy of Q(B) photoreduction at 290 and 85 K is used to investigate whether Q(B) moves upon reduction. We show that the specific interactions of the carbonyl groups of Q(B) and Q(B)(-) with the protein at a single binding site remain identical at both temperatures. Therefore, the different locations of Q(B) reported in many X-ray crystal structures probably are unrelated to functional electron transfer from Q(A)(-) to Q(B).     

Structure of the cytochrome b6f complex: quinone analogue inhibitors as ligands of heme cn

A native structure of the cytochrome b(6)f complex with improved resolution was obtained from crystals of the complex grown in the presence of divalent cadmium. Two Cd(2+) binding sites with different occupancy were determined: (i) a higher affinity site, Cd1, which bridges His143 of cytochrome f and the acidic residue, Glu75, of cyt b(6); in addition, Cd1 is coordinated by 1-2 H(2)O or 1-2 Cl(-); (ii) a second site, Cd2, of lower affinity for which three identified ligands are Asp58 (subunit IV), Glu3 (PetG subunit) and Glu4 (PetM subunit). Binding sites of quinone analogue inhibitors were sought to map the pathway of transfer of the lipophilic quinone across the b(6)f complex and to define the function of the novel heme c(n). Two sites were found for the chromone ring of the tridecyl-stigmatellin (TDS) quinone analogue inhibitor, one near the p-side [2Fe-2S] cluster. A second TDS site was found on the n-side of the complex facing the quinone exchange cavity as an axial ligand of heme c(n). A similar binding site proximal to heme c(n) was found for the n-side inhibitor, NQNO. Binding of these inhibitors required their addition to the complex before lipid used to facilitate crystallization. The similar binding of NQNO and TDS as axial ligands to heme c(n) implies that this heme utilizes plastoquinone as a natural ligand, thus defining an electron transfer complex consisting of hemes b(n), c(n), and PQ, and the pathway of n-side reduction of the PQ pool. The NQNO binding site explains several effects associated with its inhibitory action: the negative shift in heme c(n) midpoint potential, the increased amplitude of light-induced heme b(n) reduction, and an altered EPR spectrum attributed to interaction between hemes c(n) and b(n). A decreased extent of heme c(n) reduction by reduced ferredoxin in the presence of NQNO allows observation of the heme c(n) Soret band in a chemical difference spectrum.     

Reactions of oxygen radicals with the quinone ring of coenzyme Q.

Coenzyme Q, besides its role in electron transfer reactions, may act as a radical scavenger. The effect of oxygen radicals produced by ultrasonic irradiation on the quinone ring was investigated. Aqueous solutions of a Q homologue, completely lacking the side chain, were irradiated and the modifications were spectrophotometrically followed. The experimental results show that both degradation and reduction of the benzoquinone ring took place when the irradiation was performed in water. Data obtained when ultrasonic irradiation was carried out in the presence of OH. scavengers, as formate, organic and inorganic buffers, suggest: a) the responsible species for most the ubiquinol generated by sonication appeared to be the superoxide radical b) addition reactions of OH. radicals with the aromatic ring led probably to the degradation of Coenzyme Q molecules.     

Structural basis for the quinone reduction in the bc1 complex: a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the Qi site

Cytochrome bc(1) is an integral membrane protein complex essential to cellular respiration and photosynthesis. The Q cycle reaction mechanism of bc(1) postulates a separated quinone reduction (Q(i)) and quinol oxidation (Q(o)) site. In a complete catalytic cycle, a quinone molecule at the Q(i) site receives two electrons from the b(H) heme and two protons from the negative side of the membrane; this process is specifically inhibited by antimycin A and NQNO. The structures of bovine mitochondrial bc(1) in the presence or absence of bound substrate ubiquinone and with either the bound antimycin A(1) or NQNO were determined and refined. A ubiquinone with its first two isoprenoid repeats and an antimycin A(1) were identified in the Q(i) pocket of the substrate and inhibitor bound structures, respectively; the NQNO, on the other hand, was identified in both Q(i) and Q(o) pockets in the inhibitor complex. The two inhibitors occupied different portions of the Q(i) pocket and competed with substrate for binding. In the Q(o) pocket, the NQNO behaves similarly to stigmatellin, inducing an iron-sulfur protein conformational arrest. Extensive binding interactions and conformational adjustments of residues lining the Q(i) pocket provide a structural basis for the high affinity binding of antimycin A and for phenotypes of inhibitor resistance. A two-water-mediated ubiquinone protonation mechanism is proposed involving three Q(i) site residues His(201), Lys(227), and Asp(228).     

Modulation of the free energy of the primary quinone acceptor (QA) in reaction centers from Rhodobacter sphaeroides: contributions from the protein and protein-lipid(cardiolipin) interactions

The redox midpoint potential (E(m)) of Q(A), the primary quinone of bacterial reaction centers, is substantially modulated by the protein environment. Quite subtle mutations in the Q(A) binding site, e.g., at residues M218, M252 and M265, cause significant increases in the equilibrium constant for electron transfer to Q(B), which indicate relative lowering of the E(m) of Q(A). However, reports of functional linkage between the Q(A) and Q(B) sites make it difficult to partition such effects between Q(A) and Q(B) from purely relative changes. We report here measurements on the yield of delayed fluorescence emission from the primary donor (P) accompanying the thermally activated charge recombination of P(+)Q(A)(-) to form the excited singlet state of the primary donor, P*. The results show that for mutations of the Q(A) site residues, Met(M218) and Ile(M265), essentially all the substantial thermodynamic effect is localized at Q(A), with no evidence for a significant effect of these residues on the properties of Q(B) or the mutual influence (linkage) of Q(A) and Q(B). We also report a significant lowering of the E(m) of Q(A) by the native lipid, cardiolipin, which brings the E(m) in isolated reaction centers more in line with that seen in native membrane vesicles (chromatophores). Possible origins of this effect are discussed in the context of the Q(A) binding site structure.     

Comparison of structure of quinone redox site in the mitochondrial cytochrome-bc1 complex and photosystem II (QB site).

A series of nitrophenolic electron-transport inhibitors (2-substituted 4,6-dinitrophenols) of rat liver mitochondrial cytochrome-bc1 complex and of photosystem II (QB site) of spinach thylakoids was synthesized. The structure/inhibitory-activity relationship was examined to elucidate differences in the three-dimensional structure of the quinone redox site in the two systems. These inhibitors occupy the ubiquinone redox site of cytochrome-bc1 complex competitively with natural ubiquinol, probably at a Qo reaction center. The inhibitory activity tended to increase with the length of the 2-substituent, which may correspond to the isoprenoid side chain of ubiquinone and plastoquinone, increased in both experimental systems. However, the strict structural requirements of the 2-substituent for binding to the ubiquinone or plastoquinone redox site were not identical. The alkyl substituents with a branching structure at the alpha-position to the benzene ring were favorable for inhibition of the cytochrome-bc1 complex, but not of photosystem II. Molecular-orbital calculations indicated that the main chain of 2-substituents with an alpha-branching structure was almost perpendicular to the benzene-ring plane because of steric congestion between the alpha-methyl and phenolic OH groups. The main chain of 2-substituents without an alpha-branching structure was flexible. Molecular-orbital studies indicated that ubiquinol was most stable when the portion of the isoprenoid side chain adjacent to the quinol ring was perpendicular to the quinol-ring plane, because of steric congestion by the vicinal OH and methyl groups. The side chain of plastoquinol was flexible because of the lack of a vicinal methyl group. Thus, the difference in the inhibitory activities between the two systems seemed to reflect the difference in the configuration of the isoprenoid side chain of ubiquinone and plastoquinone. These results suggested that the quinone redox site of the cytochrome-bc1 complex may recognize the configuration of the side chain near the quinone ring in the strict sense, whereas that of photosystem II (QB site) may recognize it in a loose sense.     

Effects of tail-like substituents on the binding of competitive inhibitors to the Q(B) site of photosystem II

The QB quinone-binding site of photosystem II is an important target for herbicides. Two major classes of herbicides are based on s-triazine and phenylurea moieties. A small library of triazine and phenylurea compounds has been synthesized which have tail-like substituents in order to test the effects of charge, hydrophobicity and size of the tail on binding properties. It is found that a tail can be attached to one of the alkylamino groups of triazine-type herbicides or to the para position of phenylurea-type herbicides without loss of binding, provided that the tail is hydrophobic. This indicates that the herbicides must be oriented in the QB site such that these positions point toward the natural isoprenyl tail-binding pocket that extends out of the Q(B) site. In turn, the requirement that the tail must extend out of the QB site constrains the size of the other herbicide substituents in the pocket. This is in agreement with the presumed orientation and fit of ligands in the QB site. When longer hydrophobic tails are used, the binding penalty that occurs upon adding a charged substituent at the distal end is reduced. This allows the use of a series of tail substituents possessing a distal charge as an approximate molecular ruler to measure the distance from the QB site to the aqueous phase. Even a 10-carbon alkyl chain still shows a 4-fold effect from the presence or absence of a distal charge. Such a chain does not appear to be long enough to extend from the bulk aqueous phase to the QB site because binding is completely lost when a large hydrophilic domain (PEG(4000)) is attached to the distal end. Longer tails are effective only if they are sufficiently hydrophobic. An effort was made to use tailed herbicides for affinity binding of photosystem II. It was found that hydrophobic linkers promote nonspecific binding, but careful choice of solvent conditions, such as the use of excess nonionic detergent well above its critical micelle concentration, might obviate this problem during affinity-binding applications.