In Photosynthetic Reaction Centers, the Free Energy Difference for Electron Transfer Between Quinones Bound at the Primary and S

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 2658-2662, April 1989 Biophysics

In photosynthetic reaction centers, the free energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity KATHLEEN M. GIANGIACOMO* AND P. LESLIE DUTTON Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104 Communicated by Britton Chance, December 2, 1988

ABSTRACT The secondary quinone-binding site (QB site) enquire further into the specificity of the QB site for quinone of bacterial reaction centers from Rhodobacter sphaeroides is structures. We have questioned in particular the methods of generally regarded to be highly specific for its native ubiqui- assay used to establish the binding strength of a quinone to none-10 molecule. We demonstrate here that this is a miscon- the QB site and have drawn upon the detailed general ception rooted in the kinetic methods used to assay for description for QB function that has been developed by occupancy ofa quinone in the QB site. We show that observance Wraight and Stein (11). of occupancy of the QB site, revealed by kinetic assay, is The methods used to assay for the occupancy of a quinone sensitive to the free-energy difference for electron transfer in the QB site rely entirely on kinetic analysis of various between the quinone at the primary quinone-binding site (QA flash-activated electron-transfer reactions within the reaction site) and the QB site (-AG0 ). For many of the compounds center that are altered by the presence ofa functional quinone previously tested for binding at the QB site, the -AGO. between in the QB site. In these methods, an essential requirement for QA and QB is too small to permit detection of the functional quinone in the QB site. With an increased -AGO. achieved by the recognition of a functional occupant of the QB site is replacing the native ubiquinone-10 at the QA site with lower- electron transfer from the light-generated Q- to QB. potential quinones or by testing higher-potential QB candi- Wraight's model, which is summarized in Fig. 1, introduces dates, it is shown that the QB site binds and functions with the other parameters that describe the interaction and reduction unsubstituted 1,4-benzoquinone, 1,4-naphthoquinone, and of quinones at the QB site. This model includes not only the 9,10-phenanthraquinone, as well as with their various substi- rates of quinone interaction with the QB site (kon and koff), tuted forms. Moreover, quinones with the ortho-carbonyl which define the dissociation constant (Kd = koff/kon), but configuration appear to function in a similar manner to also the rates of electron transfer between quinones occu- quinones with the para-carbonyl configuration. pying the QA and QB sites (kab and kba), which define the free energy of the reaction (-AG0- = RT In kab/kba). It is clear The photochemical reaction center of photosynthetic bacte- from Fig. 1 that, under certain conditions, the dissociation ria is an integral membrane protein that contains four bac- constant measured is not the true Kd but is an apparent value teriochlorophylls (BChls), two bacteriopheophytins (BPhs), (Kd). This situation is evident when equilibrium among the and two quinones associated with discrete catalytic sites, three charge-separated states (upper line of the scheme in designated the primary and secondary quinone-binding sites Fig. 1) is attained under conditions when: (QA and QB, respectively; for review, see ref. 1). After light excitation of a special pair of bacteriochlorophylls (BChl2), kab + kba>> kad and kon[Q] + koff >> kad. Ill an electron is transferred by way of BPh to the quinone at the QA site to form the semiquinone (QA) and the state BChl'- Under these conditions, the Kd is dependent on both the Kd QQB, where BChl' is the oxidized cation radical of BChl2 and the -AG' for electron transfer from QA to QB in the (2). The QA then reduces the quinone bound at the QB site to following way: form BChl'QAQB (3, 4) In the bacterial reaction centers from Rhodobacter sphae- K' = Kd/[1 + exp(-A°G_/RT)]. [2] roides the native ubiquinone-10 (UQ-10) molecules can be reversibly removed from both the QA and QB sites (5, 6). Eq. 2 shows that as -AGC° increases the Kd for quinone at Moreover, the QA site can be functionally reconstituted with the QB site decreases and hence will increase the possibility a wide variety of other natural and synthetic quinone struc- of detecting and measuring the functional presence of a tures (6-8). In contrast, with the exception of one observa- quinone in the QB site. We have used this device to re- tion (9), parallel efforts to reconstitute QB activity have been evaluate the specificity of the QB site. successful only with quinones containing the native ubiquinone (UQ) configuration (10-13). These negative findings to the that the unlike the site, Abbreviations: BPh, bacteriopheophytin; QA, primary quinone- have led sentiment QB site, QA binding site; QB, secondary quinone-binding site; BChl2, special pair is designed to provide stringent binding requirements for the ofbacteriochlorophyll molecules; QA, semiquinone formed in the QA UQ structure (10, 12, 13). However, such a view is not site; QB, semiquinone formed in the QB site; UQ, UQ-10, and UQ-0, entirely consistent with the well-known, broad specificity of ubiquinone, -10, and -0, respectively; - AG0 , free energy difference the QB site for a variety ofherbicide structures (14-16). These for electron transfer from QA to QB; Em, in situ oxidation-reduction apparently unusual characteristics have prompted us to potential; AQ, 9,10-anthraquinone; MeAQ, 1-methyl-9,10-anthraquinone; BChl, oxidized cation radical of BChl2; cyt c, cytochrome c; BQ, 1,4-benzoquinone. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: B501 Richards payment. This article must therefore be hereby marked "advertisement" Building, 37th and Hamilton Walk, University of Pennsylvania, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Philadelphia, PA 19104. 2658 Downloaded by guest on September 24, 2021 Biophysics: Giangiacorno and Dutton Proc. Natl. Acad. Sci. USA 86 (1989) 2659 kon kab (see ref. 8) with a characteristic rate constant (kad); for example, the kad values for UQ-10 and 9,10-anthraquinone BCh12 QA + [Q] ± BCh12 QA QB >- BCh12 QAQB (AQ) are close to 7 and 70 sec-1, respectively (19). With UQ-10 at both the QA and QB sites, the observed rate constant koff kba for reduction of BChl' is 0.7 sec-1 (21, 22). Under these conditions, the fractional occupancy ofquinone at the QB site is proportional to the fraction of slow phase of the BChl2 kad kad reduction kinetics and is readily observed (10, 11). 2. Ratio ofcyt c oxidized on the second andfirstflashes. kon Z14 With QA present and QB absent in the reaction center, cyt c oxidation occurs only on the first flash (23). However, with BChl2QA + [Q]% BChl2QAQB QB functionally present, the oxidation of Q- by QB permits cyt c oxidation on a second flash. Assuming the QA is koff kinetically capable of reducing QB (see method 3), the second/first flash ratio of cyt c oxidation is governed by QB FIG. 1. Reaction scheme for electron transfer from QA to QB and occupancy and the -AG°_ between QA and (22, quinone binding at the site. QB 24). QB 3. QX to QB electron transfer observed by accompanying electrochromic shifts ofBPh. The formation of Q- induces a MATERIALS AND METHODS shift in the Qy transition of BPh that is distinct from that Reaction Center Preparation. Reaction centers were iso- accompanying the formation of Q- (3, 4). This assay is useful lated from Rb. sphaeroides strain R-26 (17). Extraction of in that it distinguishes between the states BChl'QAQn and UQ-10 from both the QA and QB sites, or the QB site only, was BChl2QAQB and permits the time resolution of electron performed (6) with slight modifications (18). transfer between QA and QB QA Replacement. The procedure of Gunner et al. (8, 18, 19) Chemicals. The quinones used in this work were obtained was used for the reconstitution of QA activity with quinones from several sources as follows: 1-methyl-9,10-anthraqui- other than the native UQ. All procedures were carried out in none (MeAQ) and 2,3-dimethyl-1,4-benzoquinone from J. 10 mM Tris HCI (pH 8.0) under aerobic conditions at 23 ± Malcolm Bruce, University of Manchester; the latter was 1°C. Detergent concentrations were maintained well below also obtained from Chang-An Yu, Oklahoma State Univer- the critical micelle concentration and sufficiently constant to sity; we gratefully acknowledge the compounds provided by have no significant effect on our results (for discussion, see our colleagues. 1,4-Benzoquinone (BQ); 1,4-naphthoqui- ref. 8). none; 9,10-anthraquinone (AQ); 9,10-phenanthraquinone; 3,5- Quinones chosen as QA replacements had in situ oxidation- di-tert-butyl-1,2-benzoquinone; and 2,3-dichloro-1,4-naph- reduction potential (Em) values lower then UQ-10 (18, 19) so thoquinone were purchased from Aldrich. When necessary, that the -AG 0 for electron transfer from a quinone at the QA these compounds were purified by recrystallization from site to a particular quinone at the QB site would be increased. petroleum ether (50-110'C). The purity of all quinones was Displacement of quinone in the QA site by a test quinone established by reverse-phase HPLC. in the QB site was minimized by choosing QA replacements The ferrous form of cyt c was prepared by reducing 1 ml of whose Kd values at the QA site were at least 60-fold tighter 10 mM cyt c (ferric form) (horse heart, type VI from Sigma) than the Kd values at the QA site for quinones chosen to with 400 ,ul offreshly prepared well-buffered 100 mM sodium reconstitute the QB site. The concentrations used to recon- dithionite followed by removal of the excess dithionite on a stitute the two quinones at the QA and QB sites were chosen Sephadex G-50 gel filtration column. The cyt c was 98% so that occupancy of the QA site by the designated QA reduced after this treatment. replacement was always >90%. Conversely, under the conditions reported here, interference of the QA replacements RESULTS with the QB site was not significant; QA replacements themselves neither displayed functional QB activity nor acted QB Site Reconstitution with BQ. As stated above, functional as competitive inhibitors of electron transfer from UQ-10 at reconstitution of the QB site with quinones possessing the the QA site to ubiquinone-2 at the QB site (unpublished native UQ headgroup configuration is well-established. The results). tailless ubiquinone-0 (UQ-0) representing the minimal UQ Spectroscopic Measurements. Reaction center protein was structure, also functions in the QB site (11). In our investi- activated by a 10-,usec xenon flash (full width at half- gations, the unsubstituted BQ was chosen as a candidate to maximum light intensity) that activated -85% of the reaction test for binding at the QB site not only because it is the centers. simplest quinone but also because its in vitro oxidation- In the visible region, the flash-induced absorbance changes reduction potential (E1/2) value in dimethylformamide is in the oxidation state of BChl1 were measured at its Qx band substantially higher (-0.14 V) than that of UQ-0 (25). Thus, using 605-540 nm while those of cytochrome c (cyt c) were if these dimethylformamide values are a reasonable guide to monitored at its a-band by using 550-540 nm (20). In the the values exhibited in the QB site (see ref. 19), then the near-infrared region, the kinetics of the flash-induced shift of -AG0- between UQ-10 at the QA site and BQ at the QB site the QY band of BPh that reports electron transfer from QA to should be favorable and the function and occupancy of BQ in QB were monitored in the single-beam mode at 750 nm (3, 4). the QB site should be detectable. However, if this is not the Data Analysis. Kinetic traces were analyzed as described case, then the use ofAQ or MeAQ to increase the -AGl- will (19). Kd values for quinones at the QB site and the rate improve the chance of detecting the reaction. constant for quinone binding (kon) at the QB site were Fig. 2A shows that when the native UQ-10 occupies the QA determined with a nonlinear least squares analysis routine site the activity of BQ at the QB site is only marginal in all from the Asystant+ software, version 1.0 (MacMillan Soft- three of the standard QB assays; at 20 ,uM BQ, QB activity is ware, New York). <18%. However, when UQ-10 at the QA site is replaced with Methods To Examine for QB Reconstitution. 1. Reduction AQ (Fig. 2B) to increase the -AG°_ by 0.15 eV, the activity kinetics offlash-generated BChl2. When quinone occupies of BQ at the QB site is increased substantially. only the QA site, the kinetics of charge recombination from In Fig. 3, again with AQ in the QA site and BQ in the QB site, BChl+Q- to BCh12QA occur as a nearly single exponential the kinetics ofelectron transfer from QA to QB were examined Downloaded by guest on September 24, 2021 2660 Biophysics: Giangiacomo and Dutton Proc. Natl. Acad. Sci. USA 86 (1989) BQ- in the QB site, is proportional to the concentration of BQ between 20 and 200 AuM. A plot of the flash-induced AA750 amplitude versus log[BQ] reveals that apparent binding of BQ to the QB site is well described by the theoretical fit to the full quadratic equation for ligand binding at a site with a Kd value of 38 AuM (Fig. 3B). In addition, at the maximum BQ concentration, the kinetics of AA750 are rapid. Although not shown in Fig. 3A, time resolution of the 2.17 mM trace revealed a half-time of 145 pusec. This half-time is similar to that found for electron transfer from UQ-10 in the QA site to UQ-0 (11) and UQ-10 (3, 4, 22) in the QB site. Fig. 3C describes the concentration dependence of electron-transfer kinetics from AQ at the QA site to BQ at the QB site and demonstrates that the binding reaction between BQ and the QB site occurs during the lifetime of Q- and that the binding rate (kon[BQ] + koff) is rate-limiting relative to the electron-transfer reaction (kab + kba). Under these conditions, the rate of binding for BQ at the QB site can be determined from the slope of the observed FIG. 2. In reaction centers from Rb. sphaeroides R-26, the binding rate (k750) versus the concentration of BQ. This rate function of BQ at the QB site when the native UQ-10 at the QA site constant is 106 M-1 sec-1 and is similar to that observed for (A) is replaced with AQ (B). The reaction solution contained 0.15 ,uM UQ-0 (11) and other small nonquinone molecules that bind at reaction centers in 10 mM Tris HCl/0.001% lauryldimethylamine the QB site (14). The fact that BQ exhibits concentration- oxide, pH 8.0, at 23°C in the absence (curve a) and presence (curve dependent electron-transfer kinetics from AQ at the QA site to b) of 30 ,uM BQ. Conditions for A and B were the same except that at site in B reaction centers depleted of their native UQ-10 were reconsti- BQ the QB suggests that, at micromolar concentrations tuted with 15 ,uM AQ. BChl' reduction kinetics were monitored at of BQ, the QB site is primarily unoccupied prior to the flash. 605-540 nm. Cyt c oxidation on the first and second flash was This observation agrees with previous observations made for monitored at 550-540 nm with a 272-msec flash interval. Conditions the binding of UQ-10 to the QB site in reaction center were as for the BChl' kinetics except that 2.03 ,uM cyt c (ferrous containing vesicles (26). form) and 30 ,uM BQ were present. Cyt c oxidation in the absence of Dependence of the Apparent Quinone Affinity on -AG0e. added QB was similar to the traces shown in Fig. SB. Electron Using the cyt c oxidation assay, Fig. 4 confirms quantitatively transfer from QA to QB was assayed at 750 nm in a reaction solution that variation of the -AG0- between QA and substantially containing 10 ,uM reaction centers in the presence of 30 ,uM BQ. The QB scale for absorbance changes indicated on the right applies to all alters the apparent affinity of the QB site for quinone. The traces presented. figure shows that by extending the BQ concentration to higher values (cf. Fig. 2A) that the measurement of a Kd value (200 by the absorbance change at 750 nm (£A750). Fig. 3A shows ,uM) is achievable for BQ even with UQ-10 at the QA site. that the amplitude of AA750, interpreted as the generation of When UQ-10 at the QA site is replaced with AQ to increase -AG°_ by 0.15 eV, the QB site exhibits a Kd value of 38 ,uM for BQ. Similarly, when MeAQ is at the QA site to increase the 9,10-AQ(QA)' (QB) -AG°_ by 0.22 eV, a Kd value of 14 ,uM for BQ is observed. Thus, although this work warrants further quantitative examination, it is clear that the reaction -AG°_ can strongly influence the Kd value of the quinone in the QB site. These results also reveal that the Em of BQ in the QB site is just 20 PM n 6 sufficiently favorable to reconstitute QB activity when UQ-10 is at QA site. x4- 37 PM G -6 -4 -2 215 &Mlog [1,4-BO](M)

392 MM3

2,l70 M &AA00.004 I.-20 - f 6 -4 -2 log 1,4-BO] M)

FIG. 3. Use of the BPh absorption shift to observe the specific -5 -4 binding and formation ofBQ- at the QB site in reaction centers ofRb. Log 1,4-benzoquinone (M) sphaeroides R-26. (A) Flash-activated formation of BQ- in the QB site was determined by monitoring absorbance changes at 750 nm at FIG. 4. Dependence ofapparent quinone binding at the QB site on the indicated concentrations of BQ using a reaction center sample the -AG°. for QA to QB electron transfer in reaction center of Rb. reconstituted at 10 jM as described in Fig. 2B. (B) Changes in the sphaeroides R-26. The -AG0- was varied by replacing the native amplitude of AA750 as a function of the concentration of BQ, taken UQ-10 at the QA site (-Em = 0.07 V) with 15 ,uM AQ (-Em = 0.22 from the traces shown in A, were fit to the full quadratic binding V) or with 15 ,uM MeAQ (-Em = 0.29 V). The theoretical lines for equation, Kdj = 38 ,uM. (C) Binding rate constant for BQ at QB was Kd were obtained as described in Fig. 3B. Reaction and conditions determined from the slope of a linear fit to k750 versus the concen- for measuring cyt c oxidation were as in Fig. 2 except that the tration of BQ. Abbreviations: 9,10-AQ, AQ; 1,4-BQ, BQ. concentration of BQ was varied. Downloaded by guest on September 24, 2021 Biophysics: Giangiacorno and Dutton Proc. Natl. Acad. Sci. USA 86 (1989) 2661

Cytochrome c Oxidation 8 UQIO(QA) AQ (OA ) (WBCh )2 Reduction Kinetics

No Adds 0

FIG. 5. Function of methyl- and ring-substituted quinones at the QB site with UQ-10 (Left) and AQ (Right) at the QA site of reaction centers from Rb. sphaeroides R-26. (A) Conditions for monitoring BChl' reduction kinetics were as in Fig. 2 in the absence (curve a) and the presence (curve b) of 25 ± 5 ,tM of each QB candidate. The indicated absorbance scale is the same for both panels. (B) Conditions for monitoring cyt c oxidation were carried out as in Fig. 2. The top two traces were typical of cyt c oxidation in the absence of added QB; all other cyt c oxidation traces were obtained from samples containing 25 ± 5 AuM of the indicated QB candidates. The time and absorbance scales are the same for both panels.

QB Site Reconstitution with Low-Potential Quinones. In Fig. DISCUSSION 5, we take advantage of the large -AG°_ provided by AQ at We have demonstrated that previous efforts to reconstitute the QA site to demonstrate that the QB site will function with the QB site of bacterial reaction centers with quinone ana- quinones that, like BQ, were believed to be nonfunctional at logues other than the native UQ were unsuccessful because the QB site. Thus, when the native UQ-10 occupies the QA of the small -AG0- between tested quinones at the QB site site, reconstitution of the QB site with 2,3-dimethyl- and the native UQ-10 at the QA site. The relatively high in situ 1,4-benzoquinone or 1,4-naphthoquinone yields marginal QB Em value that prevails for the QA/QA couple of UQ-10 activity, whether using either the BChl' reduction kinetics restricts the examination ofquinones for binding and function assay (Fig. 5A Left) or the cyt c oxidation assay (Fig. 5B at the QB site to those that also have as high or higher Em Left). However, when AQ is incorporated into the QA site, values. Unfortunately, in vitro E1/2 measurements for qui- substantial QB activity is exhibited. The amount of cyt c nones in dimethylformamide (25) and in situ Em measure- oxidized on the second flash relative to the first flash ranges from 40 to 70% for all the quinones tested (Fig. 5B Right) BA CB while the slow phase of the BChl' reduction kinetics con- ( BChl )2 Reduction Kinetics Cytochrome c Oxidation stitutes >35% of the total BChl' (Fig. SA Right). The observed variation in QB activity for these quinones probably a reflects differences in their Kd values. These experiments 0 demonstrate clearly that the QB site will bind and function tcl with the methyl-substituted and ring-extended BQs that 0 : CI ~~~~~~~~~~~~~~-t--t- aIw-- resemble the head groups of the naturally occurring quinones, plastoquinone and menaquinone. 0 QB Site Reconstitution with High-Potential Quinones. The WA -14wl- other way to generate a more favorable -AG°_ is to use C, higher-potential QB candidates while retaining the native b '4-S UQ-10 at the QA site. In Fig. 6 we show that the high-potential 2,3-dichloro-1,4-naphthoquinone and the ortho-quinones, 3,5-di-tert-butyl-1,2-benzoquinone and 9,10-phenanthraqui- a none, all exhibit substantial QB activity when monitored by the cyt c oxidation and by the BChl' kinetics assays. We have also examined the amplitude and kinetics of the I a BPh electrochromic shift at 750 nm to eliminate the possi- 0.002 A bility that these quinones may be functioning merely as -* I fT nonspecific acceptors that oxidize QA without actually en- 400 msec 20-0200 msece tering the QB site. The results from these experiments (data not shown) are very similar to those observed for BQ in Fig. FIG. 6. Function of high-potential ortho- and halogenated para- 3, thereby demonstrating that these high-potential quinones quinones at the QB site with the native UQ-10 at the QA site. (A) are indeed BChl' reduction kinetics in the absence (curve a) and presence functioning in the QB site. The experiments (curve b) of 35 Am QB. (B) Cyt c oxidation on the first and second strengthen considerably our assertion that provision of a flash in the presence of 35 Am QB Cyt c oxidation in the absence of sufficiently large -AGC° leads to easy detection of quinone added QB was similar to the trace in Fig. 5B. The conditions were as occupancy at the QB site. in Fig. 2. Downloaded by guest on September 24, 2021 2662 Biophysics: Giangiacomo and Dutton Proc. Natl. Acad. Sci. USA 86 (1989) ments for quinones at the QA site'(18) reveal that the effects the QB site in both plants and bacteria, but also perhaps as an of ring extension and most other substitutions tend to lower important parameter in devising biotechnological strategies the oxidation-reduction potential values relative to BQ. in the field. However, we have diminished this restriction by either deliberately choosing QA replacements with in situ Em values We thank Marilyn Gunner for her valuable discussions. We are that are much lower than UQ-10 or by choosing QB candi- also grateful to Roger Prince for his extensive measurements of E1/2 dates with in vitro E1/2 values very much higher than UQ-10. values for quinones in dimethylformamide. This work was supported From this work, another view of the QB site emerges. by a grant from the National Institute of Health (GM27309). Rather than being specific for a single-ringed UQ, it is clear that the site will bind and support the function of other 1. Feher, G. & Okamura, M. Y. (1978) in The Photosynthetic naturally occurring benzo- and naphthoquinone structures Bacteria, eds. Clayton, R. K. & Sistrom, W. R. (Plenum, New York), pp. 349-386. that are found in higher plants and other bacteria. In addition, 2. Rockley, M. G., Windsor, M. W., Cogdell, R. J. & Parson, the QB site will function with synthetic quinone analogues W. W. (1975) Proc. Natl. Acad. Sci. USA 72, 2251-2255. containing up to three rings, as well as with quinones that 3. Vermeglio, A. & Clayton, R. K. (1977) Biochim. Biophys. Acta have their carbonyls arranged in the ortho rather than the 461, 159-165. more familiar para configuration. 4. Wraight, C. A. (1979) Biochim. Biophys. Acta 548, 309-327. These results are at least qualitatively similar to those 5. Cogdell, R. J., Brune, D. C. & Clayton, R. K. (1974) FEBS found for the QA site (8). Work with the QA site has further Lett. 45, 344-347. demonstrated not only that there is no requirement for a 6. Okamura, M. Y., Isaacson, R. A. & Feher, G. (1975) Proc. para-carbonyl structure but also that the contribution to Nati. Acad. Sci. USA 72, 3491-3495. binding free energy from the "second" carbonyl is small (8). 7. Gunner, M. R., Tiede, D. M., Prince, R. C. & Dutton, P. L. From the work presented here, it is reasonable to consider (1982) in Function ofQuinones in Energy Conserving Systems, that the QA and sites may exhibit the same minimal ed. Trumpower, B. L. (Academic, New York), pp. 265-269. QB 8. Gunner, M. R., Braun, B. S., Bruce, J. M. & Dutton, P. L. requirements for binding. That is, binding to both the QA and (1985) in Springer Series in Chemical Physics: Antennas and QB sites may require only a partially unsaturated ring that is Reaction Centers of Photosynthetic Bacteria-Structure, In- substituted with one carbonyl. This model is remarkably teractions andDynamics, ed. Michel-Beyerle, M. E. (Springer, similar to that deduced by Trebst et al. (27) from inhibition Berlin), Vol. 42, pp. 298-304. studies done with the urea/triazine class of herbicides on the 9. Diner, B. A., Schenck, C. C. & De Vitry, C. (1984) Biochim. QB site of the reaction center of photosystem II of green Biophys. Acta 766, 9-20. plants. The binding requirements of the QB site for triazines 10. Baccarini-Melandri, A., Gabellini, N. & Melandri, B. A. (1980) and, by implication, for plastoquinone revealed from this J. Bioenerg. Biomembr. 12, 95-110. study are given as an aromatic ring substituted with an Sp2 11. Wraight, C. A. & Stein, R. R. (1983) in The Oxygen Evolving and System of Photosynthesis, eds. Inoue, Y., Crofts, A. R., hybridized group with a lone electron pair substituted Govindjee, Murata, N., Renger, G. & Satoh, K. (Academic, with a lipophilic moiety (27). New York), pp. 383-392. The significant influence of -AG°_ on the apparent affinity 12. Okamura, M. Y., Debus, R. J., Kleinfeld, D. & Feher, G. of the QB site for quinonoid compounds that we have (1982) in Function ofQuinones in Energy Conserving Systems, described is equally important for studies into herbicide ed. Trumpower, B. L. (Academic, New York), pp. 299-317. sensitivity and site-directed mutagenesis at the QA and QB 13. Baccarini-Melandri, A., Gabellini, N. & Melandri, B. A. (1982) sites. Although it is obvious that a changed amino acid in Function of Quinones in Energy Conserving Systems, eds. residue can impart changes in the strength of interaction of a Trumpower, B. L. (Academic, New York), pp. 285-298. native quinone or herbicide at the QB site, it is also the case, 14. Stein, R. R., Castellvi, A. L., Bogacz, J. P. & Wraight, C. A. (1984) J. Cell. Biochem. 24, 243-259. as we have shown, that large increases or decreases in 15. Tischer, W. & Strotmann, H. (1979) Biochim. Biophys. Acta quinone Kd, and hence herbicide activity (IC50 values), can 460, 113-125. arise from an altered -AG'-. Further, since the basic 16. Trebst, A. & Draber, W. (1979) in Advances in Pesticide electrochemical properties of both the QA and QB sites Science, ed. Geisshuhler, N. (Pergamon, New York), pp. 223- contribute to -AGO-, it follows that the source of mutation- 234. induced alterations in either the apparent affinity for quinone 17. Clayton, R. K. & Wang, R. T. (1971) Methods Enzymol. 23, or in the herbicide susceptibility at the QB site isjust as likely 696-704. to reside in the QA site as in the QB site. Thus a change in the 18. Woodbury, N. W., Parson, W. W., Gunner, M. R., Prince, amino acid complement to alter the in situ Em values for R. C. & Dutton, P. L. (1986) Biochim. Biophys. Acta 851, 6-22. 19. Gunner, M. R., Robertson, D. E. & Dutton, P. L. (1986) J. quinone in the QA site can have major effects that are Phys. Chem. 90, 3783-3795. observed in the reactions at the QB site. This prediction is 20. Dutton, P. L., Petty, K. M., Bonner, H. S. & Morse, S. D. supported by our preliminary unpublished experiments in (1975) Biochim. Biophys. Acta 387, 536-556. which AQ replacement at the QA site results in not only a 21. Chamorovsky, S. K., Remennikov, S. M., Kononenko, A. A., decreased K' for UQ-0 at the QB site, but also an increase in Venediktov, P. S. & Rubin, A. B. (1976) Biochim. Biophys. the amount of Ametryne, a QB site inhibitor, needed to effect Acta 430, 62-70. inhibition. It follows that site-directed mutagenesis at the QA 22. Kleinfeld, D., Okamura, M. Y. & Feher, G. (1984) Biochim. site could also be useful for either increasing or decreasing Biophys. Acta 766, 126-140. the observed herbicide of the site. 23. Parson, W. W. (1969) Biochim. Biophys. Acta 189, 384-396. sensitivity QB Thus, 24. Halsey, Y. D. & Parson, W. W. (1974) Biochim. Biophys. Acta mutations deliberately directed to lower or raise the Em value 347, 404-416. of quinone in the QA site will confer upon the QB site a 25. Prince, R. C., Dutton, P. L. & Bruce, J. M. (1983) FEBS Lett. controlled degree ofresistance or susceptibility to herbicides. 160, 273-276. Clearly, recognizing the influence of -AG'- on quinone 26. Wraight, C. A. (1981) Isr. J. Chem. 21, 348-354. specificity is vital not only for obtaining a comprehensive 27. Trebst, A., Donner, W. & Draber, W. (1984) Z. Naturforsch. understanding of the functional and structural properties of C 39, 405-411. Downloaded by guest on September 24, 2021