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

In photosynthetic reaction centers, the free energy difference forelectron transfer between quinones bound at the primary andsecondary quinone-binding sites governs the observedsecondary site specificityKATHLEEN 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)of bacterial reaction centers from Rhodobacter sphaeroides isgenerally regarded to be highly specific for its native ubiqui-none-10 molecule. We demonstrate here that this is a miscon-ception rooted in the kinetic methods used to assay foroccupancy of a quinone in the QB site. We show that observanceof occupancy of the QB site, revealed by kinetic assay, issensitive to the free-energy difference for electron transferbetween the quinone at the primary quinone-binding site (QAsite) and the QB site (-AG0 ). For many of the compoundspreviously tested for binding at the QB site, the -AGO. betweenQA and QB is too small to permit detection of the functionalquinone in the QB site. With an increased -AGO. achieved byreplacing the native ubiquinone-10 at the QA site with lower-potential quinones or by testing higher-potential QB candi-dates, it is shown that the QB site binds and functions with theunsubstituted 1,4-benzoquinone, 1,4-naphthoquinone, and9,10-phenanthraquinone, as well as with their various substi-tuted forms. Moreover, quinones with the ortho-carbonylconfiguration appear to function in a similar manner toquinones with the para-carbonyl configuration.

The photochemical reaction center of photosynthetic bacte-ria is an integral membrane protein that contains four bac-teriochlorophylls (BChls), two bacteriopheophytins (BPhs),and two quinones associated with discrete catalytic sites,designated the primary and secondary quinone-binding sites(QA and QB, respectively; for review, see ref. 1). After lightexcitation of a special pair of bacteriochlorophylls (BChl2),an electron is transferred by way of BPh to the quinone at theQA site to form the semiquinone (QA) and the state BChl'-QQB, where BChl' is the oxidized cation radical of BChl2(2). The QA then reduces the quinone bound at the QB site toform BChl'QAQB (3, 4)

In the bacterial reaction centers from Rhodobacter sphae-roides the native ubiquinone-10 (UQ-10) molecules can bereversibly removed from both the QA and QB sites (5, 6).Moreover, the QA site can be functionally reconstituted witha wide variety of other natural and synthetic quinone struc-tures (6-8). In contrast, with the exception of one observa-tion (9), parallel efforts to reconstitute QB activity have beensuccessful only with quinones containing the native ubiqui-none (UQ) configuration (10-13). These negative findingshave led to the sentiment that the QB site, unlike the QA site,is designed to provide stringent binding requirements for theUQ structure (10, 12, 13). However, such a view is notentirely consistent with the well-known, broad specificity ofthe QB site for a variety ofherbicide structures (14-16). Theseapparently unusual characteristics have prompted us to

enquire further into the specificity of the QB site for quinonestructures. We have questioned in particular the methods ofassay used to establish the binding strength of a quinone tothe QB site and have drawn upon the detailed generaldescription for QB function that has been developed byWraight and Stein (11).The methods used to assay for the occupancy of a quinone

in the QB site rely entirely on kinetic analysis of variousflash-activated electron-transfer reactions within the reactioncenter that are altered by the presence of a functional quinonein the QB site. In these methods, an essential requirement forthe recognition of a functional occupant of the QB site iselectron transfer from the light-generated Q- to QB.Wraight's model, which is summarized in Fig. 1, introducesother parameters that describe the interaction and reductionof quinones at the QB site. This model includes not only therates of quinone interaction with the QB site (kon and koff),which define the dissociation constant (Kd = koff/kon), butalso the rates of electron transfer between quinones occu-pying the QA and QB sites (kab and kba), which define the freeenergy of the reaction (-AG0- = RT In kab/kba). It is clearfrom Fig. 1 that, under certain conditions, the dissociationconstant measured is not the true Kd but is an apparent value(Kd). This situation is evident when equilibrium among thethree charge-separated states (upper line of the scheme inFig. 1) is attained under conditions when:

kab + kba>> kad and kon[Q] + koff >> kad. IllUnder these conditions, the Kd is dependent on both the Kdand the -AG' for electron transfer from QA to QB in thefollowing way:

K' = Kd/[1 + exp(-A°G_/RT)]. [2]

Eq. 2 shows that as -AGC° increases the Kd for quinone atthe QB site decreases and hence will increase the possibilityof detecting and measuring the functional presence of aquinone in the QB site. We have used this device to re-evaluate the specificity of the QB site.

Abbreviations: BPh, bacteriopheophytin; QA, primary quinone-binding site; QB, secondary quinone-binding site; BChl2, special pairofbacteriochlorophyll molecules; QA, semiquinone formed in the QAsite; QB, semiquinone formed in the QB site; UQ, UQ-10, and UQ-0,ubiquinone, -10, and -0, respectively; -AG0 , free energy differencefor electron transfer from QA to QB; Em, in situ oxidation-reductionpotential; AQ, 9,10-anthraquinone; MeAQ, 1-methyl-9,10-anthraqui-none; BChl, oxidized cation radical of BChl2; cyt c, cytochrome c;BQ, 1,4-benzoquinone.*To whom reprint requests should be addressed at: B501 RichardsBuilding, 37th and Hamilton Walk, University of Pennsylvania,Philadelphia, PA 19104.

2658

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Proc. Natl. Acad. Sci. USA 86 (1989) 2659

kon kabBCh12 QA + [Q] ± BCh12 QA QB >- BCh12 QAQB

kad

koff

kad

kon Z14

kba

BChl2QA + [Q]% BChl2QAQB

koff

FIG. 1. Reaction scheme for electron transfer from QA to QB andquinone binding at the QB site.

MATERIALS AND METHODS

Reaction Center Preparation. Reaction centers were iso-lated from Rb. sphaeroides strain R-26 (17). Extraction ofUQ-10 from both the QA and QB sites, or the QB site only, wasperformed (6) with slight modifications (18).QA Replacement. The procedure of Gunner et al. (8, 18, 19)

was used for the reconstitution of QA activity with quinonesother than the native UQ. All procedures were carried out in10 mM Tris HCI (pH 8.0) under aerobic conditions at 23 ±

1°C. Detergent concentrations were maintained well belowthe critical micelle concentration and sufficiently constant tohave no significant effect on our results (for discussion, seeref. 8).Quinones chosen as QA replacements had in situ oxidation-

reduction potential (Em) values lower then UQ-10 (18, 19) sothat the -AG0 for electron transfer from a quinone at the QAsite to a particular quinone at the QB site would be increased.Displacement of quinone in the QA site by a test quinone

in the QB site was minimized by choosing QA replacementswhose Kd values at the QA site were at least 60-fold tighterthan the Kd values at the QA site for quinones chosen toreconstitute the QB site. The concentrations used to recon-stitute the two quinones at the QA and QB sites were chosenso that occupancy of the QA site by the designated QAreplacement was always >90%. Conversely, under the con-ditions reported here, interference of the QA replacementswith the QB site was not significant; QA replacementsthemselves neither displayed functional QB activity nor actedas competitive inhibitors of electron transfer from UQ-10 atthe QA site to ubiquinone-2 at the QB site (unpublishedresults).

Spectroscopic Measurements. Reaction center protein wasactivated by a 10-,usec xenon flash (full width at half-maximum light intensity) that activated -85% of the reactioncenters.

In the visible region, the flash-induced absorbance changesin the oxidation state of BChl1 were measured at its Qx bandusing 605-540 nm while those of cytochrome c (cyt c) weremonitored at its a-band by using 550-540 nm (20). In thenear-infrared region, the kinetics of the flash-induced shift ofthe QY band of BPh that reports electron transfer from QA toQB were monitored in the single-beam mode at 750 nm (3, 4).Data Analysis. Kinetic traces were analyzed as described

(19). Kd values for quinones at the QB site and the rateconstant for quinone binding (kon) at the QB site weredetermined with a nonlinear least squares analysis routinefrom the Asystant+ software, version 1.0 (MacMillan Soft-ware, New York).Methods To Examine for QB Reconstitution. 1. Reduction

kinetics offlash-generated BChl2. When quinone occupiesonly the QA site, the kinetics of charge recombination fromBChl+Q- to BCh12QA occur as a nearly single exponential

(see ref. 8) with a characteristic rate constant (kad); forexample, the kad values for UQ-10 and 9,10-anthraquinone(AQ) are close to 7 and 70 sec-1, respectively (19). WithUQ-10 at both the QA and QB sites, the observed rate constantfor reduction of BChl' is 0.7 sec-1 (21, 22). Under theseconditions, the fractional occupancy ofquinone at the QB siteis proportional to the fraction of slow phase of the BChl2reduction kinetics and is readily observed (10, 11).

2. Ratio of cyt c oxidized on the second andfirstflashes.With QA present and QB absent in the reaction center, cyt coxidation occurs only on the first flash (23). However, withQB functionally present, the oxidation of Q- by QB permitscyt c oxidation on a second flash. Assuming the QA iskinetically capable of reducing QB (see method 3), thesecond/first flash ratio of cyt c oxidation is governed by QBoccupancy and the -AG°_ between QA and QB (22, 24).

3. QX to QB electron transfer observed by accompanyingelectrochromic shifts ofBPh. The formation of Q- induces ashift in the Qy transition of BPh that is distinct from thataccompanying the formation of Q- (3, 4). This assay is usefulin that it distinguishes between the states BChl'QAQn andBChl2QAQB and permits the time resolution of electrontransfer between QA and QB

Chemicals. The quinones used in this work were obtainedfrom several sources as follows: 1-methyl-9,10-anthraqui-none (MeAQ) and 2,3-dimethyl-1,4-benzoquinone from J.Malcolm Bruce, University of Manchester; the latter wasalso obtained from Chang-An Yu, Oklahoma State Univer-sity; we gratefully acknowledge the compounds provided byour colleagues. 1,4-Benzoquinone (BQ); 1,4-naphthoqui-none; 9,10-anthraquinone (AQ); 9,10-phenanthraquinone; 3,5-di-tert-butyl-1,2-benzoquinone; and 2,3-dichloro-1,4-naph-thoquinone were purchased from Aldrich. When necessary,these compounds were purified by recrystallization frompetroleum ether (50-110'C). The purity of all quinones wasestablished by reverse-phase HPLC.The ferrous form of cyt c was prepared by reducing 1 ml of

10 mM cyt c (ferric form) (horse heart, type VI from Sigma)with 400 ,ul of freshly prepared well-buffered 100 mM sodiumdithionite followed by removal of the excess dithionite on aSephadex G-50 gel filtration column. The cyt c was 98%reduced after this treatment.

RESULTS

QB Site Reconstitution with BQ. As stated above, functionalreconstitution of the QB site with quinones possessing thenative UQ headgroup configuration is well-established. Thetailless ubiquinone-0 (UQ-0) representing the minimal UQstructure, also functions in the QB site (11). In our investi-gations, the unsubstituted BQ was chosen as a candidate totest for binding at the QB site not only because it is thesimplest quinone but also because its in vitro oxidation-reduction potential (E1/2) value in dimethylformamide issubstantially higher (-0.14 V) than that of UQ-0 (25). Thus,if these dimethylformamide values are a reasonable guide tothe values exhibited in the QB site (see ref. 19), then the-AG0- between UQ-10 at the QA site and BQ at the QB siteshould be favorable and the function and occupancy of BQ inthe QB site should be detectable. However, if this is not thecase, then the use ofAQ orMeAQ to increase the -AGl- willimprove the chance of detecting the reaction.

Fig. 2A shows that when the native UQ-10 occupies the QAsite the activity of BQ at the QB site is only marginal in allthree of the standard QB assays; at 20 ,uM BQ, QB activity is<18%. However, when UQ-10 at the QA site is replaced withAQ (Fig. 2B) to increase the -AG°_ by 0.15 eV, the activityof BQ at the QB site is increased substantially.

In Fig. 3, again with AQ in the QA site and BQ in the QB site,the kinetics of electron transfer from QA to QB were examined

Biophysics: Giangiacorno and Dutton

2660 Biophysics: Giangiacomo and Dutton

FIG. 2. In reaction centers from Rb. sphaeroides R-26, thefunction of BQ at the QB site when the native UQ-10 at the QA site(A) is replaced with AQ (B). The reaction solution contained 0.15 ,uMreaction centers in 10 mM Tris HCl/0.001% lauryldimethylamineoxide, pH 8.0, at 23°C in the absence (curve a) and presence (curveb) of 30 ,uM BQ. Conditions for A and B were the same except thatin B reaction centers depleted of their native UQ-10 were reconsti-tuted with 15 ,uM AQ. BChl' reduction kinetics were monitored at605-540 nm. Cyt c oxidation on the first and second flash wasmonitored at 550-540 nm with a 272-msec flash interval. Conditionswere as for the BChl' kinetics except that 2.03 ,uM cyt c (ferrousform) and 30 ,uM BQ were present. Cyt c oxidation in the absence ofadded QB was similar to the traces shown in Fig. SB. Electrontransfer from QA to QB was assayed at 750 nm in a reaction solutioncontaining 10 ,uM reaction centers in the presence of 30 ,uM BQ. Thescale for absorbance changes indicated on the right applies to alltraces presented.

by the absorbance change at 750 nm (£A750). Fig. 3A showsthat the amplitude of AA750, interpreted as the generation of

9,10-AQ(QA)' (QB)

20 PM n 6

x4-

37 PMG -6 -4 -2

215&Mlog [1,4-BO](M)

392MM3

2,l70 M &AA00.004I.-20 - f 6 -4 -2

log 1,4-BO] M)

FIG. 3. Use of the BPh absorption shift to observe the specificbinding and formation ofBQ- at the QB site in reaction centers ofRb.sphaeroides R-26. (A) Flash-activated formation of BQ- in the QBsite was determined by monitoring absorbance changes at 750 nm atthe indicated concentrations of BQ using a reaction center samplereconstituted at 10 jM as described in Fig. 2B. (B) Changes in theamplitude of AA750 as a function of the concentration of BQ, takenfrom the traces shown in A, were fit to the full quadratic bindingequation, Kdj = 38 ,uM. (C) Binding rate constant for BQ at QB wasdetermined from the slope of a linear fit to k750 versus the concen-tration of BQ. Abbreviations: 9,10-AQ, AQ; 1,4-BQ, BQ.

BQ- in the QB site, is proportional to the concentration ofBQbetween 20 and 200 AuM. A plot of the flash-induced AA750amplitude versus log[BQ] reveals that apparent binding ofBQto the QB site is well described by the theoretical fit to the fullquadratic equation for ligand binding at a site with a Kd valueof 38 AuM (Fig. 3B). In addition, at the maximum BQ concen-tration, the kinetics of AA750 are rapid. Although not shown inFig. 3A, time resolution of the 2.17 mM trace revealed ahalf-time of 145 pusec. This half-time is similar to that found forelectron transfer from UQ-10 in the QA site to UQ-0 (11) andUQ-10 (3, 4, 22) in the QB site. Fig. 3C describes theconcentration dependence of electron-transfer kinetics fromAQ at the QA site to BQ at the QB site and demonstrates thatthe binding reaction between BQ and the QB site occurs duringthe lifetime of Q- and that the binding rate (kon[BQ] + koff) israte-limiting relative to the electron-transfer reaction (kab +kba). Under these conditions, the rate of binding for BQ at theQB site can be determined from the slope of the observedbinding rate (k750) versus the concentration of BQ. This rateconstant is 106 M-1 sec-1 and is similar to that observed forUQ-0 (11) and other small nonquinone molecules that bind atthe QB site (14). The fact that BQ exhibits concentration-dependent electron-transfer kinetics from AQ at the QA site toBQ at the QB site suggests that, at micromolar concentrationsof BQ, the QB site is primarily unoccupied prior to the flash.This observation agrees with previous observations made forthe binding of UQ-10 to the QB site in reaction centercontaining vesicles (26).Dependence of the Apparent Quinone Affinity on -AG0e.

Using the cyt c oxidation assay, Fig. 4 confirms quantitativelythat variation of the -AG0- between QA and QB substantiallyalters the apparent affinity of the QB site for quinone. Thefigure shows that by extending the BQ concentration to highervalues (cf. Fig. 2A) that the measurement of a Kd value (200,uM) is achievable for BQ even with UQ-10 at the QA site.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 ,uMfor BQ. Similarly, when MeAQ is at the QA site to increase the-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 stronglyinfluence the Kd value of the quinone in the QB site. Theseresults also reveal that the Em of BQ in the QB site is justsufficiently favorable to reconstitute QB activity when UQ-10is at QA site.

-5 -4

Log 1,4-benzoquinone (M)

FIG. 4. Dependence ofapparent quinone binding at the QB site onthe -AG°. for QA to QB electron transfer in reaction center of Rb.sphaeroides R-26. The -AG0- was varied by replacing the nativeUQ-10 at the QA site (-Em = 0.07 V) with 15 ,uM AQ (-Em = 0.22V) or with 15 ,uM MeAQ (-Em = 0.29 V). The theoretical lines forKd were obtained as described in Fig. 3B. Reaction and conditionsfor measuring cyt c oxidation were as in Fig. 2 except that theconcentration of BQ was varied.

Proc. Natl. Acad. Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 2661

8(WBCh )2 Reduction Kinetics

No Adds

Cytochrome c Oxidation

UQIO(QA) AQ (OA )

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 centersfrom 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 cytc 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 oxidationtraces were obtained from samples containing 25 ± 5 AuM of the indicated QB candidates. The time and absorbance scales are the same for bothpanels.

QB Site Reconstitution with Low-Potential Quinones. In Fig.5, we take advantage of the large -AG°_ provided by AQ atthe QA site to demonstrate that the QB site will function withquinones that, like BQ, were believed to be nonfunctional atthe QB site. Thus, when the native UQ-10 occupies the QAsite, reconstitution of the QB site with 2,3-dimethyl-1,4-benzoquinone or 1,4-naphthoquinone yields marginal QBactivity, whether using either the BChl' reduction kineticsassay (Fig. 5A Left) or the cyt c oxidation assay (Fig. 5BLeft). However, when AQ is incorporated into the QA site,substantial QB activity is exhibited. The amount of cyt coxidized on the second flash relative to the first flash rangesfrom 40 to 70% for all the quinones tested (Fig. 5B Right)while the slow phase of the BChl' reduction kinetics con-stitutes >35% of the total BChl' (Fig. SA Right). Theobserved variation in QB activity for these quinones probablyreflects differences in their Kd values. These experimentsdemonstrate clearly that the QB site will bind and functionwith the methyl-substituted and ring-extended BQs thatresemble the head groups of the naturally occurring qui-nones, plastoquinone and menaquinone.QB Site Reconstitution with High-Potential Quinones. The

other way to generate a more favorable -AG°_ is to usehigher-potential QB candidates while retaining the nativeUQ-10 at the QA site. In Fig. 6 we show that the high-potential2,3-dichloro-1,4-naphthoquinone and the ortho-quinones,3,5-di-tert-butyl-1,2-benzoquinone and 9,10-phenanthraqui-none, all exhibit substantial QB activity when monitored bythe cyt c oxidation and by the BChl' kinetics assays.We have also examined the amplitude and kinetics of the

BPh electrochromic shift at 750 nm to eliminate the possi-bility that these quinones may be functioning merely asnonspecific acceptors that oxidize QA without actually en-tering the QB site. The results from these experiments (datanot shown) are very similar to those observed for BQ in Fig.3, thereby demonstrating that these high-potential quinonesare indeed functioning in the QB site. The experimentsstrengthen considerably our assertion that provision of asufficiently large -AGC° leads to easy detection of quinoneoccupancy at the QB site.

DISCUSSIONWe have demonstrated that previous efforts to reconstitutethe QB site of bacterial reaction centers with quinone ana-logues other than the native UQ were unsuccessful becauseof the small -AG0- between tested quinones at the QB siteand the native UQ-10 at the QA site. The relatively high in situEm value that prevails for the QA/QA couple of UQ-10restricts the examination ofquinones for binding and functionat the QB site to those that also have as high or higher Emvalues. Unfortunately, in vitro E1/2 measurements for qui-nones in dimethylformamide (25) and in situ Em measure-

BA( BChl )2 Reduction Kinetics

0

tcl0 : CI0

C,

CBCytochrome c Oxidation

FIG. 6. Function of high-potential ortho- and halogenated para-quinones at the QB site with the native UQ-10 at the QA site. (A)BChl' reduction kinetics in the absence (curve a) and presence(curve b) of 35 Am QB. (B) Cyt c oxidation on the first and secondflash in the presence of 35 Am QB Cyt c oxidation in the absence ofadded QB was similar to the trace in Fig. 5B. The conditions were asin Fig. 2.

0

0

a

~~~~~~~~~~~~~~-t--t- aIw--

b

a

I a0.002 A

-* I fT400 msec

WA -14wl-

'4-S

20-0 e200 msec

Biophysics: Giangiacorno and Dutton

2662 Biophysics: Giangiacomo and Dutton

ments for quinones at the QA site'(18) reveal that the effectsof ring extension and most other substitutions tend to lowerthe oxidation-reduction potential values relative to BQ.However, we have diminished this restriction by eitherdeliberately choosing QA replacements with in situ Em valuesthat are much lower than UQ-10 or by choosing QB candi-dates with in vitro E1/2 values very much higher than UQ-10.From this work, another view of the QB site emerges.

Rather than being specific for a single-ringed UQ, it is clearthat the site will bind and support the function of othernaturally occurring benzo- and naphthoquinone structuresthat are found in higher plants and other bacteria. In addition,the QB site will function with synthetic quinone analoguescontaining up to three rings, as well as with quinones thathave their carbonyls arranged in the ortho rather than themore familiar para configuration.These results are at least qualitatively similar to those

found for the QA site (8). Work with the QA site has furtherdemonstrated not only that there is no requirement for apara-carbonyl structure but also that the contribution tobinding free energy from the "second" carbonyl is small (8).From the work presented here, it is reasonable to considerthat the QA and QB sites may exhibit the same minimalrequirements for binding. That is, binding to both the QA andQB sites may require only a partially unsaturated ring that issubstituted with one carbonyl. This model is remarkablysimilar to that deduced by Trebst et al. (27) from inhibitionstudies done with the urea/triazine class of herbicides on theQB site of the reaction center of photosystem II of greenplants. The binding requirements of the QB site for triazinesand, by implication, for plastoquinone revealed from thisstudy are given as an aromatic ring substituted with an Sp2hybridized group with a lone electron pair and substitutedwith a lipophilic moiety (27).The significant influence of -AG°_ on the apparent affinity

of the QB site for quinonoid compounds that we havedescribed is equally important for studies into herbicidesensitivity and site-directed mutagenesis at the QA and QBsites. Although it is obvious that a changed amino acidresidue can impart changes in the strength of interaction of anative quinone or herbicide at the QB site, it is also the case,as we have shown, that large increases or decreases inquinone Kd, and hence herbicide activity (IC50 values), canarise from an altered -AG'-. Further, since the basicelectrochemical properties of both the QA and QB sitescontribute to -AGO-, it follows that the source of mutation-induced alterations in either the apparent affinity for quinoneor in the herbicide susceptibility at the QB site is just as likelyto reside in the QA site as in the QB site. Thus a change in theamino acid complement to alter the in situ Em values forquinone in the QA site can have major effects that areobserved in the reactions at the QB site. This prediction issupported by our preliminary unpublished experiments inwhich AQ replacement at the QA site results in not only adecreased K' for UQ-0 at the QB site, but also an increase inthe amount of Ametryne, a QB site inhibitor, needed to effectinhibition. It follows that site-directed mutagenesis at the QAsite could also be useful for either increasing or decreasingthe observed herbicide sensitivity of the QB site. Thus,mutations deliberately directed to lower or raise the Em valueof quinone in the QA site will confer upon the QB site acontrolled degree of resistance or susceptibility to herbicides.Clearly, recognizing the influence of -AG'- on quinonespecificity is vital not only for obtaining a comprehensiveunderstanding of the functional and structural properties of

the QB site in both plants and bacteria, but also perhaps as animportant parameter in devising biotechnological strategiesin the field.

We thank Marilyn Gunner for her valuable discussions. We arealso grateful to Roger Prince for his extensive measurements of E1/2values for quinones in dimethylformamide. This work was supportedby a grant from the National Institute of Health (GM27309).

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