On the mechanism of the PMS-effected quenching of chloroplast fluorescence

On the Mechanism of the PMS-effected Quenching of Chloroplast

Fluorescence

GEORGE PAPAGEORGIOU

Nuclear Research Center “Drmocrltus,” Department of Bm1og.v. Athens, Greece

Received July 2. 197-l

Evidence is presented which suggests that IV-methylphenazonium methosulfate sup- presses the fluorescence of ~~-(:l.-f-dichlorophenyl)-l,l-dimethylurea-poisoned chloroplasts by two mechanisms: (i) indirectly. hy catalyzing the buildup of the phosphorylating potential X, across the thglakoid memhrane: (ii) directly, by interacting with excited chlorophyll molecules.

Arguments in support of direct quenching are as follows: (i) N-methylphenazonium methosulfate is an efficient quencher of the fluorescence of chlorophyll a in methanol; (ii) the dark-irreversible portion of the light-induced fluorescence lowering in the presence of N-methylphenazonium-methosulfate increases with the concentration of the cofactor, (iii) N-methylphenazonium methosulfate lowers the fluorescence of chloroplasts at an excitation that is too weak to allow formation of X b..

Ascorbate-reduced N-methylphenazonium methosulfate (PMS-SQ) is a more efficient direct quencher of chloroplast fluorescence than oxidized PMS hecause the thylakoid membrane is more permeahle to the reduced species. The permeability to these quenchers is enhanced by the light-induced protonation of the membrane, and suppressed by added Mg2+. Different permeability barriers appear to exist for the direct and for the X,-mediated quenching by N-methylphenazonium methosulfate. since the latter is known to be insensitive to the presence of Mg’*.

A number of recent studies have demon- strated a light-dependent quenching of chlorophyll fluorescence under conditions where cyclic electron transport is the pre- dominant photosvnthetic function. This effect is distinct from the fluorescence intensity fluctuations that attend the redox activity of photosystem II (l), and it re- quires strong exciting light and the presence of nonphysiological cofactors of the cyclic electron transport. such as menadi- one (2). N-methylphenazonium methosulfate (PMS)’ (2-l()), 2.3,5.6-tetramethyl-p-

’ Ahhreviations: A. e.g., A,,,, absorbance at 647 nm; CCCP, carhonyl cyanide m-chlorophenyl hydrazone; Chl. chlorophyll: DAD. ‘,3,5,6-tetramethvl-p- phenylene diamine; DCMU. 3.(3,4-dichlorophenyl)-l,l-dimethylurea; DNP, 2,Gdinitrophenol; DPIP, ‘.6-dichlorophenol indophenol: FCCP, car- bony1 cyanide p-trifluoromethoxy-phenylhydrazone: MES. (N-morpholino)ethanesulfonate; PMS, N- methylphenazonium methosulfate; PMS-SQ, a semi-

phenylene diamine (DAD) (7, 11, la), and 2,6,-dichlorophenol indophenol (DPIP) (13-15). Light-dependent quenching in the presence of these cofactors has been observed in isolated chloroplasts from higher plants (2, 446, 8, 11, 12), in photosynthetic algae (:3. 9, 13, 14), and in bacterial chro- matophores (7, 15). The quenching has been attributed to the buildup of the transmembrane electrochemical potential X,Y2 which supplies the free energy for the phosphorylation of ADP, and in particular to the protonation of the thylakoid membrane (5, 11. 12). The resulting ultrastruc- tural changes in the membrane are sup- posed to enhance the nonradiative deexci- tation of chlorophyll a (Chl a), either by

quinoid form of PMS; X,, light-induced transmemhrane electrochemical potential.

2XE is used here to denote the light-induced transmemhrane electrochemical potential.

FLUORESCENCE QIJENCHING BY PMS 391

facilitating the penetration of quenchers, PMS were prepared daily in distilled water, and their

such as molecular oxygen, to the pigment concentration was corrected on the basis of the molar

phase (6), or by altering the spatial ar- absorptivity given by Zaugg (Ref. 24; tjHfi 26,300

rangement of the pigments (8, 9). Mm1 cm I). The concentrations of carbonyl cyanide

In addition to the X,-mediated quench- m-chlorophenyl hydrazone (CCCP) and 2.4.dinitro-

ing, the cofactors of the cyclic electron phenol (DNP) were adjusted by weighing only. Micro-

transport, as oxidants, would be expected liter quantities of the quenchers were introduced

to quench also by means of direct interac- directly to the chloroplast sample in the spectropho-

tions with the excited chlorophylls. Such tometer cuvette where they were mixed with a Teflon plunger.

compounds are known to quench the Pure Chl a was prepared according to Strain and fluorescence of Chl a both in solution (16, Sherma (25) from Iyophilized cells of the blue-qreen 17) and in uI:uo (18-23). A third contribu- alga Phormidium luridurn. The middle portion of the

tion to the measured reduction of fluores- Chl a band that is eluted from the powdered sugar

cence may further arise from an inner filter column with light petroleum ether containing 0.5’r

attenuation ‘of the excitation, and/or the propanol-1 was collected. and its purity was examined

fluorescence intensity, especially when re- by absorption spectrophotometry. Only the absorp-

latively high concentrations of the cofactor tion bands of Chl a were present. The main red and

are employed. The inner filter effect ma) blue absorption maxima of the eluate were located at

not be anticipated readily from the absorp- 662 nm and at 429 nm. and A,,JA,,, was determined to he 0.87. Chl (I was transferred to methanol b>

tion spectrum of the fully oxidized cofac- evaporating the petroleum ether at 40°C and in L‘UCUU tor, as it ma:y be due to a reduced deriva- in a flash evaporator. The red and blue absorption

tive. maxima of methanolic Chl a were located at X351 nm.

In the prelient work, we reexamine the and A66,JA,32 was very close to 1. These spectral

effect of oxi,dized and ascorbate-reduced characteristics compare far-orably with those of Goed-

PMS on the fluorescence of isolated spin- heer (26). Only freshly prepared methanollc solutions

ach chloroplasts whose ability to photo- were used, since the absorption spectrum of Chl o in

evolve oxygen has been poisoned with :3- this sol\zent appears to change r,n prolonged storage.

(3,4, - dichlorophenyl) - 1.1 - dimethylurea Fluorescence was measured with a pre\-iously de-

(DCMU). The presence of this metabolic scribed instrument (211. Exciting light of regulated intensity was obtaincxd from the power-stabilized

poison ensures that the observed fluores- emission of a 150 II’ projector lamp, by means of glass cence changes do not have their origin in filters and 5 cm of a 5’; w/v solution of CuSO,, u.hich

the redox activity of photosystem II. Our served as a heat filter A spring-loaded photographic

results indicate that in addition to induc- shutter (Compur 1) allowed full intensity illumin:ltion

ing an X,-mediated quenching oxidized of the chloroplast sample within 1 ms. The fluores-

and ascorbate-reduced PMS are strong cence signal was either fed to a strip-chart recorder or

direct quenchers of excited Chl a. Further- to a Tektronix ,549 storage oscilloscope. Further :>pec-

more, it appears that at relatively moder- tral details are given in the legends ttr the figure,.

ate to high concentrations of ascorbate- Light intensities at the plane of the cuvette was

reduced PM8 the inner filter contribution measured with a calibrated Hi-Ag Eppley therm~~pile (12.junction. air type) The actual intensity reac,hing

to the measured lowering of chloroplast the sample is somewhat lower than the value? gilEn in fluorescence is present and unavoidable. the legends to the figure*. due to ahsorption and

MAT‘ERIALS AND METHODS reflections hy the glass side of the cuvette.

Absorption spectra were measured with a Bausch Broken chloroplasts were prepared conventionally and Lomb Spectronic 50.5 spectroph(‘tometer (mea-

(21) from fresh market spinach in 300 mM sucrose, 50 suring beam. 5 nm) and with a Hitachi Model :356 rn~ (N-morpholino)ethanesulfonate (MES)-KOH, dual-wavelength spectrophotometer (measuring pH 6.4, and thev were maintained as dense stocks in beam, 1 nm). The absorption spectrum of ascorbate- ice bath. Samples were derived by diluting the stock reduced PMS was obtained by adding microliter with the sucrose-MES buffer to an absorbance differ- quantities of methanolic PMS directly to the spectro- ence (A,,,- A,,,) of 0.40 and adding DCMU to 20 FM. photometer cuvette that contained 6.9 rnbf ascorbic Bovine serum albumin, 0.2% w/v. was included in the acid in methanol (ahout 200.fold excess ober PMS). buffer until it was determined to be unnecessary. To Reagent grade chemicals without further purifica- some samples 1 mM of freshly prepared ascorbic acid tion were used. Adequate incubation times were was added to reduce the PMS. Stock solutions of provided for with all additions to the c,hloroplast>.. All

392 GEORGE PAPAGEORGIOU

measurements were made after the samples were brought to room temperature.

RESULTS

Absorption spectra of oxidized and ascorbate - reduced PMS. Figure 1 illustrates the absorption spectra of methanolic solutions of oxidized and of ascorbate-reduced PMS. In the presence of excess ascorbic acid, the solution of PMS takes the deep- green color of the semiquinone free radical (PMS-SQ; Ref. 24). This compound is characterized by two absorption bands. A3,,(t = 8500M~‘cm~‘)andA,,,(t = 7800 M ’ cm- ‘), as well as by a broad region of weak absorption that extends to beyond 700 nm. Due to the interference by the strong uv absorption of ascorbic acid (A,,,), we were unable to measure the absorption of PMS-SQ below 300 nm. The absorbance of PMS-SQ was linear with the concentration and reasonably stable to per- mit determination of molar absorptivities. This was done at two wavelengths, 360 nm and 647 nm, and on the basis of the known molar absorptivitv of PMS at 338 nm (24).

A noteworthy feature of the absorption spectrum of ascorbate-reduced PMS in Fig. 1 is the absence of the characteristic absorption of the fully reduced derivative (PMSH) at 332 nm (24), although the reductant was present in great excess. PMSH, prepared as a white precipitate bs reducing PMS with excess sodium dithio- nite, was found to be insoluble in water,

petroleum ether, and methanol, while in benzene solution it reoxidized rapidly to the green semiquinone. On the strength ot these observations we shall assume that in the presence of excess ascorbate, PMS exists in the extrathylakoid space of chloroplast suspensions as a semiquinone.

Quenching of the fluorescence of Chl a in solution. A prerequisite for assigning the role of direct quenchers in vice to PMS and PMS-SQ is an analogous behavior of these compounds toward Chl a in vitro. Figure 2 shows the quenching of the fluorescence ot methanolic Chl a bv PMS to obey a linear Stern-Volmer relation, with a quenching constant, K, ~. (F ~ F’)/F’C. equal to 280 +~ 16 Mm 1 (six determinations). The corre- sponding collision rate constant k Ky7 ‘, calculated on the basis of a fluorescence lifetime for Chl a in methanol. T 6.9 nsec (27), is 4.06 x 10’” Mm ’ ,s ‘, i.e.. in the range of t,he diffusion-imposed limit for biomolecular reactions (28).

The Chl a concentration in these experiments (A,,, r 0.35) corresponds to an average intermolecular distance of 965 A. which is much greater than the distance for 50% probability of transfer (R, 69 A; Ref. 29). It is, therefore. unlikely that intermolecular transfer of excitation energy contributes to the observed quenching. We confirmed this expectation by measuring the effect of PMS on a lo-fold concentrated solution of Chl a in methanol (A,,,5 - :i..51. Although, here, energy transfer is 100 times

Fm. 1. Absorption spectra of’PMS and PMS-SQ in methanol. The dashed line reproduces part of the PMS-SQ spectrum on a 10.fold expanded ordinate scale. PMS-SQ was obtained by adding microliter quantities of methanolic PMS to a spectrophotometer cuvette that contained 2.9 ml of 6.9 mM ascorbic acid in methanol. Molar absorptivities are based on the value given for PMS h> Zaugg (Ref. 24; E,,, = 26,300 Mu ’ cm ‘).

FLUORESCENCE QUENCHING BY PMS 393

PMS, ml.’

FIG. 2. Stern-Volmer plot of the quenching of the fluorescence of Chl a in methanol (A,,, 0.35) by PMS. Fluorescence excitation, 620 nm; half-hand width, 12 nm; Intensity 4900 ergs.cm “-see ‘. Fluo- resence observation, 675 nm; half-hand width 6.6 nm; guard filters Corning C.S. 2.58 and Baird-Atomic. Inc.. interference filter 67.5 nm. F and F’ denote unquenched and quenched fluorescence. respectively. Exactly the sane F/F’ ratios were obtained with a IO-fold concentrated solution of Chl a in methanol (A,,, 3.5).

more prohallle, the measured quenching constant is exactly the same as that of the dilute solution.

Due to the strong inner filter absorption. we were unable to measure the quenching parameters of PMS-SQ quantitatively. Taking into account the “front -face” fluorescence ‘detection geometry of our flu- orimeter, we estimated using Duysens’ method (30) and absorptivity values from Fig. 1 that 20 ELM PMS-SQ attenuates the true fluorescence signal by 5%. while at 50 PM attenuation is l%Q. Qualitatively, however, the measured fluorescence lowering in the presence of PMS-SQ is greater than the inner filter e:;timates, suggesting that the semiquinone quenches directlv. At the concentration used in these experiments ascorbic acid had no effect on the fluorescence of Chl a in methanol.

In contrast to PMS, the photophosphorylation uncoupler CCCP. which with the related compound FCCP has been shown to suppress the variable fluorescence of Chl a in civo at moderate to strong excitation (9. 3L34), does not quench the fluorescence of methanolic Chl a up to the tested concentration of 2 mM. Clearly. therefore, the in LliLw quenching by CCCP does not involve direct interactions of this uncoupler with the excited bulk chlorophylls. On

the other hand, the uncoupler DNP. which has been shown to quench the fluorescence of Chl a in uivo (9), is also a quencher for Chl a in methanol (KQ = 35 M-I). In this capacity, it behaves as a typical nitroaromatic quencher of porphyrin fluorescence (16, 21).

Quenching of chloroplast fluorescence under strong excitation. The X,-mediated quenching of chloroplast fluorescence re- quires strong exciting light (5), but the experimentally measured lowering with PMS may also be due, partly, to direct quenching and to inner filter effects. We shall evraluate the contributions of these quenching modes by reference to Fig. 3. which is composite of actual oscilloscopic traces.

F, is the steady-state fluorescence 01 DCMU-poisoned chloroplasts under continuous intense excitation; it consists roughly of 65“G variable yield and 355 constant yield fluorescence. The lower level F, is attained after the introduction ot PMS to the illuminated chloroplasts and reflects the combined effect of all quenching modes. Because of the low concentration of PMS (10 PM) in these experiments, the inner filter contribution to the fluorescence lowering does not exceed 2’7 and it can be disregarded. During a dark period, that follows the light period, part of the lost capacity for fluorescence is recovered. This is shown by the trace from F,? to FH, which connects transient spikes (rise time 1 msec) obtained bv reexciting the chloroplasts at the end of‘increasingly longer dark inter- vals. The level Fs is reproducible in suc:ces- sive dark-light cycles, proving that the photolabile PMS was not destroyed b!; the blue exciting light of’ the experiment. We shall consider the dark-reversible difference FN ~ F, as the true measure of’ the light-induced quenching that manifests the presence of X,, and the dark-irreversible difference F, - F, as the true measure of direct quenching.

Figure 4 illustrates PMS concentration curves for F, FH, F, Fs, and FH - Fs; Fig. 5 shows similar results obtained in the presence of excess ascorbate. The concentration curves for the difference F, ~ FR, that measures direct quenching, are bi-


r r FR .---

,

-

TIME

Frc. 3. PMS-induced kinetics of the fluorescence of DCMU-poisoned chloroplasts during repetitive cycles of light and darkness. The reaction mixture contained in 3 ml: chloroplasts A,,, = 0.4; DCMU. 20 pM; sucrose, 300 my MES .KOH, pH 6.4, 50 mM. Fluorescence excitation through Corning filter C.S. 4-72 and 5 cm of 5% CuSO,; intensity 29,100 ergs.cm %ec-‘. Fluorescence observation. 680 nm, with guard filter Corning C.S. 2.58. F,. steady level of fluorescence of DCMU-poisoned chloroplasts in continuous light; Fs, low fluorescence level obtained after the introduction of 10 pM PMS to the illuminated chloroplast sample; F,, fluorescence recorded in about 1 ms after the onset of excitation on a PMS-containing sample that has been given a long dark rest, Similar kinetics were recorded when 1 rnM ascorbic acid was included in the mixture. The figure combines several oscilloscopic traces.

FIG. 4. The variation of the differences F, - FR, F, F, and F, Fs with the concentration of PMS in the chloroplast sample, Experimental details are those of Fig. 3; cf. also Materials and Methods.

phasic. The transition occurs at about the concentration where the greater part of the quencher-sensitive variable fluoresence is destroyed (3-4 FM; cf. curves F, Fs). Due to the second phase, the differences F/~-F, are not maximized, but they con- tinue to increase with the concentration of oxidized and ascorbate-reduced PMS. At

all concentrations, the reduced species is the stronger direct quencher. In contrast, the concentration curves for the difference F, - Fs show a typical saturation pattern. As suggested by the faster rise of F,-F,? in Fig. 5, ascorbate-reduced PMS is more effective than oxidized PMS in catalyzing the X E-mediated fluorescence lowering; at


Y z 04

2

ii2 8 02

3 LL

0 0 2 4 6 8 10 12 14 I6

ASCORBATE - REDUCED, PMS, PM

FIG. 5. The variation of the differences F, FR, F, F,,. and F, F, with the concentration of ascorbate-reduced PMS in the chloroplast sample. Experimental details are those of Fig. 3: ascorbic acid, 1 mM; cf. also Materials and Methods.

saturating concentrations, however, both cofactor species are equally effective.

We may assume that PMS catalyzes energy-coupled cyclic electron transport by binding reversibly to appropriate sites of the thylakoid membrane. If AF = FH - Fs measures the resulting energy-dependent quenching, the following variant of the Lineweaver-Burk equation should be obeyed.

1 1 K, 1 ~~ - -_-, z AF,,,,, + AF,,,,, C

where C is the molarity of the cofactor. and K, = [M] [Q 1/[MQ] the apparent dissociation constant of the complex cofactor (Q) ~ membrane site (M). Figure 6 shows that plots of (FR -- Fs) - 1 against Cm’ are linear when PMS is. less than 6-8 pM. At higher concentrations, the effect of direct quenching becomes prominent and causes the double-reciprocal plots of Fig. 6 to deviate from linearity. Maximal X,-mediate quenching (ordinate intercept) is about 36%, accounting for nearly one-half of the variable fluorescence. The greater effectiveness of reduced over oxidized PMS in catalyzing the X,-mediated quenching of chloroplast fluorescence is evidenced by the apparent dissociation constants of the membrane complexes of the two species (abscissa inte.rcepts); these are 0.86 pM and 2.08 pM for ascorbate-reduced and oxidized PMS, respectively.

Quenching of chloroplast fluorescence under u:eah excitation. By employing low- intensity excitation it is possible to stud! direct quenching by PMS independtly of the X,-mediated effect, which is negligible under these conditions. Figure 7 presents the results of a relevant experiment in terms of Stern-Volmer plots. The chloroplasts were given weak red excitation (620 nm; 350 ergs.cm- 2. set ‘) and the fluorescence was monitored at 685 nm. Confirm- ing a previous report (5). we observed only slight quenching at cofactor concentrations that would have sufficed, for maximal X,-mediated suppression of Chl a fluorescence, and for nearly maximal direct quenching, in strong exciting light. At higher cofactor concentrations, however, the quenching effect becomes more prominent, but ascorbate-reduced PMS (cf. blow-up of the absorption spectrum of PMS-SQ in Fig. 1). introduces an inner filter component to the observed fluorescence lowering.

The quenching ratios F/F’ in Fig. 7 for ascorbate-reduced PMS (curves A,A’) are corrected for inner filter attenuation. on the assumption that the samples are t,rue solutions. The correction breaks down in the higher concentration range, since the samples are scattering and this intensifies the inner filter effect by increasing the path lengths travelled by the excitation and the fluorescence. No correction is required for oxidized PMS, since it has no absorption

396 GEORGE PAPAGEORGIOIl

itself a quencher c:f the fluorescence of Chl a in Llillo.

DISCUSSION

Our results indicate that low concentrations of PMS suppress the fluorescence of DCMU-poisoned chloroplasts by means of two processes: (i) bv catalyzing the buildup of the electrochemical potential X, across the thylakoid membrane: and (ii) by in teracting directly with excited bulk chlorophylls. It is probable that PMS has to ovrercome different membrane permeahil- ity barriers for each of these processes. At higher cofactor concentrations. the inner filter contribution to the measured fluorescence lowering becomes increasingly evident. especially in the case of excess ascorbate that converts PMS to a semiquinone characterized hy a broad absorption spectrum.

Confirming the role of the transmern- hrane electrochemical potential X,. as a suppressor of Chl a fluorescence (5. 11 ), we observed the PMS-induced quenching to be more extensive in strong exciting light.

PMS, PM-’

Ftc. 6. Double-reciprocal plot of the difference F, ~ Fs versus the concentration of PMS, in the presence and in the absence of 1 rn~ ascorbic acid. Experimental details as in Fig. 3: cf. also Materials and Methods.

beyond 520 nm (curves B.B’). It is noteworthy, that in the low concentration range, where inner filter errors are mini- mal, oxidized and ascorhate-reduced PMS are equally effective as direct quenchers.

Mg2+ and other divalent cations have been shown to influence the accessibility ot the chlorophylls in situ to neutral quenching molecules, such as nitroaromatic compounds (20, 21), presumably through their action on the ultrastructure of the thylakoid membrane (35). On the other hand. the X,-mediated quenching appears to be insensitive to the presence of Mg2+ (8, 12). Figure 7 (curves A’, B’) shows that 20 mM

MgCl, lowers the quenching effectiveness of both oxidized and ascorbate-reduced PMS. At 20 mM the effect of MgCl, is maximal, since on increasing its concentration to 100 mM we obtained exactly the same quenching curves.

Reflecting the absence of X,-mediated quenching, the PMS-induced fluorescence lowering in weak excitation is not reversed by a dark rest of the chloroplasts, nor by 5 PM CCCP. On the other hand. when the 620-nm excitation was raised to 10.900 ergs,cm-“.s-‘, the fluorescence lowering induced by 20 pM PMS was inhibited 40-70s’ by 5 PM CCCP. This uncoupler, however, exerts a complex influence, being

P’48 UN

FIG. 7. Stern-Volmer plots of the quenching of the fluorescence of DCMU-poisoned chloropiasts. in the presence (A. A’) and in the absence (R. B’I of 1 rn~ ascorbic acid. and in the presence (A’. I!‘) and in the absence (A, H) of 20 mM MgCl,. Fluorescence excitation, as in Fig. 2; intensity, 850 ergs .cm I-see ’ Fluorescence observation. as in Fig. :i. Curves A and A’ are corrected for inner filter errors t)?; PMS-SQ on the basis of’ the molar ahsorptivities given in Fig. 1, and by assuming a nonscattering sample. Similar results, as those of curves A’ and B’ were obtained when the concentration of Mg” was raised to 100 mM.


and to be partly reversed by darkness and uncouplers of the photophosphorylation such as CCCP. On the other hand, the direct quenching interactions between the cofactor and excited chlorophylls were made evident by the following observations: (i) PMS is a highly efficient quencher of excited Chl a in solution; (ii) oxidized and ascorbate-reduced PMS quench the fluorescence of chloroplasts at an excitation that is too weak to generate the electrochemical potential X,; and (iii) the dark-irreversible portion of the PMS- induced fluorescence lowering in strong light increases with the concentration of the factor.

The approach of the rate of quenching collisions between excited Chl a and PMS in methanol to the theoretical limit. indicates that there is no preferred direction for an effective encounter of these two molecules. This is consonant with the delocalization of a positive charge over the entire PMS cation and the delocalization of the excitation in the porphyrin ring of Chl a. An important consequence is that in UI’LYI the quencher can interact with any one of its neighboring chlorophylls that may be- come excited. Energy migration greatly enhances the quenching effectiveness, since excitons generated by photon capture at distant chlorophylls have a chance to reach the PMS site. The linearity of the Stern-Volmer plot obtained for Chl a in methanol (‘Fig. 2) suggests that the quenching is (due to a single process. We are inclined to believe that “static quenching” (36) does not contribute to the measured fluorescence lowering since we did not detect new absorption bands when PMS was added to the solution of Chl a in methanol, or to chloroplasts. It appears, therefore, the PMS quenches “dynamical- Iv” by interacting only with the excited pigments. This conclusion is consistent with the report of Mohanty et al. (8) that PMS shortens the fluorescence lifetime of Chl a in uiLw

Due to strong inner filter effects, we were unable to measure the quenching by PMS- SQ quantitatively, but our results suggest to us that the semiquinone is a quencher in vitro, and pos.sibly also in cico (Figs. 5 and

7). Another uncoupler of the photophosphorylation, DNP, was found to quench the fluorescence of methanolic Chl a, but less efficiently than PMS (I& = 35 51-l). This may evidence a preferred orientation of DNP for an effective collision with Chl a. Finally. CCCP which exerts various pro- found quenching effects on the fluorescence of Chl a in uklo (9, 3-34) was completely inert toward Chl a in methanol. Clearly. therefore, the in. uivo effect of CCCP is entirely indirect.

The PMS-induced fluorescence lowering in strong light (Fig. 3) can be kinetically resolved into an X,-mediated component (F, F,) and a direct quenching component (F,-FR). The first is maximized at low concentrations of PMS, or ascorbate- reduced PMS and obeys typical enzyme kinetics (Fig. 6); in contrast. the second does not saturate with the concentration of the cofactor, although its concentration curves are biphasic. As the common ordinate intercept in Fig. 6 indicates, oxidized and ascorbate-reduced PMS cause the same maximal (36(F) X,-mediated quenching, although below saturation the reduced species is always more effective (Figs. 446). These properties may signify faster penetration by the reduced species of a membrane permeability barrier, probably because it is less positive than oxidized PMS. A similar conclusion was reached by Hauska (37) who found ascorhate-reduced PMS to be a better catalyst of the cyclic electron transport than oxidized PMS. Once, however, the catalysis sites are reached, the PMS redox couples (which we presume to be PMYPMS-SQ) are poised by the light-driven cyclic electron transport. According to this interpretation, maximal XL-mediated quenching implies the occupation of all the available catalytic sites by PMS, while the faster permeation by the reduced species manifests itself in the smaller value of the apparent dissociation constant of its membrane complex (abscissa intercepts, Fig. 6).

Direct quenching is more pronounced in strong light, and ascorbate-reduced PMS is more effective than its oxidized counter- part in causing it. In weak exciting light, on the other hand, both oxidized and ascor-


bate-reduced PMS appear to be equally effective direct quenchers (Fig. 7, A and B). Mg2+, which reportedly does not influence the cofactor-induced fluorescence lowering in strong light (8. la), is shown here to diminish the direct quenching by PMS in weak light (Fig. 7 A’ and B’). We would like to interpret these observations in terms of known properties of the thylakoid membrane, whose structural elements may impose stereochemical and electro- static barriers on the mobility of charged molecules.

Inner chloroplast membranes are known to carry bound negative charge (38, 39). which should impede the freedom of mo- tion of the PMS cation and of the semiquinone radical. During the formation of the electrochemical potential XE, only a frac- tion of the protons taken up from the extrathylakoid space appear free inside the thylakoid (40). Presumably, the remainder lowers the membrane-bound negative charge and causes local conformational changes (39). Accordingly, in strong light, the PMS-catalyzed protonation of the thylakoid membrane may facilitate the penetration of oxidized and ascorbate-reduced PMS to the pigment phase, and thus lead to more extensive quenching than would have been realized in weak light.

The fact that the X,-mediated quenching in strong light is insensitive to Mg2+ (8, 12), while direct quenching in weak light is sensitive, may indicate different permeability barriers for each quenching mechanism. Finally. the suppression of the quenching effectiveness both of oxidized and of ascorbate-reduced PMS by Mg2+ may imply that this metal cation primarily modifies the stereochemical properties, rather than the electrical properties of the membrane.

ACKNOWLEDGMENTS

I am indebted to Miss J. Isaakidou and to Mrs. M. Tsimilli-Michael for technical assistance in some of the experiments reported in this paper.

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