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A further study of P430: A possible primary electron acceptor of photosystem I
ARCHIVES
OF
RIOCHEMISTRY
Further
Study
AND
RIOPHYSICS
of P430:
147, 99-108 (1971)
A Possible
Primary
of Photosystem TETSUO Charles F. Kettering
HIYAMA
AND
Research Laboratory,
Electron
Acceptor
I’ BACON
KE
Yellow Springs,
Ohio &387
Received June 1, 1971; accepted July 22, 1971 The nature of P430, which is represented by an absorption change observed in isolated photosystem- particles, haa further been investigated using spinach subchloroplast particles: (1) The kinetic relationship has been established between three components involved in an artificial cyclic electron flow: P430 -+ N, N, N’, N’-tetramethyl-p-phenylenediamine + P700 hv T (2) The recovery (reoxidation) of P430 was a pseudo-first-order reaction with respect to the artificial acceptors, e.g., methyl viologen and safranine T. (3) The back flow of electrons was a direct reduction of the photooxidized P700 by the photoreduced P430. (4) The quantum yield and effective wavelengths for P430 photoreduction were identical with those for P700 photooxidation. The results further support P430 as the primary electron acceptor of photosystem I in green plant photosynthesis.
insight into the primary events in photosystem I of green-plant photosynthesis.
In previous communications (1, 2) we have reported an absorption change around 430 nm in several photosystemparticles isolated from blue-green algae and spinach chloroplasts. This spectral component, tentatively designated as “P430” from its absorption maximum, was found to be of particular interest since its onset time upon flash excitation was comparable to that of P700 and its recovery was accelerated by photosystemelectron acceptors, including ferredoxin. It was further shown that the
METHODS
AND MATERIALS
Throughout the present work, photosystemparticles fractionated from spinach by digitonin treatment according to Anderson and Boardman (3) were used. The particles (D-144) were stored at -80” in sucrose solution as described by Hauska et al. (4).
Absorption changes were measured by a singlebeam spectrophotometer described previously (5). The measuring light was provided by a quartziodine lamp (GE-1958, 150 W) passing through a 500-mm Bausch & Lomb monochromator. In the region of 400-560 nm, two blue filters (Corning 4-96) were placed between the cuvette (10 X 10 mm, four-side polished) and the phototube (EM1 9558) to protect the latter from the red actinic flash. For the region of 56&800 nm, another monochromator was employed in place of the guard filters to minimize the interference from fluorescence as well as the actinic light. The intensity of measuringlight was found to be crucial for good reproducibility, particularly in the red region, where a relatively high intensity of measuring light was needed due to the lower sensitivity of the phototube in this region and loss of light in the additional monochromator. It should
photoreduction of some dyes kinetically corresponded to the recovery of photobleached P430. These results were explained by proposing that P430 is reduced in the primary photochemical event in photosystern I and then donates electrons to either a dye or ferredoxin. In the present paper, detailed evidence is presented which would lend further support to the above proposition and provide some 1 Contribution No. 430 from the Charles F. Kettering Research Laboratory, Yellow Springs, OH. Work supported in part by a National Science Foundation Grant GM-8460. 99
100
HIYAMA
be noted that theoretically even a weak measuring light could cause some absorption changes. In practice, however, a compromise intensity can be chosen that would cause little effect and yet provide a tolerable signal-to-noise ratio which may be improved by signal averaging. Typical measuringbeam intensities used were 100 erg crnm2 set+ at 700 nm and 20 erg cmp2 set-’ at 430 nm as measured by a Kettering Model 68 radiometer. The excitation light was provided by a xenon flash lamp (GE, FT.403) passed through appropriate filters; an interference filter (65&730 nm) plus Corning 2-58 and 2-64 for the red; an interference filter (Baird-Atomic, 400-480 nm) plus Corning 4-96 for the blue. For the quantum yield measurement, IO-nm wide interference filters (Baird-Atomic) were employed together with Corning 2-58 to obtain narrow-band actinic flash. The reaction mixture was cross-illuminated through one side of the cuvette at right angle to the measuring beam. The excitation energy was measured by a calibrated photodiode (Lite-Mike, EG&G) and an oscilloscope. As described earlier (5)) the flash duration was measured to be 20 psec at half-peak height. The intensities used routinely were 1.8 X lo-? J cme2 for the red and 2.2 X 1OW J cn-2 for the blue at the surface of the cuvette. The red flash was about eight times stronger than the saturation intensities in most cases, while the blue one was slightly above the saturation point in order to minimize the fluorescence. The photocurrent from the phototube was amplified by a differential amplifier balanced with fixed off-set voltage, usually 1.0 or 2.0 V. The amplified difference signal was fed into a signal averager (Fabri-tek Model 1062 Instrument Computer), which also triggered the flash with an appropriate timing. The signals induced by successive flashes were digitized and accumulated in the memory device of the signal averager. The averaged signal from several measurements stored in the instrument could then be displayed and recorded on a chart paper by an X-Y recorder. In principle, using the signal-averaging technique, all absorption changes induced by a flash should be reversible (e.g., cyclic or noncyclic reaction with excess ascorbate and oxygen; see the text); the entire system should return to the original state before the next flash is applied. In practice, the incoming signal in each experiment was monitored and confirmed, and an appropriate interval between flashes was employed. Routinely, a lo-see interval was used, and sometimes 20 set was employed when necessary. Notably, even with this precaution, some irreversible or extremely slow recovering changes might have been excluded from the final results in using this technique, as pointed out earlier (6). For the calculation of quantum yield, the trans-
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mittance of the reaction mixture was measured b. a Cary Model 14 spectrophotometer equipped with an end-on phototube located close to the cuvettes. The light scattering, however, was insignificant at 671 and 703 nm in I)-144 particles; the regular photometer set-up with W-cm distance from t,he cuvette to the photot,ube gave almost identical results. For the extinction coefficients of P700, 64 mM-’ cm-’ at 700 nm and 45 ITIM-’ cm-l at 430 nm were used. These values were separately determined for D-144 particles using TMPD as the donor, as will be mentioned later. Details and discussion on some discrepancy from previously reported values (7) will be reported in a forthcoming paper. All optical measurements were carried out at 22” in a cuvette with optical path length of 10.0 mm. Chlorophyll concentration was calculated as chlorophyll a from the optical density of the reaction mixture at 680 nm using an extinction coefficient of 8.4 X low2 ml pg-’ cm-l, which was separately determined in 80% acetone extract of D-144 particles, based on Mackinney’s coefficient (8). Digitonin, tricine, and ascorbic acid were obtained from Sigma Chemical Co.; N,N,N’,N’tetramethyl-p-phenylene diamine (TMPD2) from Eastman Kodak; safranine T from Aldrich Chemicals; methyl viologen from Mann Research. RESULTS
TMPD-Mediated
AND
DISCUSSION
Electron Flow Photosystem I
Around
As shown previously, the oxidized form of TlMPD (Wurster’s blue; abbreviated as TMPD+ hereafter) could serve as a secondary electron acceptor for photosystem I (2). Since the dye had a considerably high absorption at 575 nm, which is an isosbestic point in the P700 difference spectrum, it became possible to observe absorption changes due to the dye with little interference from P700 changes. The isosbestic point was separately determined by using phenazine methosulfate (PMS) as an electron carrier which had practically no absorption at this wavelength (see below). Figure 1 shows typical absorption changes 2 Abbreviations. TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine; TMPD+, oxidized TMPD; PMS, phenasine methosulfate; P430-, reduced form of P430; P700+, oxidized form of P700; MV, methyl viologen; MV-, reduced MV; Asc, ascorbate; DHAsc, dehydroascorbate; ST, safranine T; ST-, reduced ST.
A FURTHER
PM TMPD 67 I~PD(ox!O6
TMPD 67 TMPD 67 ASC 167 167 To” 330 “;C 2
2
2
c-4
800ms
c----l 20 msec FIG. 1. Flash-induced absorption changes in digitonin-fractionated photosystemparticles (D-144). Chlorophyll concentration, 12 rg/ml in 50 mM Trickle-NaOH, pH 8.0. For the reaction mixture used in the left column, 67 PM TMPD and 0.6 pM TMPD+ (formed by aeration) were added. In the middle column, additional 0.67 mM ascorbic acid was present. Both reaction mixtures were made anaerobic by evacuation before measurement. In the right column, 330 MM methyl viologen and air were introduced into the reaction mixture used for the middle column. For 703 and 575 nm; 4 and 16 blue flashes, respectively, were applied; for 442.5 nm, 32 red flashes. The arrow shows the point when flash was fired.
induced by a 20-psec xenon flash in spinach photosystem-I particles (D-144). As shown in the left column of Fig. 1, in the presence of 0.6 PM TMPD+ 3 and A7 p&zTMPD under anaerobic conditions, the light flash caused an abrupt decrease in absorption at 575 nm followed by a recovery which was kineCcally identical with that of P700 measured at 703 nm, with half decay t’ime of 300 msec. At a shorter t,ime scale shown at the bottom of Fig. 1, it was revealed that the abrupt de3 TMPD+ was obtained by a brief aeration of the reaction mixture before evacuation and determined by the difference in the absorption spectra before and after reduction by ascorbate at the end of the experiment.
STUDY
OF P430
101
crease at 575 nm actually took place with a half tjime of 4 msec. This half time was almost idenCca1 with that of P430 recovery (shown immediately above the 575..nm trace) measured at 442.5 nm, another isosbestic point4 of P700. These results indicat,e that TJIPD+ was directly reduced by photoreduced P430 and that photooxidized P700 was directly reduced by TMPD, thus creating a simple cyclic electron flow around photosystem I.” In t,he middle column of Fig. 1, TMPD+ was reduced to TnIPD by ascorbat.e, as confirmed by absorption spect’rum of the reaction mixture. Under these conditions, the recovery of 1’700 became faster (~~1~E 45 msec). These results may be explained as follows: Upon losing the secondary acceptor, TJIPDf, electrons from the primary acceptor had no ot,her place to go except back to P700+, the only available oxidant, under these conditions. The nearly ident’ical half decay times of P700 and P430 under these conditions indicate a direct int’eraction of these t,wo components. The above notion would further be supported by the experiments shown in the right column of Fig. 1. Upon addition of methyl viologen under aerobic conditions, P700 recovery became slow again wit’h a half time of 300 msec, a positive change at 575 nm appeared, and the I’430 decay became much fast,er (21/2g 1 msec). The observed change at 575 nm may represent the oxidation of TMPD followed by re-reduction by ascorbate. The oxidation of T-\IPD represent’ed by the positfive signal at, 575 nm could be coupled to P700 recovery, although the magnitude of observed T,\/IPD oxidation seemed too small t,o be coupled to P700 reduction. The undersized absorption increase at 57fi nm, however, was likely caused by a relatively fast re-reduction of TMPD by ascorbate (tllz g 4 It was found that the isosbestic points of ~700 tended to shift from preparation to preparation by a few nanometers. Usually the wavelength region of 442444 nm was scanned prior to measurements. 5 From the extinction coefficient of TMPD* at 575 nm 110.7 mM-* cm-l; also see Ref. (12)], the differential ext)inction coefficients for P700 and P430 could be calculated. The value quoted in Methods and Materials section and in a later section was so obtained.
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HIYAMA
0.2 set). This implication was further supported by the results shown in Fig. 2, in which the absorption increase at 57.5 nm became larger with increasing TMPD concentration (see Fig. 2B and C), whereas it became smaller with increasing ascorbsbte concentration (Fig. 2C and D). Further addition of a small amount of PMS virtually eliminated the change at 575 nm (Fig. 2E). PMS practically had no absorption at 575 nm either in the oxidized or reduced form and a much larger rate constant for P700 reduction (1.5 X 10’ k1-l see-I) (9) than that of TMPD (1.7 X lo4 h1-l se+, see below). Thus, PMS substituted TMPD for P700 reduction and eliminated the absorption change due to TMPD. Indeed, an isosbestic point of P700 was found to be at 575 nm in separate experiment’s employing PhIS as the elect’ron carrier. Note that absorption changes due to methyl viologen were also expected in this wavelength region. This contribution, however, seemed almost negligible judging from t,he above results, presumably because its autooxidation rate was extremely high under present experimental condit’ions. Direct reduction of P700 by TMPD was further demonstrated in Fig. 3, where the 703nm t -----
A
“v-s . t ----B
l-
L
575nm
t c-c--
t T :: 8.
TMPD ASC
33/~M 670
TMPD ASC -QMV
33 670 67
+TMPD ASC MV
100 670 67
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half-time of P700 recovery was plotted against TMPD concentration on a fulllogarithmic scale, as in the case of PRIS (9). The result’s resasonably fitted the theoretical curve (dashed line) assuming that the react,ion was pseudo-first-order with the rate constant of 1.7 X 10” M-’ see-‘. The flash-induced reactions shown in the right column of Fig. 1 thus can be explained as a noncyclic electron flow from ascorbate to oxygen mediated by TMPD, photosystem I (P700 and P430, activated by photon via antenna chlorophylls, etc.), and methylviologen. The results may be summarized as follows : For the cyclic
reaction
hu P430 x P439P766 --3 p706+ P430- + TMPD+ P790+ + TMPD For the Fig. l),
back
(the left column
(1)
(2) +
P430 + TMPD P796 + TMPD’
reaction
(the
P430 J% P430P700 -% P7cMlf p790+ f P430-
-
For the noncyclic
reaction
100 1300 67
TMPD ASC MV *PMS
100 1300 67 I.7
FIG. 2. Identity of the 575-nm change. Experimental conditions were the same as in Fig. 1 except for the concentrations of redox agents, which were added successively aa indicated on the right. Open arrows indicate either newly supplemented or increased ingredients.
middle
(3) (4) column
of
(1)
(59
P430 -$ P799 P43C- + P700+ + MV- + s HzOz
P43cr p799+ MV TMPD % 02 -----) %
TMPD+
+ x AC-
P709 + P430 (the right
(5) column), (1)
(2) --f -
Hz0
P430 + MV(6) P766 + TMPD+ (4) MV + % HoOz (7) + % 02 @I see footnote 6. TMPD + g DHAsc (9)
Sum: s Asc + s 02 TMPD +ASC MV
of Fig. 1))
hv
4i’ Hz0 + $$ DHAsc
Nature of the Back Reaction As suggested in the previous section, P430- apparently reduced P700+ directly in the absence of any secondary acceptors. An experiment shown in Fig. 4 would lend further support: In the absence of an artificial secondary acceptor, the recovery of P430 at 444 nm was kinetically indistinguishable from those of P700 at 703 or 433 e The reaction was presumably catalase present in D-144 particles
catalyzed (4).
by
A FURTHER
STUDY
103
OF P430 CONTROL
)r
t MV. 60pt.i
ro3-+---
*
+
(nm) +q‘+-
-----q----
-+.ooo2AA
If--
433-
FIG. 3. Effect of TMPD concentration on P700 recovery. P700 recovery was measured at 703 nm in the presence of 67 m methyl viologen. Other conditions were the same as in Figs. 1 and 2. The theoretical curve (broken line) was drawn asauming 1.7 X lo4 M-I see-1 for the pseudo-first-order rate constant.
nm. It should be noted, however, that at 433 nm the observed change was due to both P700 and P430. Simply because P700 and P430 were kinetically identical under these particular conditions, the observed kinetics at 433 nm were identical with those at 703 and 444 nm. On the other hand, in the presence of methyl viologen (shown in the right column of Fig. 4), P430 decay at 444 nm became much faster, while P700 decay at 703 nm became rather slower. At 433 nm, the P430 change was now hardly visible because of its extremely high recovery rate in the presence of methyl viologen. Although the possibility of the back reaction in photosystem I has been suggested by Kok et al. (10) and Rumberg and Witt (9), the present result would be the first direct demonstration of such a reaction. The recovery kinetics of P700 and P430 in the back reaction were apparently nonexponential, as the semilogarithmic plot was nonlinear. However, a reciprocal plot (Fig. 5) applicable to a special case of secondorder reaction7 where concentrations of two reactants are equivalent as described in reaction Fi was found to fit the observed kinetics. This would readily indicate the simul’ The rate equation in this case can be described as follows: kt = (l/C) - (l/Co), where k is the second-order rate constant; C, concentration of the reactants at time t; CO, concentration of the reactants at time t = 0 (11).
------
*
y/-
I
,
0
200
+
I
0 m*ec
200
FIQ. 4. Effect of methyl viologen on the flashinduced absorption changes. The reaction mixture contained: D-144 particles, 9.5 pg/ml chlorophyll; ascorbic acid, 670 PM; 2,6-dichlorophenol-indophenol, 33 PM; tricine-NaOH, 50 II-M, pH 8.0; and 60 FM methyl viologen where indicated. Eight blue and red flashes were applied for 703 and 433 nm changes, respectively; 32 red flashes for the 444-nm change.
P436 + P7Od J-P430
+ P700
II
1 0
50
100
150
msec FIG. 5. Plot of the reciprocals of absorption changes of P700 and P430 VS. time. Experimental conditions were the same as indicated in Fig. 4. No methyl viologen was added. The reciprocals of absorption changes were normalized to give a value of unity at 30-msec point both for P700 (A&a? ,,) and P430 (AA 444,,). The scale was arbitrary. The theoretical curve was drawn assuming 3 X 1OgI@ see-1 for the second-order rate constant.
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taneous production of equimolar PTOO+ and P430- by the primary photochemical event in photosystem I. The second-order rate constant for t’he back reaction was 3 X log x1-l set-‘.
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Efect oJ’ AI ethyl I’iolocgen Concentration
As mentioned in the first section, absorption changes attributable to the photoreduct’ion of methyl viologen was not’ detected under present experimental conditions, probably due to its high rate of autLight-Induced Reduction of Safranine T oxidation. The following results, however, It was shown previously (2) that t’he half would lend an indirect evidence for the methyl viologen reduction by reduced P430. decay times of P430 reasonably corresponded Time courses of t,he P430 recovery at 444 to the half rise times of the concomitant nm shown in Fig. 7 revealed that the decay reduction of safranine T at several different concentrations. The matter has been re- was exponential in the presence of the examined in terms of the reaction order. accept)or. As in the case of safranine T, a The half decay times of P430 and the half full-logaritjhmic plot of half time US.concent’ration yielded a curve agreeing with a rise times of safranine T reduction induced by a flash were plotted against the dye con- pseudo-first’-order reaction (Fig. 8). The rate constant at 22’ was 9.6 f 0.7 X lo6 11-l centrations on a full-logarithmic scale (Fig. see-l. 6). The results reasonably fitted the theoretiTable I summarizes the pseudo-firstcal curve for a pseudo-first-order reaction with t,he rate constant of 1 f 0.7 X IO* 311-l order rat,e constants for P430- reoxidation by several acceptors obtained by similar se+ at’ 22”. methods under t,he same conditions. T is autoxidizable, Since safranine though not at a rate as high as methyl Effect of Excitation Energy viologen, the reaction performed aerobically Red flashes (650-730 nm) at several could be described similarly by Reactions intensities obtained by means of neutral(l), (‘2), and (4-9) as in the case of methyl density filters were employed to study viologen, with the latter replaced by saturation profiles of bot’h P700 and P430. safranine T. The result’s shown in Fig. 9 suggest a roughly
FIG. 6. Effect of safranine T (ST) concentrations on P430 decay. Reduction of safranine T was measured at 520 nm; P430 decay, at 408 nm. The theoretical curve was drawn assuming 1 X lo8 M-I set-1 for the pseudo-first-order rate constant. The reaction mixture contained: D-144 particles, 13 pg/ml chlorophyll; 13 FM TMPD ; 0.67 mu ascorbic acid; 50 mu Tricine-NaOH, pH 8.0; and indicated concentrations of safranine T. Sixteen red flashes were used for each data point.
FIG. 7. Time courses of P430 decay in the presence of methyl viologen. The reaction mixture contained: D-144 particles, 7 pg/ml chlorophyll; 33 PM TMPD; 0.67 mM ascorbic acid; 50 rnM Tritine-NaOH, pH 7.8; and indicated concentrations of methyl viologen (MV). Absorption changes were measured at 444 nm. Thirty-two red flashes were used for each trace.
A FURTHER
STUDY
identical quantum requirement for both I?700and P430. An approximate estimate of quantum yield at a relative intensity of 2 % gave a value close to unity for both P700 and P430. ‘QA more elaborate study using narrow-band interference filters shown in Fig. 10 revealed that at the excitation wavelength of 703 nm the quantum requirement was almost one, whereas it increased to two at 671 nm, for both P700 and P430. These results would lend additional support to the assumption that both P700 and P430 are bleached by the same long-wavelength pigment system. Di$erence
lo.!?
OF P430
green algae and spinach chloroplasts have been presented in the previous paper (2). In the present report, though the method for extracting the P430 spectrum from overall absorption changes induced by flashes was essentially the same, a few precautions were taken to minimize possible errors and interference from other components involved. As described in the Methods and Materials section, t’he intensity of measuring light
Spectra
The difference spectra of P430 in several photosystem-I particles isolat’ed from blue-
-I
I
IMV),*LM FIG. 8. Effect of methyl viologen concentration on P430 decay. The experimental conditions were the same as indicated in Fig. 7 except with different. chlorophyll concentrations; open circles, 7 rg/rnl; closed circles, 18 @g/ml. The theoretical curve (dotted line) was drawn assuming 9.6 X lo6 M-' set-’ for the pseudo-first-order rate constant.
FIG. 9. Effect of flash intensity on P700 and P430 changes. Absorption changes induced by 20psec red (650-730 nm) flashes were measured at 430 nm both for P700 and P430. For P700,67 PM benzyl viologen was added to accelerate P430 recovery. The absorption change due to P700 thus obtained w&s then subtracted from total change without benzyl viologen. Reaction mixture contained: D-144 particles, 7.7 pg/rnl chlorophyll; 33 PM TMPD; 670 PM ascorbic acid; 150 mM KCl; 50 mM K-phosphate, pH 7.2. Sixteen flashes were used for each point. The flashes were spaced at 20-see intervals. Triangles, total changes; open circles, P700; closed circles, P430; squares, ratios of absorption change between P700 and P430 at 430 nm. Absolute intensity at loOyc was 1.8 X lo-? J cmm2.
TABLE I PSEUDO-FIRST-ORDER RATE CONSTANTSFOR P430- OXIDATION Acceptors l,l’-Trimethylene-2,2’-dipyridylium-4,4’-methyl l,l’-Trimethylene-2,2’-dipyridylium dibromide Methyl viologen Benzyl viologen Safranine T Methylene blue TMPD+
Ed (mV) dibromide
- 656” -521a -44@ - 359c - 29oc $llc +260c
y Ref. (19). * No visible effect on P430 recovery has been observed at the concentration c Ref. (Zo).
k(22”)
(ah’
SIX-~)
-6
-106 9.6 X -1.8 X 1 x -1.5 X -4 x
of 67 PM.
lo6 107 108 lo8 108
106 could
HIYAMA
affect
the
magnitude
of absorption
changes, particularly in the samples containing methyl viologen, where an intense measuring light could result in a lower dark level of reduced P700 available for photooxidation because of a lower recovery rate of P700 at low TMPD concentrations. Another precaution was to minimize the interference from the absorption changes due to dyes added to the reaction mixture
&I O 0
I
I
I
I
4 NUMBER ABSORBED
0 OF
I
I 12
PHOTONS (X Id31
FIG. 10. Quantum requirement of P7OO and P430. Both P700 and P430 were measured at 430 nm. Beneyl viologen was used for the separation of the two components as described in Fig. 9. Other conditions were the same as in Fig. 9 except for chlorophyll concentration (17 rg/ml). See the text for other details. Open circles, P700; closed circles, P430; excited by 703-nm flash. Open triangles, P700; closed triangles, P430; excited by 671-nm flash.
I
400
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as donors and acceptors. TMPD+ has broad absorption peaks throughout 500630 nm [AEhoo = 4, and AE5,6 = 10.7 rnM-l cm-’ (la)], while around 430 nm it shows lower absorption (AE + 0.6 rnM+ cm-l). PMS, on the other hand, had a minor peak around 430 nm (AE + 2.5 mM-’ cm-l) besides its main peak at 387 nm [AE = 25 rn& cm-’ (13)], while it, had practically no absorption at wavelengths longer than 480 nm. Taking these into consideration, an appropriate electron donor was selected for each spectral region as described in the legend of Fig. 11. Cytochromes are present in D-144 particles, though in most cases photochemically inactive (14, 15). Those contributions in the present experiments, however, seemed to be quite small if any, since, as in the case of Plectonema particles described previously (a), flash-induced absorption changes in spinach particles virtually consisted of only two components when contributions from dyes were negligible; i.e., P430, whose recovery was much accelerated by secondary acceptors, and P700, whose recovery was exponential when the back reaction was blocked by secondary acceptors. It should also be noted that little absorption changes attributable to the “515 nm-change” first
I
500 450 WAVELENGTH (nm)
FIG. 11. Difference spectra of P700 and P430 (390680 nm). The basic reaction mixture consisted of D-144 particles, chlorophyll, 12 pg/ml; 0.67 mM ascorbic acid; and 100 mu Tricine-NaOH, pH 8.0. For P700, 67 pM methyl viologen was added in addition to the donors; open circles, 33 PM TMPD; solid circles, 33 pM TMPD and 1.7 PM PMS; triangles, 0.33 p~ PMS. To obtain the P430 difference spectrum (squares) P700 changes were subtracted from the total changes obtained with the reaction mixture containing TMPD-ascorbate as donor and no acceptors. Sixteen red flashes were used for the total-change measurement, and four for P700 changes.
A FURTHER
STUDY
discovered by Duysens (16) were seen in D-144 part,icles as mentioned earlier (2). As seen in Fig. 11, there was a broad absorption increase around this wavelength which
FIG. 12. Difference spectra of P700 and P430 (600-730 nm). The basic reaction mixture was the same as in Fig. 11 except the chlorophyll concentration was 10 pg/ml. Open circles, total changes with 33 PM TMPD; closed circles, with 67 PM methyl viologen present; squares, differences (open circles minus closed circles). Sixteen blue flashes were used for the total-change measurements, and eight for the methyl viologen-supplemented reaction; flashes spaced at 20-set intervals.
107
OF P430
took place as fast as P700. The recovery was also kinetically identical with that of P700 under various conditions, indicating that these positive changes were due to P700 itself. The spectrum of P430 depicted with above stated precautions (Fig. 11) shows a less prominent peak around 430 nm than those previously report’ed (2). It is rather obvious that, without t,hose precautions, the previous spectra apparently suffered an underestimation of P700 and, consequently, an overstatement of P430, though the overall shape may not have been appreciably deformed. Differential extinction coefficient of P430 at 430 nm calculated from the ratio of absorption change to P700 was 12 f 3 mM-1 cm-l, assuming that A,!%s~~~ of I’700 is 45 rnW’ cm-‘. In the red region, on the other hand, no absorption changes corresponding to P430 changes in the blue region have yet been observed. Figure 12 shows spectra of t,otal changes, of those with methyl viologen sup-
-600 -400 -200 0 +200+400FIG. 13. An electron
P700” flow scheme around
photo-system
I. See text for details.
108
HIYAMA
plemented, and of the differences between those two. For the moment, it could be said that in the region of 600-730 nm little absorption changes can be ascribed to P430. CONCLUDING
REMARKS
Figure 13 is a schematic diagram showing electron flow around photosystem I in spinach D-144 particles in the presence of several artificial electron carriers. Although only methyl viologen and safranine T are included in the diagram for the sake of simplicity, other acceptors listed in Table I could be in their place.8 Where applicable, the pseudo-first- or second-order rate constants are indicated in the diagram. Where the constants have not yet been determined, half times of the reaction under routine experimental conditions are shown. The redox potential of I’430 was tentatively set around -500 mV, based on the results of Kok et al. (17), Zweig and Avron (18), and Black (19) for the primary acceptor of photosystem I. A low rate constant of l , l’-t’rimethylene-2,2’-dipyridylium dibromide (E’o = - 521 mV) and little 1 , 1’.t,rimethylene-2,2’-dipyrieffect by dylium-4,4’-methyl dibromide (E’o = -656 mV) shown in Table I seemed to agree with this potential value, although the actual value has yet to be determined by direct measurement with P430. REFERENCES 1. HIYAMA, T., BND KE, B., Biophys. 11,31a (1971).
Sot. Abstr.
8 Preliminary results indicated that some electron carriers, e.g., 2,6-dichlorophenolindophenol and PMS, could not simply substitute TMPD in this scheme; at least part of the re-reduction of P7OO seemed to be mediated by a third factor located between P700 and dyes. This matter is currently under investigation.
AND
NE
2. HIYAMA, T., AND KE, B., Proc. Nat. Acad. Sci. U.S.A. 68, 1010 (1971). 3. ANDERSON, J. M., AND BOARDMAN, N. K., Biochim. Biophys. Ada 112, 403 (1966). 4. HAUSKA, G. A., MCCARTY, R. E., .\ND RACKER, E., Biochim. Biophys. Acta 197, 206 (1970). 5. KE, B., TRF,HARNE, R. W., AND MCKIBBEN, C., Rev. Sci. Instrum. 36, 296 (1964). 6. CHANCE, B., KIHARA, T., DEV~VLT, D., HILDRETH, W., NISHIMURA, M., .\ND HIYAMA, T., “Progress in Photosynthesis Research,” Vol. III, p. 1321. Tiibingen, 1969. 7. Kn, B., OGAW-A, T., HIYAMA, T., AND VERNON, L. P., Biochim. Biophys. Acta 226,53 (1971). 8. MCKINNEY, G., J. Biol. Chem. 132, 91 (1940). 9. RUMBERG, B., AND WITT, H. T., 2. Naturforsch. B 19,693 (1964). 10. KOK, B., RURAINSKI, H. J., AND HARMON, E. A., PZant PhysioZ. 39, 513 (1964). 11. EASTMAN, E. D., AND ROLLEFSON, G. K., “Physical Chemistry,” p. 381. McGraw-Hill, New York, 1947. 12. MICHAELIS, L., SCHUBERT, M. P., AND GRANICK, S., J. Amer. Chem. Sot. 61, 1981 (1939). 13. JONES, K. M. “Data for Biochemical Research” (R. M. C. Dawson, D. C. Elliot, W. H. Elliot, and K. M. Jones, Eds.), p. 436. Oxford Univ. Press, New York, 1969. 14. BOARDMAN, N. K., AND ANDERSON, J. M., Biochim. Biophys. Acta 143, 187 (1967). 15. ANDERSON, J. M., FORK, D. C., AND AMESZ, J., Biochem. Biophys. Res. Commun. 23, 874 (1966). 16. DUYSENS, L. N. M., Science 120, 353 (1954). 17. KOK, B., RURAINSKI, H. J., AND OWENS, 0. V. H., Biochim. Biophys. Acta 109, 347 (1965). 18. ZWUXG, G., AND AVRON, M., Biochem. Biophys. Res. Commun. 19,397 (1965). 19. BLACK, C. C., Biochim. Biophys. Acta lu), 332 (1966). Poten20. CLARK, W. M., “Oxidation-Reduction tials of Organic Systems.” Williams & Wilkins, Baltimore, 1960.
OF
RIOCHEMISTRY
Further
Study
AND
RIOPHYSICS
of P430:
147, 99-108 (1971)
A Possible
Primary
of Photosystem TETSUO Charles F. Kettering
HIYAMA
AND
Research Laboratory,
Electron
Acceptor
I’ BACON
KE
Yellow Springs,
Ohio &387
Received June 1, 1971; accepted July 22, 1971 The nature of P430, which is represented by an absorption change observed in isolated photosystem- particles, haa further been investigated using spinach subchloroplast particles: (1) The kinetic relationship has been established between three components involved in an artificial cyclic electron flow: P430 -+ N, N, N’, N’-tetramethyl-p-phenylenediamine + P700 hv T (2) The recovery (reoxidation) of P430 was a pseudo-first-order reaction with respect to the artificial acceptors, e.g., methyl viologen and safranine T. (3) The back flow of electrons was a direct reduction of the photooxidized P700 by the photoreduced P430. (4) The quantum yield and effective wavelengths for P430 photoreduction were identical with those for P700 photooxidation. The results further support P430 as the primary electron acceptor of photosystem I in green plant photosynthesis.
insight into the primary events in photosystem I of green-plant photosynthesis.
In previous communications (1, 2) we have reported an absorption change around 430 nm in several photosystemparticles isolated from blue-green algae and spinach chloroplasts. This spectral component, tentatively designated as “P430” from its absorption maximum, was found to be of particular interest since its onset time upon flash excitation was comparable to that of P700 and its recovery was accelerated by photosystemelectron acceptors, including ferredoxin. It was further shown that the
METHODS
AND MATERIALS
Throughout the present work, photosystemparticles fractionated from spinach by digitonin treatment according to Anderson and Boardman (3) were used. The particles (D-144) were stored at -80” in sucrose solution as described by Hauska et al. (4).
Absorption changes were measured by a singlebeam spectrophotometer described previously (5). The measuring light was provided by a quartziodine lamp (GE-1958, 150 W) passing through a 500-mm Bausch & Lomb monochromator. In the region of 400-560 nm, two blue filters (Corning 4-96) were placed between the cuvette (10 X 10 mm, four-side polished) and the phototube (EM1 9558) to protect the latter from the red actinic flash. For the region of 56&800 nm, another monochromator was employed in place of the guard filters to minimize the interference from fluorescence as well as the actinic light. The intensity of measuringlight was found to be crucial for good reproducibility, particularly in the red region, where a relatively high intensity of measuring light was needed due to the lower sensitivity of the phototube in this region and loss of light in the additional monochromator. It should
photoreduction of some dyes kinetically corresponded to the recovery of photobleached P430. These results were explained by proposing that P430 is reduced in the primary photochemical event in photosystern I and then donates electrons to either a dye or ferredoxin. In the present paper, detailed evidence is presented which would lend further support to the above proposition and provide some 1 Contribution No. 430 from the Charles F. Kettering Research Laboratory, Yellow Springs, OH. Work supported in part by a National Science Foundation Grant GM-8460. 99
100
HIYAMA
be noted that theoretically even a weak measuring light could cause some absorption changes. In practice, however, a compromise intensity can be chosen that would cause little effect and yet provide a tolerable signal-to-noise ratio which may be improved by signal averaging. Typical measuringbeam intensities used were 100 erg crnm2 set+ at 700 nm and 20 erg cmp2 set-’ at 430 nm as measured by a Kettering Model 68 radiometer. The excitation light was provided by a xenon flash lamp (GE, FT.403) passed through appropriate filters; an interference filter (65&730 nm) plus Corning 2-58 and 2-64 for the red; an interference filter (Baird-Atomic, 400-480 nm) plus Corning 4-96 for the blue. For the quantum yield measurement, IO-nm wide interference filters (Baird-Atomic) were employed together with Corning 2-58 to obtain narrow-band actinic flash. The reaction mixture was cross-illuminated through one side of the cuvette at right angle to the measuring beam. The excitation energy was measured by a calibrated photodiode (Lite-Mike, EG&G) and an oscilloscope. As described earlier (5)) the flash duration was measured to be 20 psec at half-peak height. The intensities used routinely were 1.8 X lo-? J cme2 for the red and 2.2 X 1OW J cn-2 for the blue at the surface of the cuvette. The red flash was about eight times stronger than the saturation intensities in most cases, while the blue one was slightly above the saturation point in order to minimize the fluorescence. The photocurrent from the phototube was amplified by a differential amplifier balanced with fixed off-set voltage, usually 1.0 or 2.0 V. The amplified difference signal was fed into a signal averager (Fabri-tek Model 1062 Instrument Computer), which also triggered the flash with an appropriate timing. The signals induced by successive flashes were digitized and accumulated in the memory device of the signal averager. The averaged signal from several measurements stored in the instrument could then be displayed and recorded on a chart paper by an X-Y recorder. In principle, using the signal-averaging technique, all absorption changes induced by a flash should be reversible (e.g., cyclic or noncyclic reaction with excess ascorbate and oxygen; see the text); the entire system should return to the original state before the next flash is applied. In practice, the incoming signal in each experiment was monitored and confirmed, and an appropriate interval between flashes was employed. Routinely, a lo-see interval was used, and sometimes 20 set was employed when necessary. Notably, even with this precaution, some irreversible or extremely slow recovering changes might have been excluded from the final results in using this technique, as pointed out earlier (6). For the calculation of quantum yield, the trans-
AND
KE
mittance of the reaction mixture was measured b. a Cary Model 14 spectrophotometer equipped with an end-on phototube located close to the cuvettes. The light scattering, however, was insignificant at 671 and 703 nm in I)-144 particles; the regular photometer set-up with W-cm distance from t,he cuvette to the photot,ube gave almost identical results. For the extinction coefficients of P700, 64 mM-’ cm-’ at 700 nm and 45 ITIM-’ cm-l at 430 nm were used. These values were separately determined for D-144 particles using TMPD as the donor, as will be mentioned later. Details and discussion on some discrepancy from previously reported values (7) will be reported in a forthcoming paper. All optical measurements were carried out at 22” in a cuvette with optical path length of 10.0 mm. Chlorophyll concentration was calculated as chlorophyll a from the optical density of the reaction mixture at 680 nm using an extinction coefficient of 8.4 X low2 ml pg-’ cm-l, which was separately determined in 80% acetone extract of D-144 particles, based on Mackinney’s coefficient (8). Digitonin, tricine, and ascorbic acid were obtained from Sigma Chemical Co.; N,N,N’,N’tetramethyl-p-phenylene diamine (TMPD2) from Eastman Kodak; safranine T from Aldrich Chemicals; methyl viologen from Mann Research. RESULTS
TMPD-Mediated
AND
DISCUSSION
Electron Flow Photosystem I
Around
As shown previously, the oxidized form of TlMPD (Wurster’s blue; abbreviated as TMPD+ hereafter) could serve as a secondary electron acceptor for photosystem I (2). Since the dye had a considerably high absorption at 575 nm, which is an isosbestic point in the P700 difference spectrum, it became possible to observe absorption changes due to the dye with little interference from P700 changes. The isosbestic point was separately determined by using phenazine methosulfate (PMS) as an electron carrier which had practically no absorption at this wavelength (see below). Figure 1 shows typical absorption changes 2 Abbreviations. TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine; TMPD+, oxidized TMPD; PMS, phenasine methosulfate; P430-, reduced form of P430; P700+, oxidized form of P700; MV, methyl viologen; MV-, reduced MV; Asc, ascorbate; DHAsc, dehydroascorbate; ST, safranine T; ST-, reduced ST.
A FURTHER
PM TMPD 67 I~PD(ox!O6
TMPD 67 TMPD 67 ASC 167 167 To” 330 “;C 2
2
2
c-4
800ms
c----l 20 msec FIG. 1. Flash-induced absorption changes in digitonin-fractionated photosystemparticles (D-144). Chlorophyll concentration, 12 rg/ml in 50 mM Trickle-NaOH, pH 8.0. For the reaction mixture used in the left column, 67 PM TMPD and 0.6 pM TMPD+ (formed by aeration) were added. In the middle column, additional 0.67 mM ascorbic acid was present. Both reaction mixtures were made anaerobic by evacuation before measurement. In the right column, 330 MM methyl viologen and air were introduced into the reaction mixture used for the middle column. For 703 and 575 nm; 4 and 16 blue flashes, respectively, were applied; for 442.5 nm, 32 red flashes. The arrow shows the point when flash was fired.
induced by a 20-psec xenon flash in spinach photosystem-I particles (D-144). As shown in the left column of Fig. 1, in the presence of 0.6 PM TMPD+ 3 and A7 p&zTMPD under anaerobic conditions, the light flash caused an abrupt decrease in absorption at 575 nm followed by a recovery which was kineCcally identical with that of P700 measured at 703 nm, with half decay t’ime of 300 msec. At a shorter t,ime scale shown at the bottom of Fig. 1, it was revealed that the abrupt de3 TMPD+ was obtained by a brief aeration of the reaction mixture before evacuation and determined by the difference in the absorption spectra before and after reduction by ascorbate at the end of the experiment.
STUDY
OF P430
101
crease at 575 nm actually took place with a half tjime of 4 msec. This half time was almost idenCca1 with that of P430 recovery (shown immediately above the 575..nm trace) measured at 442.5 nm, another isosbestic point4 of P700. These results indicat,e that TJIPD+ was directly reduced by photoreduced P430 and that photooxidized P700 was directly reduced by TMPD, thus creating a simple cyclic electron flow around photosystem I.” In t,he middle column of Fig. 1, TMPD+ was reduced to TnIPD by ascorbat.e, as confirmed by absorption spect’rum of the reaction mixture. Under these conditions, the recovery of 1’700 became faster (~~1~E 45 msec). These results may be explained as follows: Upon losing the secondary acceptor, TJIPDf, electrons from the primary acceptor had no ot,her place to go except back to P700+, the only available oxidant, under these conditions. The nearly ident’ical half decay times of P700 and P430 under these conditions indicate a direct int’eraction of these t,wo components. The above notion would further be supported by the experiments shown in the right column of Fig. 1. Upon addition of methyl viologen under aerobic conditions, P700 recovery became slow again wit’h a half time of 300 msec, a positive change at 575 nm appeared, and the I’430 decay became much fast,er (21/2g 1 msec). The observed change at 575 nm may represent the oxidation of TMPD followed by re-reduction by ascorbate. The oxidation of T-\IPD represent’ed by the positfive signal at, 575 nm could be coupled to P700 recovery, although the magnitude of observed T,\/IPD oxidation seemed too small t,o be coupled to P700 reduction. The undersized absorption increase at 57fi nm, however, was likely caused by a relatively fast re-reduction of TMPD by ascorbate (tllz g 4 It was found that the isosbestic points of ~700 tended to shift from preparation to preparation by a few nanometers. Usually the wavelength region of 442444 nm was scanned prior to measurements. 5 From the extinction coefficient of TMPD* at 575 nm 110.7 mM-* cm-l; also see Ref. (12)], the differential ext)inction coefficients for P700 and P430 could be calculated. The value quoted in Methods and Materials section and in a later section was so obtained.
102
HIYAMA
0.2 set). This implication was further supported by the results shown in Fig. 2, in which the absorption increase at 57.5 nm became larger with increasing TMPD concentration (see Fig. 2B and C), whereas it became smaller with increasing ascorbsbte concentration (Fig. 2C and D). Further addition of a small amount of PMS virtually eliminated the change at 575 nm (Fig. 2E). PMS practically had no absorption at 575 nm either in the oxidized or reduced form and a much larger rate constant for P700 reduction (1.5 X 10’ k1-l see-I) (9) than that of TMPD (1.7 X lo4 h1-l se+, see below). Thus, PMS substituted TMPD for P700 reduction and eliminated the absorption change due to TMPD. Indeed, an isosbestic point of P700 was found to be at 575 nm in separate experiment’s employing PhIS as the elect’ron carrier. Note that absorption changes due to methyl viologen were also expected in this wavelength region. This contribution, however, seemed almost negligible judging from t,he above results, presumably because its autooxidation rate was extremely high under present experimental condit’ions. Direct reduction of P700 by TMPD was further demonstrated in Fig. 3, where the 703nm t -----
A
“v-s . t ----B
l-
L
575nm
t c-c--
t T :: 8.
TMPD ASC
33/~M 670
TMPD ASC -QMV
33 670 67
+TMPD ASC MV
100 670 67
AND
KE
half-time of P700 recovery was plotted against TMPD concentration on a fulllogarithmic scale, as in the case of PRIS (9). The result’s resasonably fitted the theoretical curve (dashed line) assuming that the react,ion was pseudo-first-order with the rate constant of 1.7 X 10” M-’ see-‘. The flash-induced reactions shown in the right column of Fig. 1 thus can be explained as a noncyclic electron flow from ascorbate to oxygen mediated by TMPD, photosystem I (P700 and P430, activated by photon via antenna chlorophylls, etc.), and methylviologen. The results may be summarized as follows : For the cyclic
reaction
hu P430 x P439P766 --3 p706+ P430- + TMPD+ P790+ + TMPD For the Fig. l),
back
(the left column
(1)
(2) +
P430 + TMPD P796 + TMPD’
reaction
(the
P430 J% P430P700 -% P7cMlf p790+ f P430-
-
For the noncyclic
reaction
100 1300 67
TMPD ASC MV *PMS
100 1300 67 I.7
FIG. 2. Identity of the 575-nm change. Experimental conditions were the same as in Fig. 1 except for the concentrations of redox agents, which were added successively aa indicated on the right. Open arrows indicate either newly supplemented or increased ingredients.
middle
(3) (4) column
of
(1)
(59
P430 -$ P799 P43C- + P700+ + MV- + s HzOz
P43cr p799+ MV TMPD % 02 -----) %
TMPD+
+ x AC-
P709 + P430 (the right
(5) column), (1)
(2) --f -
Hz0
P430 + MV(6) P766 + TMPD+ (4) MV + % HoOz (7) + % 02 @I see footnote 6. TMPD + g DHAsc (9)
Sum: s Asc + s 02 TMPD +ASC MV
of Fig. 1))
hv
4i’ Hz0 + $$ DHAsc
Nature of the Back Reaction As suggested in the previous section, P430- apparently reduced P700+ directly in the absence of any secondary acceptors. An experiment shown in Fig. 4 would lend further support: In the absence of an artificial secondary acceptor, the recovery of P430 at 444 nm was kinetically indistinguishable from those of P700 at 703 or 433 e The reaction was presumably catalase present in D-144 particles
catalyzed (4).
by
A FURTHER
STUDY
103
OF P430 CONTROL
)r
t MV. 60pt.i
ro3-+---
*
+
(nm) +q‘+-
-----q----
-+.ooo2AA
If--
433-
FIG. 3. Effect of TMPD concentration on P700 recovery. P700 recovery was measured at 703 nm in the presence of 67 m methyl viologen. Other conditions were the same as in Figs. 1 and 2. The theoretical curve (broken line) was drawn asauming 1.7 X lo4 M-I see-1 for the pseudo-first-order rate constant.
nm. It should be noted, however, that at 433 nm the observed change was due to both P700 and P430. Simply because P700 and P430 were kinetically identical under these particular conditions, the observed kinetics at 433 nm were identical with those at 703 and 444 nm. On the other hand, in the presence of methyl viologen (shown in the right column of Fig. 4), P430 decay at 444 nm became much faster, while P700 decay at 703 nm became rather slower. At 433 nm, the P430 change was now hardly visible because of its extremely high recovery rate in the presence of methyl viologen. Although the possibility of the back reaction in photosystem I has been suggested by Kok et al. (10) and Rumberg and Witt (9), the present result would be the first direct demonstration of such a reaction. The recovery kinetics of P700 and P430 in the back reaction were apparently nonexponential, as the semilogarithmic plot was nonlinear. However, a reciprocal plot (Fig. 5) applicable to a special case of secondorder reaction7 where concentrations of two reactants are equivalent as described in reaction Fi was found to fit the observed kinetics. This would readily indicate the simul’ The rate equation in this case can be described as follows: kt = (l/C) - (l/Co), where k is the second-order rate constant; C, concentration of the reactants at time t; CO, concentration of the reactants at time t = 0 (11).
------
*
y/-
I
,
0
200
+
I
0 m*ec
200
FIQ. 4. Effect of methyl viologen on the flashinduced absorption changes. The reaction mixture contained: D-144 particles, 9.5 pg/ml chlorophyll; ascorbic acid, 670 PM; 2,6-dichlorophenol-indophenol, 33 PM; tricine-NaOH, 50 II-M, pH 8.0; and 60 FM methyl viologen where indicated. Eight blue and red flashes were applied for 703 and 433 nm changes, respectively; 32 red flashes for the 444-nm change.
P436 + P7Od J-P430
+ P700
II
1 0
50
100
150
msec FIG. 5. Plot of the reciprocals of absorption changes of P700 and P430 VS. time. Experimental conditions were the same as indicated in Fig. 4. No methyl viologen was added. The reciprocals of absorption changes were normalized to give a value of unity at 30-msec point both for P700 (A&a? ,,) and P430 (AA 444,,). The scale was arbitrary. The theoretical curve was drawn assuming 3 X 1OgI@ see-1 for the second-order rate constant.
104
HIYAMA
taneous production of equimolar PTOO+ and P430- by the primary photochemical event in photosystem I. The second-order rate constant for t’he back reaction was 3 X log x1-l set-‘.
AND
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Efect oJ’ AI ethyl I’iolocgen Concentration
As mentioned in the first section, absorption changes attributable to the photoreduct’ion of methyl viologen was not’ detected under present experimental conditions, probably due to its high rate of autLight-Induced Reduction of Safranine T oxidation. The following results, however, It was shown previously (2) that t’he half would lend an indirect evidence for the methyl viologen reduction by reduced P430. decay times of P430 reasonably corresponded Time courses of t,he P430 recovery at 444 to the half rise times of the concomitant nm shown in Fig. 7 revealed that the decay reduction of safranine T at several different concentrations. The matter has been re- was exponential in the presence of the examined in terms of the reaction order. accept)or. As in the case of safranine T, a The half decay times of P430 and the half full-logaritjhmic plot of half time US.concent’ration yielded a curve agreeing with a rise times of safranine T reduction induced by a flash were plotted against the dye con- pseudo-first’-order reaction (Fig. 8). The rate constant at 22’ was 9.6 f 0.7 X lo6 11-l centrations on a full-logarithmic scale (Fig. see-l. 6). The results reasonably fitted the theoretiTable I summarizes the pseudo-firstcal curve for a pseudo-first-order reaction with t,he rate constant of 1 f 0.7 X IO* 311-l order rat,e constants for P430- reoxidation by several acceptors obtained by similar se+ at’ 22”. methods under t,he same conditions. T is autoxidizable, Since safranine though not at a rate as high as methyl Effect of Excitation Energy viologen, the reaction performed aerobically Red flashes (650-730 nm) at several could be described similarly by Reactions intensities obtained by means of neutral(l), (‘2), and (4-9) as in the case of methyl density filters were employed to study viologen, with the latter replaced by saturation profiles of bot’h P700 and P430. safranine T. The result’s shown in Fig. 9 suggest a roughly
FIG. 6. Effect of safranine T (ST) concentrations on P430 decay. Reduction of safranine T was measured at 520 nm; P430 decay, at 408 nm. The theoretical curve was drawn assuming 1 X lo8 M-I set-1 for the pseudo-first-order rate constant. The reaction mixture contained: D-144 particles, 13 pg/ml chlorophyll; 13 FM TMPD ; 0.67 mu ascorbic acid; 50 mu Tricine-NaOH, pH 8.0; and indicated concentrations of safranine T. Sixteen red flashes were used for each data point.
FIG. 7. Time courses of P430 decay in the presence of methyl viologen. The reaction mixture contained: D-144 particles, 7 pg/ml chlorophyll; 33 PM TMPD; 0.67 mM ascorbic acid; 50 rnM Tritine-NaOH, pH 7.8; and indicated concentrations of methyl viologen (MV). Absorption changes were measured at 444 nm. Thirty-two red flashes were used for each trace.
A FURTHER
STUDY
identical quantum requirement for both I?700and P430. An approximate estimate of quantum yield at a relative intensity of 2 % gave a value close to unity for both P700 and P430. ‘QA more elaborate study using narrow-band interference filters shown in Fig. 10 revealed that at the excitation wavelength of 703 nm the quantum requirement was almost one, whereas it increased to two at 671 nm, for both P700 and P430. These results would lend additional support to the assumption that both P700 and P430 are bleached by the same long-wavelength pigment system. Di$erence
lo.!?
OF P430
green algae and spinach chloroplasts have been presented in the previous paper (2). In the present report, though the method for extracting the P430 spectrum from overall absorption changes induced by flashes was essentially the same, a few precautions were taken to minimize possible errors and interference from other components involved. As described in the Methods and Materials section, t’he intensity of measuring light
Spectra
The difference spectra of P430 in several photosystem-I particles isolat’ed from blue-
-I
I
IMV),*LM FIG. 8. Effect of methyl viologen concentration on P430 decay. The experimental conditions were the same as indicated in Fig. 7 except with different. chlorophyll concentrations; open circles, 7 rg/rnl; closed circles, 18 @g/ml. The theoretical curve (dotted line) was drawn assuming 9.6 X lo6 M-' set-’ for the pseudo-first-order rate constant.
FIG. 9. Effect of flash intensity on P700 and P430 changes. Absorption changes induced by 20psec red (650-730 nm) flashes were measured at 430 nm both for P700 and P430. For P700,67 PM benzyl viologen was added to accelerate P430 recovery. The absorption change due to P700 thus obtained w&s then subtracted from total change without benzyl viologen. Reaction mixture contained: D-144 particles, 7.7 pg/rnl chlorophyll; 33 PM TMPD; 670 PM ascorbic acid; 150 mM KCl; 50 mM K-phosphate, pH 7.2. Sixteen flashes were used for each point. The flashes were spaced at 20-see intervals. Triangles, total changes; open circles, P700; closed circles, P430; squares, ratios of absorption change between P700 and P430 at 430 nm. Absolute intensity at loOyc was 1.8 X lo-? J cmm2.
TABLE I PSEUDO-FIRST-ORDER RATE CONSTANTSFOR P430- OXIDATION Acceptors l,l’-Trimethylene-2,2’-dipyridylium-4,4’-methyl l,l’-Trimethylene-2,2’-dipyridylium dibromide Methyl viologen Benzyl viologen Safranine T Methylene blue TMPD+
Ed (mV) dibromide
- 656” -521a -44@ - 359c - 29oc $llc +260c
y Ref. (19). * No visible effect on P430 recovery has been observed at the concentration c Ref. (Zo).
k(22”)
(ah’
SIX-~)
-6
-106 9.6 X -1.8 X 1 x -1.5 X -4 x
of 67 PM.
lo6 107 108 lo8 108
106 could
HIYAMA
affect
the
magnitude
of absorption
changes, particularly in the samples containing methyl viologen, where an intense measuring light could result in a lower dark level of reduced P700 available for photooxidation because of a lower recovery rate of P700 at low TMPD concentrations. Another precaution was to minimize the interference from the absorption changes due to dyes added to the reaction mixture
&I O 0
I
I
I
I
4 NUMBER ABSORBED
0 OF
I
I 12
PHOTONS (X Id31
FIG. 10. Quantum requirement of P7OO and P430. Both P700 and P430 were measured at 430 nm. Beneyl viologen was used for the separation of the two components as described in Fig. 9. Other conditions were the same as in Fig. 9 except for chlorophyll concentration (17 rg/ml). See the text for other details. Open circles, P700; closed circles, P430; excited by 703-nm flash. Open triangles, P700; closed triangles, P430; excited by 671-nm flash.
I
400
AND
KE
as donors and acceptors. TMPD+ has broad absorption peaks throughout 500630 nm [AEhoo = 4, and AE5,6 = 10.7 rnM-l cm-’ (la)], while around 430 nm it shows lower absorption (AE + 0.6 rnM+ cm-l). PMS, on the other hand, had a minor peak around 430 nm (AE + 2.5 mM-’ cm-l) besides its main peak at 387 nm [AE = 25 rn& cm-’ (13)], while it, had practically no absorption at wavelengths longer than 480 nm. Taking these into consideration, an appropriate electron donor was selected for each spectral region as described in the legend of Fig. 11. Cytochromes are present in D-144 particles, though in most cases photochemically inactive (14, 15). Those contributions in the present experiments, however, seemed to be quite small if any, since, as in the case of Plectonema particles described previously (a), flash-induced absorption changes in spinach particles virtually consisted of only two components when contributions from dyes were negligible; i.e., P430, whose recovery was much accelerated by secondary acceptors, and P700, whose recovery was exponential when the back reaction was blocked by secondary acceptors. It should also be noted that little absorption changes attributable to the “515 nm-change” first
I
500 450 WAVELENGTH (nm)
FIG. 11. Difference spectra of P700 and P430 (390680 nm). The basic reaction mixture consisted of D-144 particles, chlorophyll, 12 pg/ml; 0.67 mM ascorbic acid; and 100 mu Tricine-NaOH, pH 8.0. For P700, 67 pM methyl viologen was added in addition to the donors; open circles, 33 PM TMPD; solid circles, 33 pM TMPD and 1.7 PM PMS; triangles, 0.33 p~ PMS. To obtain the P430 difference spectrum (squares) P700 changes were subtracted from the total changes obtained with the reaction mixture containing TMPD-ascorbate as donor and no acceptors. Sixteen red flashes were used for the total-change measurement, and four for P700 changes.
A FURTHER
STUDY
discovered by Duysens (16) were seen in D-144 part,icles as mentioned earlier (2). As seen in Fig. 11, there was a broad absorption increase around this wavelength which
FIG. 12. Difference spectra of P700 and P430 (600-730 nm). The basic reaction mixture was the same as in Fig. 11 except the chlorophyll concentration was 10 pg/ml. Open circles, total changes with 33 PM TMPD; closed circles, with 67 PM methyl viologen present; squares, differences (open circles minus closed circles). Sixteen blue flashes were used for the total-change measurements, and eight for the methyl viologen-supplemented reaction; flashes spaced at 20-set intervals.
107
OF P430
took place as fast as P700. The recovery was also kinetically identical with that of P700 under various conditions, indicating that these positive changes were due to P700 itself. The spectrum of P430 depicted with above stated precautions (Fig. 11) shows a less prominent peak around 430 nm than those previously report’ed (2). It is rather obvious that, without t,hose precautions, the previous spectra apparently suffered an underestimation of P700 and, consequently, an overstatement of P430, though the overall shape may not have been appreciably deformed. Differential extinction coefficient of P430 at 430 nm calculated from the ratio of absorption change to P700 was 12 f 3 mM-1 cm-l, assuming that A,!%s~~~ of I’700 is 45 rnW’ cm-‘. In the red region, on the other hand, no absorption changes corresponding to P430 changes in the blue region have yet been observed. Figure 12 shows spectra of t,otal changes, of those with methyl viologen sup-
-600 -400 -200 0 +200+400FIG. 13. An electron
P700” flow scheme around
photo-system
I. See text for details.
108
HIYAMA
plemented, and of the differences between those two. For the moment, it could be said that in the region of 600-730 nm little absorption changes can be ascribed to P430. CONCLUDING
REMARKS
Figure 13 is a schematic diagram showing electron flow around photosystem I in spinach D-144 particles in the presence of several artificial electron carriers. Although only methyl viologen and safranine T are included in the diagram for the sake of simplicity, other acceptors listed in Table I could be in their place.8 Where applicable, the pseudo-first- or second-order rate constants are indicated in the diagram. Where the constants have not yet been determined, half times of the reaction under routine experimental conditions are shown. The redox potential of I’430 was tentatively set around -500 mV, based on the results of Kok et al. (17), Zweig and Avron (18), and Black (19) for the primary acceptor of photosystem I. A low rate constant of l , l’-t’rimethylene-2,2’-dipyridylium dibromide (E’o = - 521 mV) and little 1 , 1’.t,rimethylene-2,2’-dipyrieffect by dylium-4,4’-methyl dibromide (E’o = -656 mV) shown in Table I seemed to agree with this potential value, although the actual value has yet to be determined by direct measurement with P430. REFERENCES 1. HIYAMA, T., BND KE, B., Biophys. 11,31a (1971).
Sot. Abstr.
8 Preliminary results indicated that some electron carriers, e.g., 2,6-dichlorophenolindophenol and PMS, could not simply substitute TMPD in this scheme; at least part of the re-reduction of P7OO seemed to be mediated by a third factor located between P700 and dyes. This matter is currently under investigation.
AND
NE
2. HIYAMA, T., AND KE, B., Proc. Nat. Acad. Sci. U.S.A. 68, 1010 (1971). 3. ANDERSON, J. M., AND BOARDMAN, N. K., Biochim. Biophys. Ada 112, 403 (1966). 4. HAUSKA, G. A., MCCARTY, R. E., .\ND RACKER, E., Biochim. Biophys. Acta 197, 206 (1970). 5. KE, B., TRF,HARNE, R. W., AND MCKIBBEN, C., Rev. Sci. Instrum. 36, 296 (1964). 6. CHANCE, B., KIHARA, T., DEV~VLT, D., HILDRETH, W., NISHIMURA, M., .\ND HIYAMA, T., “Progress in Photosynthesis Research,” Vol. III, p. 1321. Tiibingen, 1969. 7. Kn, B., OGAW-A, T., HIYAMA, T., AND VERNON, L. P., Biochim. Biophys. Acta 226,53 (1971). 8. MCKINNEY, G., J. Biol. Chem. 132, 91 (1940). 9. RUMBERG, B., AND WITT, H. T., 2. Naturforsch. B 19,693 (1964). 10. KOK, B., RURAINSKI, H. J., AND HARMON, E. A., PZant PhysioZ. 39, 513 (1964). 11. EASTMAN, E. D., AND ROLLEFSON, G. K., “Physical Chemistry,” p. 381. McGraw-Hill, New York, 1947. 12. MICHAELIS, L., SCHUBERT, M. P., AND GRANICK, S., J. Amer. Chem. Sot. 61, 1981 (1939). 13. JONES, K. M. “Data for Biochemical Research” (R. M. C. Dawson, D. C. Elliot, W. H. Elliot, and K. M. Jones, Eds.), p. 436. Oxford Univ. Press, New York, 1969. 14. BOARDMAN, N. K., AND ANDERSON, J. M., Biochim. Biophys. Acta 143, 187 (1967). 15. ANDERSON, J. M., FORK, D. C., AND AMESZ, J., Biochem. Biophys. Res. Commun. 23, 874 (1966). 16. DUYSENS, L. N. M., Science 120, 353 (1954). 17. KOK, B., RURAINSKI, H. J., AND OWENS, 0. V. H., Biochim. Biophys. Acta 109, 347 (1965). 18. ZWUXG, G., AND AVRON, M., Biochem. Biophys. Res. Commun. 19,397 (1965). 19. BLACK, C. C., Biochim. Biophys. Acta lu), 332 (1966). Poten20. CLARK, W. M., “Oxidation-Reduction tials of Organic Systems.” Williams & Wilkins, Baltimore, 1960.