Determined by Radiation Inactivation - NCBI

Plant Physiol. (1987) 85, 158-163 0032-0889/87/85/01 58/06/$0 1.00/0

Functional Size of Photosynthetic Electron Transport Chain Determined by Radiation Inactivation' Received for publication October 27, 1986 and in revised form May 20, 1987

RUN SUN PAN, LEE FENG CHIEN, MAY YUN WANG, MAI YU TSAI, RONG LONG PAN*, AND BAN DAR HSU Institute of Radiation Biology, College ofNuclear Sciences, National Tsing Hua University, Hsin Chu

30043, Taiwan, Republic ofChina ABSTRACI Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H20 to methylviologen) was 623±37 kilodaltons; for photosystem II (H20 to dimethylquinone/ ferricyanide) 174 ± 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 ± 11 kilodaltons. The difference between 364 t 22 (the sum of 174 1 11 and 190 ± 11) kilodaltons and 623 ±37 kilodaltons is pally explined to be due to the presence of two molecules of cytochrome bJf complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and orgaization of polypeptides for photosynthetic electron transport chain is proposed.

The photosynthetic electron transport chain of higher plants consists of two photosystems, PSI and PSII, which are believed to operate in a linear sequence within the thylakoid membrane of chloroplasts (7). Photosystem II carries out the photooxidation of water resulting in production of 02 and a weak reductant that can reduce plastoquinone (PQ) and thereby donate electrons to PSI. PSI, on the other hand, generates a relatively weak oxidant but produces a reductant capable of reducing NADP+. A linear electron flow from water to NADP+ is summarized in the following sequence: H20 PSII PQ- Cyt b6/fPC - PSI --Fd -* NADP+ 02 where PC is plastocyanin, Fd is ferredoxin, and double arrows indicate that several other electron carriers are involved in the sequence. Partially purified preparations of PSI and PSII, supporting high rates of electron transport (6, 26), have been used extensively in the study of electron flow through reaction center complexes. SDS-PAGE has revealed considerable information about the polypeptide composition of both of these partially purified preparations (8, 25, 26). In addition, treatments such as partial trypsin Supported by the grants from National Science Council, Republic of China (NSC73-0203-B007-05) to B. D. H. and (NSC73-0204-B007-05) to R. L. P. I

digestion, salt or alkaline tris wash, and detergent solubilization have allowed for the assignment of many of these polypeptides as structural and/or functional components of either PSI (6, 26) or PSII (8, 25). Even so, further investigation into the identity, stoichiometry, and molecular organization of the polypeptides involved in these electron transfer reactions is still an area of intense current interest. Recently, the technique of radiation inactivation has been used effectively in estimating the native mol wt of membrane bound components such as enzymes, transporters, and receptors (13, 16). This method involves irradiating membrane preparations with ionizing radiation such as electron beam, x-rays, or yrays and analyzing the dose response of inactivation. The molecular mass can then be estimated by applying target theory analysis. This technique offers several advantages not always available with more conventional biochemical methods for determining the molecular size of membrane bound proteins. In particular, the procedure can be performed on the proteins in situ, the measurements are independent of the degree of purity, and the molecular mass obtained is the minimum functional size. In this work, we report the functional size of photosynthetic electron transport chain and its components from radiation inactivation measurements. The functional sizes of various parts of photosynthetic electron transport chain are compared to the relative molecular mass of polypeptides obtained by other more conventional biochemical methods. MATERIALS AND METHODS

Preparation of Thylakoid Membranes. Thylakoid membranes were isolated from commercial spinach (Spinacia oleracea L.) as described elsewhere (18) and finally suspended in a storage medium containing 25 mM Hepes (pH 7.5), 15% glycerol, 2.5% dimethyl sulfoxide, 10 mM NaCl, and 2 mg/ml BSA. The chloroplast preparations were then stored at -70°C until irradiation. The irradiated samples were either ready for immediate activity assay or stored again at -70°C until used. Hydroxylamine- and tris-treatment of chloroplasts were used to deplete oxygen evolving capacity according to the method of Ort and Izawa (24) and Lee et al. (18). The Chl concentration of the isolated chloroplasts was measured according to the method of Arnon (2). Irradiation Procedure. Irradiation was performed with a 'Co irradiator (1200 Ci) at our institute. The dose rate was 1.14 ± 0.08 Mrad/h which was determined by the method of Hart and Fricke ( 11) using Fe2+/Fe3+ or Ce4+/Ce3+ couple. Internal standardization of radiation exposure was carried out with glucose 6-P dehydrogenase (Mr = 104 kD), lactate dehydrogenase (Mr = 140 kD), catalase (Mr = 232 kD), glutamate dehydrogenase (Mr = 320 kD), and 03-galactosidase (Mr = 464 kD). Enzymes at concentration of 200 ,gg/ml were suspended in the storage me158

FUNCTIONAL SIZE OF PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN dium as described above. Both chloroplasts (1 mg Chl/ml) and standard enzymes were irradiated at -18 to -25°C maintained by a cryothermostat (Cryothermostat model WK6, Colora). The control samples were run concurrently under the same condition but without irradiation. Activity Assays. Electron transport rates were measured using either a Clark-type 02 electrode (Rank Brother) or a laboratory built spectrophotometer described earlier (18). The 02 electrode measurements were performed at 20C in a 2 ml mixture containing 25 mm Tris-Cl (pH 8.0), 5 mM MgCl2, 10 mM NaCl, 5 mM methylamine, and an appropriate amount of electron donor or acceptor. The Chl concentration was 25 Ag Chl/ml. Spectrophotometric measurements of electron transport were made under the same condition except that the Chl concentration was reduced to 8 ,ug/ml and 0.1 mm DCIP2 was used as the electron acceptor. Reaction rates were monitored by DCIP photoreduction at A59o and calculated based on an oxidized minus reduced extinction coefficient of 20.6 mm-' *cm-'. The activity of glucose 6-P dehydrogenase was determined spectrophotometrically as the rate ofdecrease at A30 in a reaction medium containing 50 mm Tris-Cl (pH 7.8), 3 mM MgCl2, 3 mm NAD, 3 mm glucose 6-P, and 1 gg/ml glucose 6-P dehydrogenase. Catalase activity was measured spectrophotometrically, in a mixture containing 50 mm phosphate buffer (pH 7.4), 20 mm H202, and 0.2 Ag/ml of catalase, as the rate of decrease at A240. The activity of,B-galactosidase was determined by measuring the increase of A420 in a mixture containing 100 mm phosphate buffer (pH 7.5), 2 mM o-nitrophenyl-/3-D-galactopyranoside, 1 mM MgCl2, and 6.0 ug/ml ,B-galactosidase. The activity of glutamate dehydrogenase was determined spectrophotometrically as the rate of decrease at A340 in a reaction solution containing 20 mm phosphate buffer (pH 7.8), 0.188 mM NADPH, 1.0 mM ADP, 1.0 M ammonium acetate, 20 mM ,8-ketoglutarate, and 1.0 ,ug/ml glutamate dehydrogenase. The activity of lactate dehydrogenase was determined spectrophotometrically as the rate of decrease at A340 in a medium containing 50 mm phosphate buffer (pH 7.0), 0.6 mM pyruvate, 1.67 mM NADH, and 66.6 gug/ml lactate dehydrogenase. The extinction coefficients used were 0.0394 mm-' *cm-' for H202, 6.32 mm-' *cm-' for NAD, and 3.5 mM-'. cm-' for o-nitrophenyl-B3-D-galactopyranoside. All assays of standard enzymes were carried out at room temperature. Calculation of Target Size. Mol wt (target size) were calculated from the equation of Beauregard and Potier (3): log Mr = 5.89 - log D37,, -0.0028 t where Mr is the funtional size in daltons, t is the temperature (°C) during irradiation, D37,1 is the dose of radiation in megarads required to reduce the activity to 37% of that found in unexposed control at temperature t (°C). The inactivation of many enzymes gives an exponential increase in D37 with decreasing temperature (3). The equation of Beauregard and Potier has already included the temperature correction factor (3). In addition, a method to obtain a comparative molecular mass of enzymes using f,-galactosidase as reference was also made (for details, see "Results and Discussion" section). Materials. Glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroides, EC 1.1.1.49), catalase (Bovine liver, EC 1.1 1.1.6), #B-galactosidase (Escherichia coli, grade VIII, EC 3.2.1.23), glutamate dehydrogenase (bovine liver, EC 1.4.1.3), lactate dehy2Abbreviations: DCIP, 2,6-dichlorophenolindophenol; Asc, ascorbate; DAD, diaminodurene (2,3,5,6-tetramethyl-p-phenylenediamine); DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DMQ, 2,5-dimethyl-benzoquinone; DPC, 1,5-diphenylcarbazide; FeCN, ferricyanide; MV, methylviologen; OEC, oxygen evolving complex; SiMo, a12-molybdosilicic acid; TMPD, N,N,N',N'-tetramethyl-p-phenylenedi-

amine.

159

drogenase (rabbit muscle, type XI, EC 1.1. 1.27), NADPH (type I), o-nitrophenyl-,3-D-galactopyranoside, and ,B-ketoglutarate were purchased from Sigma. TMQH2 was prepared according to Izawa and Pan (14). Silicomolybdic acid (a- 12-molydosilicic acid; H4SiMo12040) was kindly supplied by Climax Molybdenum Co. (Ann Arbor, MI). DBMIB was a generous gift from Dr. A. Trebst. All chemicals were of reagent grade and used without further purification. RESULTS AND DISCUSSION Molecular Mass of Standard Enzymes. In order to verify that the radiation inactivation analysis is a feasible technique to determine the functional size of photosynthetic electron transport chain, we estimated the molecular mass of a number of standard enzymes under our conditions. When the standard enzymes were irradiated in the frozen state, the enzyme activity declined in an exponential manner with increasing radiation dose as illustrated in Figure 1A. The D37 of these standard enzymes was determined using linear regression (r = 0.99). The molecular mass was thus calculated according to the equation of Beauregard and Potier (3). Table I shows the molecular mass of standard enzymes estimated by this way and that by other conventional biochemical methods. Since slopes of Figure 1A depend linearly on mol wt of standard enzymes, we obtained a plot for their relationship (Fig. 1B) using j3-galactosidase as reference (Mr = 464 kD). RG is the ratio ofthe slopes of dose-response plots for any selected enzyme to that of,8-galactosidase (Fig. IA). Thus, the molecular mass of any unknown protein may be determined by extrapolating the slope ratio (RG) as in Figure lB. For instance, RG for photosynthetic electron transport from H20 to MV is 1.34 indicating a molecular mass (i.e. target size) of 622 kD for this electron transport sequence (for details, see below). The precision of molecular mass determined by these manners comparing with that by conventional biochemical methods is less than +20% (Table I). Furthermore, within an appropriate range of molecular mass standards, it is even more precise for proteins with larger mol wt. In this study of photosynthetic electron transfer, we used the equation of Beauregard and Potier (3) for the calculation of molecular mass. Functional Size of Photosynthetic Electron Transport Chain. When isolated chloroplasts were exposed to high energy -y-ray irradiation, the photosynthetic electron transport rate was reduced with increasing radiation dose. Figure 2A depicts the different radiation sensitivities of various partial electron transport chains. In each case, the activity of these reactions decreased exponentially with the radiation dosage (Fig. 2B). From analysis of data using linear regression (r = 0.99), it was found that the radiation dose (D37) of 1.40 ± 0.1 1 Mrad was required to decrease the activity to 37% of control value for reaction from water to MV. This D37 value implies a functional size (Mr) of 623 ± 37 kD according to the equation of Beauregard and Potier (3). This functional size represents the mass of components associated with electron transport involving both photosystems as well as the intervening carriers. The functional sizes of the PSI and PSII were measured separately by isolating their activities using appropriate electron donors, electron acceptors, and specific inhibitors. The mass for PSII (H20 -* DMQ/FeCN) was determined as 174 ± 11 kD while that for PSI (DAD/Asc -* MV) as 190 ± 11 kD (Fig. 2B). Table II lists the functional sizes of various partial photosynthetic electron transport chains. Functional Size of Photosystem II. The molecular mass of PSII was about 174 ± 11 kD obtained from both reactions 3 (H20 -- DMQ/FeCN) and reaction 4 (H20-- DCIP) (Table II) in the presence of DBMIB. The similar functional size for the reactions supported by DMQ and DCIP implies that these oxidants intercept electrons at the same site on PSII. On the other

PAN ET AL.

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Plant Physiol. Vol. 85, 1987

trophoresis (25). It is therefore interesting to measure the molec-

ular mass of OEC and PSII reaction center by radiation inactivation in order to further investigate the composition and stoichiometry of their functional subunits. We used NH2OH- and tris-treated chloroplasts, employing DPC, benzidine, and catechol as electron donors which are believed to bypass OEC (15, 24), and measure the activity of PSII reaction center per se. We determined a mass of 156 ± 9 kD for the electron transfer from DPC to DCIP, i.e. without the functioning OEC. The difference of about 20 kD, by comparing reaction 5 (DPC -- DCIP) with 3 (H20 -- DCIP), should represent the molecular mass involved in oxygen evolving system. In the same manner, we obtained an estimate of 42 kD for the functional size of OEC from comparison of H20 to MV (reaction 1) with catechol or benzidine to MV (reaction 6 or 7). Several polypeptides of molecular mass 33, 24, and 18 kD can be washed out from PSII particles by alkaline tris or NaCl treatment (8, 25) resulting in the inhibition of oxygen evolving system. Several lines of evidence have suggested that 33 kD polypeptide contains manganese and is necessary for 02 evolution: (a) removal of all 33 kD polypeptide results in a total loss of 02 evolution (21) and (b) after removal of 18 and 24 kD proteins, the PSII preparatiois3 containing 33 kD polypeptide may still evolve oxygen in the presence of Ca2l and Cl- ions (22). It is generally believed that 24 kD is required to keep C1in its proper place since the addition of the 24 kD polypeptide to membrane depleted of this protein lowers their Cl- concentration requirement for oxygen evolution (1). It is also shown that 24 kD protein maintains a high affinity binding site for Ca2l (18). On the other hand, 18 kD polypeptide is believed not to play a crucial role in oxygen evolution since its complete absence does not always affect oxygen evolution (17, 22). The stoichiometry of these subunits of OEC is not settled yet (8). Murata et al. (21) have obtained a ratio of 1:1: per reaction center while

u

d)

a, >

'U 40

cc

Dose , M rad 700

snn

AGAL

2

300

GDH CAT

100

G6DH

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Larsson et

RG FIG. 1. A, Relationship between dose and response for various ard enzymes. Reaction conditions were described as in "Materials Methods." (U), glucose 6-P dehydrogenase; (A), lactate dehydrogenase; (A), catalase; (0), glutamate dehydrogenase; (0), (l-galactosidase. points are means of at least four assays with line fitted by regression analysis (r = 0.99). B, The plot for molecular mass versus RG. ratio of the slope of any enzymes in dose-response plot (A) to galactosidase. The molecular mass of any unknown protein obtained by extrapolating its RG from dose-response plot. (lgalactosidase; GDH, glutamate dehydrogenase; CAT, catalase; lactate dehydrogenase; G6PDH, glucose 6-P dehydrogenase; (0), stand-

and

All

data

RG is the

that ,Bof may

be

GAL,

LDH,

H20

MV.

hand, reaction 2 (H20

--

SiMo/DCMU) gives

a

slightly

smaller

molecular mass, 150 ± 9 kD. It has been generally held that SiMo intercepts electrons before DCMU binding site whereas DMQ and DCIP accept at this polypeptide. Ben-Hayyim and Neumann (5) suggested that SiMo might modify the chloroplast membrane such that in its presenceQA is accessible to exogenous oxidants like ferricyanide. However, it has been shown recently (9) the SiMo competes with DCMU at the binding site rather than modifying the membrane integrity thereby creating illusion of inhibitor insensitivity. At present, radiation inactivation technique is unable to distinguish these alternatives. Nevertheless, with evidence above and our results, we speculate that the molecular mass of PSII + QEC is in the range of about kD and the presence of SiMo may alter the 'physical PSII and probably lowers the functional size as well. A number of polypeptides have been assigned to OEC PSII reaction center using analysis of a variety of chloroplast preparations by sodium dodecyl sulfate-polyacrylamide the

170

state'

of

and

gel elec-

al.

suggested two copies of each per reaction center

(17). From our results using radiation inactivation and the evidence of other workers using other conventional biochemical methods,

we conclude that only a small fraction of these peripheral membrane proteins (20-40 kD versus 73 = [32 + 24 + 18] kD) may be involved catalytically in oxygen evolution. The rest of other polypeptides washed out by tris treatment may only play either regulatory or structural roles. We believe the 33 kD polypeptide is the most likely candidate as an obligatory component of oxygen evolution. We further calculated the functional size of PSII reaction center from comparison of reaction 3, 4, and 5 (TableI) and obtained a molecular mass of about 150 kD. Recently, a PSII complex containing only 32 kD (DI), 34 kD (D2) and 9 kD polypeptides was isolated by Nanba and Satoh (23). The heterodimer of the 9 kD and an often unresolved smaller polypeptide (about 4.5 kD) are the apoproteins of Cyt b559. However, the function ofthis Cyt has not been demonstrated in primary charge separation. The 32 kD polypeptide (DI) has been identified as a herbicide-binding protein as well as the binding site of secondary quinone electron acceptor (25). The function of 34 kD (D2) subunit is still in dispute. Nevertheless, in the presence of sodium dithionite this isolated D1-D2-Cyt b559 complex exhibited the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of PSII reaction center. Meanwhile, the homology of amino acid and nucleotide sequence between the Di and D2 subunits and the L and M subunits of reaction center from purple photosynthetic bacteria(19) has led to a suggestion that the site of primary charge separation in PSII is located on this complex (19, 24). Under this model, the sum of molecular mass of these three proteins is 75 (= 34 + 32 + 9) kD which is half of the functional size of PSII (150 kD) we

FUNCTIONAL SIZE OF PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN

161

Table I. Functional Size of Various Standard Enzymes The functional size of various standard enzymes were measured using either the equation of Beauregard and Potier (3) or the extrapolation from the plot of molecular mass versus RG (Fig. 1B). (M"d): standard molecular mass determined by conventional biochemical methods. (MT,BP): molecular mass measured by the equation of Beauregard and Potier (3). (Mr,R): molecular mass obtained by the intrapolation from Figure l B. Enzymes

,-Galactosidase Glutamate dehydrogenase Catalase Lactate dehydrogenase Glucose 6-P dehydrogenase

Mr,t

Mr,BP

kD 464 320 232 140 104

kD 481 337 214 120 125

A(Mr,BP- Mr,std) kD +17 +17 -22 -20 +21

800 cm

600

400

3000

0

X

x 200 * 0

1000

5 Dose . Mrad

10

1.1

0.7

Mr,R

A(Mr,R - Mr,t)

kD

kD

%

1.00 0.70

464 325

0 +5

0 +1.56

0.45 0.25 0.26

209 116 121

-23 -24 +17

-9.91 -17.14 +16.35

employed (Table II). The mol wt for electron flow from DAD/ Asc to MV was 190 ± 11 kD, from DCIP/Asc to MV, 120 ± 7 kD, and from TMPD/Asc to MV, 73 + 4 kD, respectively. It is believed that DAD/Asc and DCIP/Asc donate electrons at (or near) Cyt b6/f-plastocyanin complex although these reactions are not sensitive to quinol oxidase inhibitor (e.g. DBMIB). Our data imply that the donating sites for DAD/Asc and DCIP/Asc are different with DAD/Asc donating electrons at a site before that for DCIP/Asc. The difference of mass between these two sites is about 70 kD. Meanwhile, the data suggest that TMPD/Asc donates electrons at a site much closer to PSI reaction center (P700) than that for DAD/Asc and DCIP/Asc. The PSI-l00 complex of Mullet et al. (20) is composed of 60 to 70 kD P7oo-binding apoprotein, five to six polypeptides of 10 to 20 kD molecular mass including primary and secondary electron acceptor of PSI and PSI associated antenna light harvesting Chl protein which consists of three or four polypeptides of mass between 21 and 24 kD. Further treatment of the PSI110 complex with Triton or charged detergents to release the light harvesting protein complex from PSI reaction center yielded the PSI-65 complex. It appears the PSI-65 complex contains only the reaction center apoprotein (65 kD) and several low mol wt

1000

E

% +3.66 +5.31 +7.76 -14.29 +20.19

RG

B

0.5

0.3

polypeptides. It is generally agreed that the mol wt of PSI reaction center is about 60 to 70 kD (4). We obtained a mol wt of 73 ± 4 kD for TMPD/Asc -- MV. It is possible that this reaction represents the electron transport from a component very close to reaction center to a small primary electron acceptor. This functional size argues against the proposal that the 70 kD polypeptide of PSI core complex is in a dimeric form (4). The reaction center of 60 to 70 kD and the small primary electron acceptor (Fe-S center) with mol wt of 10 kD may form the smallest unit for functional PSI core complex. The larger functional size for DCIP/Asc and DAD/Asc than that for TMPD/Asc as electron donor to PSI FIG. 2. Relationship between photosynthetic electron transport rate implies that DCIP/Asc and DAD/Asc may donate electrons to and radiation dose. Reaction conditions were described as in 'Materials P700+ through other components like plastocyanin and Cyt b6/f and Methods." (0), H20 MV; (0), DAD/Asc MV; (A), H20 complex. Bengis and Nelson (4) suggested that a 20 kD subunit DMQ/FeCN. All data points are means of at least triplicate assays with from Swiss chard was required for the electron transport from line fitted by regression analysis (r 0.99). Semilogarithm plot of (A). plastocyanin to P700+, although Takabe et al. (27) believed that The intersects on abscissa at 37% activity of control give the D37 dose plastocyanin interacts directly with P700'. The larger functional values. size with DAD/Asc rather than DCIP/Asc as electron donors 0.1

0

5

Dose

10

,Mrad

--

=

determined by radiation inactivation. With this evidence, we propose that the PSII reaction center may be in a dimeric form. However, we cannot exclude the possibility that PSII reaction center is a monomer and can only function properly in the presence of other components ofthe same molecular mass under our conditions. A tentative working hypothesis for the composition of PSII reaction center and OEC is shown in Table III. The Functional Size of Photosystem I. The mass of electron transport chain via PSI varied depending on the electron donor

suggests that DAD/Asc may donate electrons at a site near the Cyt b6/f complex but after the inhibition site of DBMIB, while DCIP/Asc may donate electrons at a site close to plastocyanin and PSI reaction center. Since TMQH2 donation to PSI is DBMIB sensitive (14), it is believed that TMQH2 donates electrons before the DBMIB inhibition site. We therefore sought to determine the functional size of election transport chain from TMQH2 to MV (Table II). Surprisingly, we obtained a smaller molecular size for this reaction than for DAD/Asc (121 ± 7 versus 190 ± 11 kD). This

162

Plant Physiol. Vol. 85, 1987

PAN ET AL.

Table II. Functional Size ofPhotosynthetic Electron Transport Chain The reaction conditions were described as in "Materials and Methods." The reaction rates were measured as 02 uptake for those with MV as electron acceptor, while the rest (except reaction 5) were measured as 02 evolution. The activity of reaction 5 was obtained spectrophotometrically by DCIP photoreduction at A590. The functional sizes were determined according to the equation of Beauregard and Potier (3).

Reactions

Control Rate

D37

Functional

Chmoleq/mg

Mrad

kD

1.4 ± 0.1 5.8 ± 0.3 5.0 ± 0.3 5.0 ± 0.3 5.6 ± 0.3 1.5±0.1 1.5 ±0.1 4.6 ± 0.3 7.8 ± 0.5 12.0 ± 0.7 7.2 ± 0.4

623 ± 37 150 ± 9

C'hl.h 800

1. H20 - MV 2. H20 -* Si/Mo/FeCN, DCMU 3. H20 -- DMQ/FeCN, DBMIB 4. H20 DCIP/DBMIB 5. DPC - DCIP/DBMIB 6.Catechol -MV 7. Benzidine - MV 8. DAD/Asc MV/DCMU, DBMIB 9. DCIP/Asc -MV/DCMU, DBMIB 10. TMPD/Asc -. MV/DCMU, DBMIB 11. TMQH2- MV/DCMU

240 432 420 65 80 90 4840 1980 800 2700

174 ± 11 174 ± 11 156 ± 9 581±35 581 ± 35 190 ± 11 112±7 73 ± 4 121 ± 7

Table III. Possible Composition and Stoichiometry ofPolypeptidesfor Photosynthetic Electron Transport Chain Functional size of partial reactions was determined by radiation inactivation (this work). The molecular mass for OEC (20-40 kD) was calculated from the comparison between reactions 4 and 5 or 1 and 7 (see text). The mol wt of possible components were obtained from other work as indicated in references. Symbols: Fe- S, Rieske iron-sulfur center; PC, plastocyanin; RC, reaction center. of

Functional Possible Mr SystemsReactionsSize ComponePossible Components

Systems

1. PSII + OEC PSII

Reactions

-

H20- DCIP DPC -. DCIP

kD 174 ± 11 156 ± 9

PSII

DI (PSII + OEC) - (PSII)

20

-

40

OEC

or

2. PSI

(PSII + OEC + PSI) - (PSII + PSI) TMPD - MV DAD--MV

3. OEC + PSII + PSI

H20 -* MV

Reference

kD

D2 OEC

Sum of MR

of Possible Components

73 ± 4

PSI RC Fe-S PSI RC+Fe-S PC (?) PSII+OEC PSI

190 ± 11

623±37

Cyt b6/f measurement has been made at least 20 times. The reasons for the contradiction of molecular size to conventional sequence for electron transport from TMQH2 to MV is still unknown. We are currently engaged in further studies to elucidate this discrepancy. According to the conventional Z-scheme, reaction 1 (H20 MV) consists of PSI (i.e. reaction 8: DAD/Asc -- MV) and PSII (i.e. reaction 3: H20 -* DMQ). The molecular mass of reaction 1 is much larger than the sum of reaction 3 + reaction 8 by 259 kD (= 623 - [190 + 174] kD). We believe that this value is the molecular mass of components aligned between PSII and PSI (i.e. between the DAD/Asc donating site and the DCMU binding site). Several laboratories (12) have isolated from pea and spinach chloroplasts a Cyt b6/f complex that consists of four or five polypeptides of mol wt 37, 34, 22, 19, and 16 kD. Thus, the sum of molecular mass of subunits (128 kD) above is close to half of 259 kD value calculated by radiation inactivation. On this basis, -

This work This work This work; 23

34 (2x) 32 (2x) 33, or 23 or 16 (?)

132

60 - 70 10

70 - 80

This work 5

190

Thiswork

614

This work This work This work; 10

This work; 25

73 10 107 174

190 125 (2x)

we speculate that Cyt b6/f complex may be in a dimeric form in accordance with the suggestion of Graan and Ort (10). Nevertheless, we cannot exclude the possibility that Cyt b6/f complex is in a monomeric form and that other unknown components may account for the larger target size. Acknowlegments-We thank Dr. Y. K. Lai for reading the manuscript and

valuable discussions and Ms. J. H. Chou for her expert secretarial assistance. We also thank Mr. F. C. Song and S. T. Sheu for irradiation of samples. The generosity of Radiation Application Division, Institute of Nuclear Energy Research (Long Tam) to provide 'Co as a gift to Radioisotope Section at our college is gratefully

acknowledged.

LITERATURE CITED 1. ANDERSON B, C CRITCHLEY, U RYIUE, C JANSSON, C LARSSON, JM ANDERSON 1984 Modification of the chloride requirement for photosynthetic 02 evolution: the role ofthe 23 kDa polypeptide. FEBS Lett 168: 113-117 2. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Plant Physiol 24:

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