[39] Acceptors and donors and chloroplast electron transport

[39]

CHLOROPLAST ELECTRON TRANSPORT

413

A second technical difficulty arose in the stability of electrode sensitivity, which drifted with time after imposition of a chosen polarizing potential. Polarograms of current signal versus potential showed a marked hysteresis when traversed first toward higher and then toward lower potentials. For example, the current at 0.6 V after a period of 0.7 V was almost twice that observed after a period of 0.5 V. The following precedures suggested by Gilman. 5 We conditioned the electrode surface through a treatment consisting of about 10 rain of timed 50/rain alternations of polarizing potential between 0.2 and 0.8 V. This can be accomplished by utilizing a timer coupled to a single-pole double-throw microswitch which alternately connects the electrodes to two points in the divider chain to provide the desired voltages. Practical experiences has shown that H2S, CO, and HCN behave as electrode poisons and greatly reduce sensitivity. Photoflo, a wetting agent, that is routinely added to KC1 solutions by the manufacturers can degrade electrode performance. It is, therefore, suggested that the KCI solution be prepared from purified salt. A poisoned electrode can generally be reconditioned by thorough cleaning. A polishing compound (No. 600 or finer) can be used to polish the platinum electrode. Dilute ammonium hydroxide can be used to remove the silver chloride from the silver electrode, followed by recoating. Acknowledgments

I am grateful to Dr. L. W. Jones and Dr. S. Lien for sharing with me their recent experiences with the electrode. S. Gilman, in " E l e c t r o a n a l y t i c a l C h e m i s t r y " (A. J. Gard, ed.), Vol. 2, p. 111. D e kke r, N e w Yo rk , 1967.

[39] A c c e p t o r s a n d D o n o r s f o r C h l o r o p l a s t E l e c t r o n Transport

By S. IZAWA The methodology of chloroplast electron-transport studies using exogenous electron acceptors and donors was last reviewed in 1972 by Trebst ~ in this series. As a sequel to it, this article is concerned mainly

I A. Trebst, Vol, 24, p. 146.

METHODS IN ENZYMOLOGY, VOL. 69

Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181969-8

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with the new developments that have been made in the field since around 1970.1a One of the notable recent developments is the introduction of a new group of Hill oxidants which are capable of intercepting electrons from photosystem II (PS-II) at very high rates even in unfragmented chloroplasts. 2 Represented by oxidized p-phenylenediamines, these PS-II electron acceptors (or "class III acceptors") are all lipid-soluble oxidants with high redox potentials (E0'> + 0.1 V). Together with the introduction of a number of new electron-transport inhibitors, s'4 the introduction of class III acceptors has opened up a new approach toward the mechanisms of photosynthetic electron transport and associated phosphorylation. Furthermore, the recognition of lipophilicity as an essential character of PS-II electron acceptors has stimulated the interests of investigators in the membrane topology of the electron-transport system and thereby contributed much to our understanding of the structure-function of the thylakoid membrane. Recent review articles by Trebst 5 and by Hauska 6 include discussions of these and other new studies involving the use of artificial electron donors and acceptors. This article deals only with experiments which utilize the envelopefree "class II" chloroplasts 7 (or "type D " chloroplasts according to Hall's terminologyS), the standard material for electron-transport and photophosphorylation studies. These chloroplasts consist simply of sheets of thylakoids (lamellae) which usually retain all the components of the photosynthetic electron-transport chain except for ferredoxin which is leached out during chloroplast isolation. Figure 1 represents a simplified model of photosynthetic electron transport in isolated chloro-

~a Abbreviations: DAD, diaminodurene (2,3,5,6-tetramethyl-p-phenylenediamine); DADox, oxidized form of DAD (duroquinonediimide); DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone); DCIP, 2,6-dichlorophenolindophenol; DCIPH2, reduced form of DCIP; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; PD, p-phenylenediamine; PDox, oxidized PD (p-benzoquinonediimide); PMS, phenazine methosulfate (N-methylphenazonium methosulfate); TMPD, N,N,N',N'Xtetramethylp-phenylenediamine. 2 S. Saha, R. Ouitrakul, S. Izawa, and N. E. Good, J. Biol. Chem. 346, 3204 (1971). 3 S. Izawa, in "Photosynthesis I" (A. Trebst and M. Avron, eds.), Encycl. Plant Physiol., New Ser., Vol. 5, p. 266. Springer-Verlag, Berlin and New York, 1977. 4 A. Trebst, this volume, p. 675. 5 A. Trebst, Annu. Rev. Plant Physiol. 25, 423 (1974). e G. Hauska, in "Photosynthesis I" (A. Trebst and M. Avron, eds.), Encycl. Plant Physiol., New Ser., Vol. 5, p. 253. Springer-Verlag, Berlin and New York, 1977. r D. Spencer and H. Unt, Aust. J, Biol. Sci. 18, 197 (1965). s D. O. Hall, Nature (London), New Biol. 235, 125 (1972),

[39]

CHLOROPLASTELECTRONTRANSPORT

415

3 H20 - ~ - - P 6 8 0 . P S - ] I - Q - i ~ P _ ~ - I - - ~ f - P C - P 7 0 0 Tris /

®

e ~

®

e'l

®

Hg

• PS-I.X L

®

FIG. 1, A model of photosynthetic electron transport in isolated chloroplasts showing sites of electron donation and acceptance (arrows) and sites of inhibition (broken lines). The symbols and abbreviations used are: P680, the reaction center chlorophyll of photosystem II (PS-II); Q, the primary electron acceptor of PS-II; PQ, the plastoquinone pool; f, cytochrome f; PC, plastocyanin; P700, the reaction center chlorophyll of photosystem I (PS-I); X, the primary electron acceptor of PS-I; DCMU, 3-(3,4-dichlorophenyl)-l,1dimethylurea; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone).

plasts (for details, see Trebst 5 and Golbeckg). The numbered arrows indicate regions where electron donation and acceptance by exogenous redox agents are believed to take place. (In the following sections, electron donation and acceptance will be discussed in reference to these regions.) The broken lines indicate sites where electron transfer can be blocked by specific inhibitors. 4 Acceptors of Electrons from Photosystem I (Region 1) Of all parts of the thylakoid membrane-bound photosynthetic electron-transport chain, the reducing end of PS-I (X in Fig. l) seems to be the part which is most easily accessible to exogenous electron acceptors. Presumably, this region is exposed on the outer surface of the membrane. ~,5,6 Furthermore, the primary electron acceptor of PS-I (X), when reduced, is an extremely strong reductant (E0' ~> - 0 . 5 V). ~°m One must, therefore, assume that any known Hill oxidant, regardless of its standard redox potential and of its solubility properties, should be able to accept electrons from PS-I. All but a few of the PS-I electron acceptors listed below are already covered by the previous review, 1 some of them under "PS-II acceptors." The regrouping here is based on information which

9 j. H. Golbeck, S. Lien, and A. San Pietro, in "Photosynthesis I" (A. Trebst and M. Avron, eds.), Encycl. Plant Physiol. New Ser., Vol. 5, p. 94. Springer-Verlag, Berlin and New York, 1977. 10 R. Malkin, in "Photosynthesis I" (A Trebst and M. Avron, eds.), Encycl. Plant. Physiol. New Ser., Vol. 5, p. 179. Springer-Verlag, Berlin and New York, 1977. 11 B. Ke, Biochim. Biophys. Acta 301, 1 (1973).

416

METHODOLOGY

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has become available in the past several years. Some additional comments are also given to well-established PS-I electron acceptors. NADP/Ferredoxin

This reconstituted natural electron-acceptor system is completely PSI specific, but it tends to be inefficient and requires a relatively large amount of ferredoxin (50-100/zg/ml) for rate saturation.12 Whether or not the terminal NADP reduction step is rate-limiting may be examined by comparing the rate of NADP reduction or of associated 02 production with the rate of methyl viologen reduction as observed by 02 consumption (see next section). If it is rate-limiting, the electron flow does not respond properly to exogenous agents such as inhibitors, uncoupling agents, and ADP/phosphate, which is misleading. TM Methods. Besides the method given previously 1 the NADP Hill reaction can be measured as 02 evolution or by directly observing the absorbance increase (340 nm) of a reaction mixture containing a relatively small amount of chloroplasts (e.g., 5-10/zg chlorophyll/ml). An example of a reaction mixture for O~ assay under phosphorylating conditions is 0. l M sucrose, 40 mM tricine-NaOH buffer (pH 8.0), 5 mM MgC12, 1 m M ADP, l0 mM NazH32PO4, 0.2 mM NADP, 100/~g ferredoxin/ml, and chloroplasts equivalent to 25/zg chlorophyll/ml. Autoxidizable Electron Carriers

y,y'-Dipyridyl salts such as methyl viologen (Eo' -0.44 V) and lowpotential quinones such as anthraquinone sulfonates (Eo' ~ - 0 . 2 V) are representatives of this group of electron acceptors. They accept electrons exclusively from PS-I, and pass them on to ambient Oz to reduce it to the level of superoxide radical, which dismutates to HzO2 and 02. Thus, with catalase-free chloroplast preparations or in the presence of a catalase inhibitors (e.g., 1 mM KCN or NAN3), the transport of electrons from water to the end of PS-I can be measured as 02 uptake (Mehler reaction). The rate of 02 uptake corresponds exactly to the rate of concurrent (but masked) 02 evolution, a3 For more details, see the previous review. 1 These low potential acceptors are also very convenient to use with artificial electron donors, but their use for this purpose requires caution (see section for PS-II electron donors). is H. E. Davenport, in "Biological Structure and Function" (T. W. Goodwin and O. Lindberg, eds.), Vol. 2, p. 449. Academic Press, New York, 1961. 13 In the absence of any of these artificial low potential acceptors, washed chloroplasts normally show a Mehler reaction activity up to 50/zequiv/hr-mg chlorophyll. See, for example, J. M. Gould and S. Izawa, Eur. J. Biochem. 37, 185 (1973).

[39]

CHLOROPLAST ELECTRON TRANSPORT

417

Methods. The reaction mixtures described for ferricyanide (next section) can be modified for methyl viologen experiments by simply replacing the ferricyanide with O. 1 mM methyl viologen.

Potassium Ferricyanide Thermodynamically, ferricyanide (Eo' +0.42 V) has a capacity to accept electrons directly from PS-II, and indeed it does so efficiently in subchloroplast particles. TM These facts led many workers to the belief that ferricyanide reduction is always a pure PS-II reaction. However, recent inhibition experiments with plastocyanin inhibitors 15-t7 and with DBMIB 18't9 strongly indicate that in unfragmented thylakoid membranes ferricyanide in fact acts more as a PS-I electron acceptor than as a PS-II acceptor. PS-II reduction may account for only - 5 - 1 0 % of the total ferricyanide reduction in chloroplasts with high membrane integrity 1~,~9 and 20-40% in average chloroplast preparations? 7,1s The accessibility of the PS-II reduction sites (region 2 in Fig. 1) to highly polar oxidants such as ferricyanide seems to be very limited unless the thylakoid membrane is disrupted. This appears to be also true of the cytochrome/plastocyanin region, z° Methods. The ferricyanide Hill reaction may be measured as 02 evolution or by following the absorbance decrease of the reaction mixture at 420 nm (absorption peak of ferricyanide; miUimolar extinction, 1.00 mM -~ cm-0 but the optical assay tends to be inaccurate when large changes in the light-scattering properties of chloroplasts occur during electron transport. An example of a reaction mixture useful for both 02 assay and optical assay under phosphorylating conditions is 0.1 M sucrose, 40 mM tricine-NaOH buffer (pH 8.3), 3 mM MgC12, 10 mM Na2H3~PO4, 1 m M ADP, 0.4 mM potassium ferricyanide, and chloroplasts equivalent to 25/zg chlorophyll/ml. The maximum rate of electron transport can be measured at pH 7.0 using phosphate or MOPS buffer 21 and methylamine hydrochloride as an uncoupling agent.

14 j. M. Anderson and N. K. Boardman, Biochim. Biophys. Acta 112, 403 (1966). 1~ R. Ouitrakul and S. Izawa, Biochim. Biophys. Acta 305, 105 (1973). 10 M. Kimimura and S. Katoh, Biochim. Biophys. Acta 325, 167 (1973). lr W. G. Nolan and D. G. Bishop, Arch. Biochem. Biophys. 166, 323 (1975). 18 H. B6hme, S. Reimer, and A. Trebst, Z. Naturforsch., Teil B 26, 341 (1971). ~9 S. lzawa, J. M. Gould, D. R. Ort, P. Felker, and N. E. Good, Biochim. Biophys. Acta 305, 119 (1973). 20 p. Horton and W. A. Cramer, Biochirn. Biophys. Acta 368, 348 (1974). 21 N. E. Good and S. lzawa, Vol. 24, Part B, p. 53.

418

METHODOLOGY

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2,6-Dichlorophenolindophenol (DCIP) and R e l a t e d Oxidants

DCIP (Eo' = +0.22 V, pKa' 5.7) is another oxidant which was often treated as a PS-II electron acceptor and which has recently been shown to behave more as a PS-I acceptor when used with unfragmented chloroplasts. 16,1s,19 This situation was already predicted by the kinetic experiments of Kok et al. 22 The blue, ionized form of the dye, which predominates at neutral to basic pH's is hydrophilic and, like ferricyanide, seems to have only limited access to the PS-II reduction sites. However, in fragmented chloroplasts 14 or at acidic pH's where the red, lipid-soluble undissociated form predominates, the dye will certainly intercept electrons from PS-II more freely. It is also this lipid-soluble form of the dye that seems to be responsible for the well-known uncoupler action of DCIP. z In line with these observations, it has been reported that the highly hydrophilic sulfonated DCIP does not uncouple and behaves as a typical PS-I electron acceptorY 3 One of the quinonimide dyes synthesized and tested by Hill, 24 compound XVI or reduced dichroin, also showed properties of a nonuncoupling PS-I electron acceptor. Methods. DCIP photoreduction can be easily assayed spectrophotometrically by following the absorbance decrease of the reaction mixture at or near 600 nm (absorption peak of DCIP at pH > 7; millimolar extinction, 21 mM -a cm-1). An example of a reaction mixture is 0.1 M sucrose, 30 mM HEPES-NaOH buffer (pH 7.5), 30/zM DCIP (A600 0.6) and chloroplasts equivalent to 5 /~g chlorophyll/ml. 02 assay is possible but not very practical.

Acceptors of Electrons from Photosystem II (Region 2) The Hill reaction supported by lipid soluble oxidants, such as oxidized phenylenediamines and quinones, show properties which deviate from those of the standard Hill reaction with water-soluble electron acceptors such as ferrianide. The electron transport is distinctively more rapid, less responsive to addition of ADP and phosphate, and is less efficient in supporting phosphorylation. Extreme deviations are found with oxidized p-phenylenediamine and oxidized diaminodurene. They are photoreduced at rates which are even faster than the rate of fully uncoupled 22B. Kok, S. Malkin, O. Owens, and B. Forbush,Brookhaven Symp. Biol. 19, 446 (1966). 2aG. Hauska, A. Trebst, and W. Draber, Biochim. Biophys. Acta 305, 632 (1973). 24R. Hill,Bioenergetics 4, 432 (1972).

[39]

CHLOROPLASTELECTRONTRANSPORT

419

ferricyanide reduction. The electron flow, which is almost completely independent of the presence or absence of ADP and phosphate, does support phosphorylation but with only half the efficiency of conventional noncyclic photophosphorylation (Fig. 2). To explain these findings, Saha e t a l . z proposed a model of chloroplast electron transport which postulated that the electron transport chain contained two energy coupling sites and an intermediate reduction site. The intermediate reduction site (X' in the scheme below), which was placed between the two sites of energy coupling ( - ) , was assumed to be burried in the lipid membrane and therefore only accessible to lipid-soluble Hill oxidants. The terminal reduction site (X) was assumed to be exposed on the external surface of the membrane and therefore accessible to all Hill oxidants. Lipophilic oxidants H20 ~ PS'II

fast

~'

• )X'(buried)

All oxidants / slow

~ PS-I ---~X(exposed)

This model acquired strong support when it was demonstrated that the photoreduction of lipid-soluble oxidants and associated phosphorylation are only partially inhibited by the plastocyanin inhibitor KCN 15 and by the plastoquinone antagonist DBMIB. 19,25 These inhibitors not only abolished strictly PS-I-requiring reactions, such as the methyl viologen Hill reaction; in well-coupled chloroplasts they nearly abolished ferricyanide photoreduction as well, indicating the relative inaccessibility of the intermediate reduction site (X') to hydrophilic oxidants. Evidently the lipophilic quinoid compounds do have access to, and accept electrons efficiently from the site(s). However, the fact that their photoreduction is partially inhibited by KCN and DBMIB (20 to 50% inhibition depending on the oxidant) strongly suggests that substantial portions of the reduction take place at PS-I when the pathway of electrons from the intermediate reduction site(s) to PSI-I is open. (This two-site reduction model has been discussed in some detail. 15) Oxidized p-phenylenediamines and substituted quinones are now in frequent use for investigations of various PS-II-associated phenomena. In such experiments DBMIB-poisoned or KCN-treated chloroplasts are routinely employed to ensure that PS-I is not involved in the reaction under study (see Yocum, this volume. 26)

25 A. Trebst and S. Reimer, Biochirn. Biophys. Acta 305, 129 (1973). z6 C. F. Yocum, this volume, Article [54].

420

METHODOLOGY

[39]

Oxidized p-Phenylenediamines and Related Oxidants At neutral to basic pH's, p-phenylenediamine (PD) (Eo' = +0.36 V) and 2,3,5,6-tetramethyl-p-phenylenediamine (diaminodurene or DAD) (E0' = +0.22 V) can be oxidized quantitatively or nearly quantitatively by ferricyanide to p-benzoquinonediimide (PDox) and duroquinonediimide (DADox), respectively. HsC

CHs

HzN~

HsC

CHs

Nil2 ~ ~ H N ~ N H

HsC

CHs

(DAD)

HsC

+ 2e-+

2H +

CHs

~ADox)

PDox and DADox are both excellent PS-II electron acceptors, z PDox can support vast rates of electron transport (up to 2000 tzEq/hr mg chlorophyll) which, according to inhibition data, 15 is mostly (70-80%) due to PS-II reduction. Unfortunately, PDox is chemically highly unstable. The relatively stable DADox is, therefore, the preferred oxidant for routine work even though DADox-SUpported PS-II electron transport and phosphorylation are appreciably slower than those supported by PDox. Interestingly, photoreduction of PDo~ and DADo~ is markedly inhibited, rather than stimulated, by uncoupling agents, z7-3° (An indication of this uncoupler effect is seen in Fig. 2.) The phenomenon has been interpreted to mean that energy-linked proton translocation is contributing to the high rates of photoreduction in coupled chloroplasts, either by facilitating the shuttling of the amine/imine forms of the acceptor across the thylakoid membrane, 2s or by inducing the internal accumulation of the imine form. z° Other useful PS-II electron acceptors of this category include oxidized forms of 2,5-diaminotoluene, 4,4'-diaminodiphenylamine, TMPD, 2 N,Ndimethyl-p-phenylenediamine, N,N-diethyl-p-toluidine, 2-methyl-5methoxy-p-phenylenediamine, etc. al Not surprisingly, many of these and other amines which act in their oxidized forms as PS-II electron acceptors are known to donate electrons to PS-I. al Methods. Because of their chemical instability, oxidized p-phenylenediamines (PDox, DADox, etc.) are prepared in a buffered reaction me2r j. M. Gould and D. R. Ort, Biochim. Biophys. Acta 325, 157 (1973). 2s A. Trebst and S. Reimer, Biochim. Biophys. Acta 325, 546 (1973). 29 W. S. Cohen, D. E. Cohen, and W. Bertsch, FEBS Lett. 49, 350 (1975). 30 j. M. Guikema and C. F. Yocum, Biochemistry 15, 362 (1976). 31 A. Trebst and S. Reimer, Z. Naturforsch., Teil C 28, 710 (1973).

CHLOROPLAST ELECTRON TRANSPORT

[39]

......

A

421

/ .~" "'*"-,e FeCy I.o1-- - ~ / . . . . . . . . .

~,1/

150C

T

.~ 15oc

/<7

u

0

E

0.5

oAo,.

o.stC- ~..~,--~- --

1.0

,

8

1

9

/(-.\

&

I000

IOOO

,/

E'I(DADoxl

/ E.T.(FeCyl

\

>

50- 500

50C

_=- /~ATP

IJ.I

=.~/~/~ O, Fe'C 0hi

~5 DAbox(mM)

I

1.0

°"" i~"" ATP(FeCy:"

7

8

9

pH

F]G. 2. The Hill reaction and associated phosphorylation with oxidized diaminodurene (DADox) as the electron acceptor. The reaction mixture for (A) contained 40 mM TricineNaOH buffer (pH 8.2), 1 mM ADP, 5 mM K2Hz2PO4, 3 mM MgCI2, 5 mM methylamineHCI (MA) (if added), 0.4 mM potassium ferricyanide plus varied concentrations of DAD.x, and chloroplasts equivalent to 20/xg chlorophyU/ml. At DAD,,x = 0, only ferricyanide was present as the electron acceptor. In the pH profile experiments of (B), the data for the regular ferricyanide Hill reaction are given for comparison. The buffers used are 2-(Nmorpholino) ethanesulfonic acid (MES)-NaOH for pH 6.5, N-2-hydroxyethylpiperazineN'-ethanesulfonic acid (HEPES)-NaOH for pH 7 and 7.5, Tricine-NaOH for pH 8, and N-tris(hydroxymethyl)-3-aminopropanesulfonicacid (TAPS)-NaOH buffer for pH 8.5 and 9. All buffers were at 40 mM. The ferricyanide concentration was 0.4 mM. When present, DAD.x (see text) was 0.6 mM. Other conditions were the same as in (A). Electron transport was assayed as ferricyanide reduction observing the absorbance changes of the reaction mixture at 420 nm. The intensity of actinic light, approximately 500 kergs sec -1 cm -z (>600 nm). (Adapted from S. Saha, R. Ouitrakul, S. lzawa, and N. E. Good, J. Biol. Chem. 246, 3204 (1971).)

d i u m , i m m e d i a t e l y b e f o r e t h e r e a c t i o n , b y m i x i n g d i h y d r o c h l o r i d e salts o f t h e a m i n e s ( t y p i c a l final c o n c e n t r a t i o n , 0.4 m M ) a n d e x c e s s f e r r i c y a n i d e (1.2 m M ) . A f t e r m i x i n g , t h e m e d i u m s h o u l d r e m a i n c o l o r l e s s e x c e p t f o r t h e p a l e y e l l o w d u e to t h e p a r t o f f e r r i c y a n i d e w h i c h h a s r e m a i n e d u n r e d u c e d . ( I m p u r e p - p h e n y l e n e d i a m i n e s t e n d to f o r m d e e p - b r o w n oxidation by-products.) For details of the experimental procedure and pur i f i c a t i o n o f p - p h e n y l e n e d i a m i n e s , s e e Y o c u m , this v o l u m e Y 6

422

METHODOLOOY

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Substituted Benzoquinones The ability of p-benzoquinone (E0' = +0.29 V) to penetrate biological membranes was already recognized by Warburg 32 who introduced this Hill oxidant more than 30 years ago. It is still used quite often, especially for the purpose of investigating the photosynthetic electron-transport activity of plant and algal cells. However, in terms of the ability to intercept electrons from PS-II in isolated chloroplasts, 2,5-dimethyl-pbenzoquinone (E0' = +0.18 V) is far superior to p-benzoquinone. 2 Furthermore, the dimethylquinone is chemically much more stable than the unsubstituted quinone and can be used freely at pH 8 (standard pH for phosphorylation experiments) where the latter will denature in seconds. As a PS-II electron acceptor, 2,5-dimethylquinone is also much more convenient to use than oxidized p-phenylenediamines (PDox, DADox, etc.) although the latter oxidants do support faster rates of PS-II electron transport and phosphorylation. 2,5-Dichloro-p-benzoquinone is also a good PS-II acceptor. 2 Trebst's group, who routinely use 2,6-dimethyl-pbenzoquinone (Eo' = +0.18 V) 25 rather than the 2,5-dimethyl analog, have recently introduced an autooxidizable PS-II electron acceptor, dimethylmethylenedioxy-p-benzoquinone, ns which allows one to assay its reduction by PS-II as 02 consumption. No doubt many more interesting PS-II electron acceptors will be found in the future, for instance among those various quinones and their derivatives which Trebst and his associates used in their early work on chloroplast photophosphorylation? 4 Methods. This author uses 2,5-dimethylquinone routinely for PS-II electron transport and phosphorylation studies. The quinone can be used alone (rate-saturating concentration, 0.5 mM) or in combination with ferricyanide (0.5 mM) to observe the reaction as O3 evolution or as ferricyanide reduction (see section for ferricyanide under PS-I electron acceptors). DBMIB as PS-H Electron Acceptor The plastoquinone analog DBMIB (E0' ~ +0.17 V), now widely used as an electron-transport inhibitor, 4 acts as a PS-II electron acceptor when

32 O. Warburg and W. L0ttgens, Naturwissenschaften 38, 301 (1944). 33 A. Trebst, S. Reimer, and F. Dallcker, Plant Sci. Lett. 6, 21 (1976). 34 A. Trebst and H. Eck, Z. Naturforsch., Teil B 16, 44 (1961); see also A. Trebst, H. Eck, and S. Wagner, in "Photosynthetic Mechanisms of Green Plants" (B. Kok and A. T. Jagendorf, eds.), p. 174. Natl. Acad. Sci.--Natl. Res. Counc., Washington, D.C., 1963.

[39]

CHLOROPLASTELECTRONTRANSPORT

423

used at relatively high concentrations (optimum, 10-20 ~M). 19'35 The advantage of using DBMIB as the oxidant is that the reaction observed is a pure PS-II reaction because electron transfer between PS-II and PSI is completely blocked by the DBMIB itself. The disadvantage is that the reaction tends to be slow ( < 2 0 0 / z E q / h r mg chlorophyll) presumably because of the side effects which high concentrations of DBMIB exert on PS-II. The reaction supports phosphorylation as well as proton translocation. 35 M e t h o d s . DBMIB reduction can be measured as 02 evolution (pH < 8), 02 consumption (pH > 8.2) or spectroph0tometrically using ferricyanide as the terminal acceptor. Ferricyanide and D C I P

As already discussed, these ionic oxidants accept electrons mostly from PS-I in undisturbed thylakoid membrane but they can be reduced in large part by PS-II when the membrane is disturbed (see discussion o f PS-I electron acceptors). E l e c t r o n A c c e p t o r s for t h e D C M U - I n s e n s i t i v e Hill R e a c t i o n ( R e g i o n 3) Photoreduction of heteropoly compounds such as silicotungstate, phosphotungstate, phosphomolybdate, and silicomolybdate, has been shown to be totally or partially insensitive to DCMU. ar-3s Fluorescence experiments suggest that these oxidants may be capable of accepting electrons from the primary electron acceptor of PS-II (Q in Fig. 1). 39"4° Since silicomolybdate-washed chloroplasts can also photoreduce such c o m m o n oxidants as DCIP and ferricyanide, it seems that silicomolybdate perturbs the thylakoid membrane in some way and that this membrane modification renders the primary acceptor accessible to exogenous oxidants including silicomolybdate itself. 41 In line with this notion, it has recently been shown that mildly trypsin-treated chloroplasts can photo35j. M. Gould and S. Izawa, Eur. J. Biochem. 37, 185 (1973); see also J. M. Gould and S. Izawa, Biochim. Biophys. Acta 333, 509 (1974). a6 G. Girault and J. M. Galmiche, Biochim. Biophys. Acta 333, 314 (1974). ar R. T, Giaquinta, R. A. Dilley, F. L. Crane, and R. Barr, Biochern. Biophys. Res. Comrnun. 59, 985 (1974). a8 R. Barr, F. L. Crane, and R. T. Giaquinta, Plant Physiol, 55, 460 (1975). as R. T. Giaquinta and R. A. DiUey,Biochim. Biophys, Acta 387, 288 (1975). 40 B. Zillinskas and Govindjee, Biochim. Biophys. Acta 387, 306 (1975). 41G. Ben-Hayyim and J. Neumann, FEBS Lett. 56, 240 (1975).

424

METHODOLOGY

[3 9]

reduce ferricyanide in a DCMU-insensitive reaction, although in this case rather high concentrations of ferricyanide (>10 mM) is required. 42 The section below describes the chemical properties and the reaction characteristics of silicomolybdate, the most frequently used oxidant of this category.

Silicornolybdic Acid (12-Molybdosilicic acid) In 1 N HC1, silicomolybdic acid (H4Mo12040) accepts up to 8 electrons in a four-step reaction (E0' = +0.56, +0.43, +0.19, +0.02 V). 43 The redox properties of this substance in neutral pH regions (where the complex is gradually hydrolyzed) are not clear, except for the fact that the fully oxidized form (usual form) is still a strong enough oxidant to be visibly reduced by comparable concentrations of ferrocyanide (E0' = +0.42 V). This strong poly acid forms insoluble complexes with various organic bases, such as amines, quarternary ammonium salts, amides, etc. It also tends to form insoluble complexes and salts with various metals (including K ÷) and their chelates. 43 It precipitates such commonly used cationic oxidants as methylviologen and N-methylphenazonium ion ("PMS")44 and seems to aggregate gramicidin. Clearly the use of silicomolybdate for electron transport and phosphorylation experiments calls for special caution. Silicomolybdate is also a multiple-character Hill oxidant. Its behavior apparently depends on the extent of the damage it causes to the chloroplast membrane. Thus in the presence of high concentrations of bovine serum albumin (a membrane protective agent) silicomolybdate behaves exactly like ferricyanide, supporting well-coupled, completely DCMU-sensitive electron transport. Only when the protection is weakened, does it begin to extract electrons from PS-II in a DCMU-insensitive reaction which m a y 44-47 o r may not 37,39 support phosphorylation depending on the extent of protection and on other as yet unclarified conditions. In the total absence of protective agents, 0.1 mM silicomolybdate strongly uncouples and quickly abolishes all electron transport activities. 44 Methods. The following are modifications of the reaction mixture and procedure which have been used in this author's laboratory to demon42 G. Renger, FEBS Lett. 69, 225 (1976). 43 G. A. Tsigdinos and C. J. Hallada, J. Less-Commun. Met. 36, 79 (1974); see also G. A. Tsigdinos, Bull. Cdb-12a. Climax Molybdenum Co., Greenwich, Connecticut, 1969. 44 S. P. Berg and S. Izawa, Biochim. Biophys. Acta 460, 206 (1977). 45 S. Izawa and S. P. Berg, Biochem. Biophys. Res. Commun. 72, 1512 (1976). 46 L. Rosa and D. O. Hall, Biochirn. Biophys. Acta 449, 23 (1976). 4r G. Ben-Hayyim and J. Neumann, Eur. J. Biochem. 72, 57 (1977).

[39]

CHLOROPLAST ELECTRON TRANSPORT

425

strate the phosphorylation associated with DCMU-insensitive silicomolybdate reduction. Reaction mixture (2 ml): 0.1 M sucrose, 40 mM H E P E S - N a O H buffer, zl 2 mM MgC12, 5% (v/v) glycerol, 5 mM Na2Hn2PO4, 0.75 mM ADP, and tobacco chloroplasts containing 50 txg chlorophyll/ml. The reaction is initiated by simultaneously adding 10 tzl of silicomolybdic acid solution (50 mg/ml in dimethyl sulfoxide-water, 1 : 1, v/v) and turning on the light. The reaction is measured as 02 evolution. Barr e t a l . 38 have assayed silicomolybdate reduction following the absorbance increase at 750 nm ("molybdate blue'" formation). Donors of Electrons to Photosystem I (Regions 4-6) Electron donors which support DCMU-insensitive, PS-I-mediated reactions are covered extensively in the previous review. 1 These PS-I electron donors can now be subdivided into at least three groups based on the sensitivity of the reaction (electron transport or phosphorylation) to KCN or other plastocyanin inhibitors and to the plastoquinone antagonist DBMIB. 1. Reactions insensitive to KCN: the probable site of electron donation at P700 (region 4 of Fig. l). 2. Reactions sensitive to KCN but insensitive to DBMIB: the probable site of electron donation in the cytochrome flplastocyanin region (region 5). 3. Reactions sensitive to DBMIB: the probable site of electron donation in the plastoquinone region (region 6). Reaction systems for PS-I-mediated noncyclic electron transport require three basic components besides the electron donor to be tested: (a) a PS-II blocking agent, (b) an electron reservoir for the donor, and (c) a low-potential electron acceptor. DCMU (1-5 I~M) is usually employed as the PS-II blocking agent, although any one of well-established PS-II inhibitors 4"47a may be used. As for the electron reservoir, ascorbate (Dor L-; 0,5-2 mM) is practically the only choice. Ascorbate is satisfactory both in terms of its high enough reducing potential (E0' -- +0.06 V) to keep most of PS-I electron donors nearly completely in their reduced forms (which prevents or minimizes electron cycling) and in terms of its poor ability to serve itself as a direct electron donor to the transport chain. Because of its susceptibility to superoxide oxidation, ascorbate does pose a problem when it is used with autoxidizable electron acceptors (e.g., viologens and anthraquinones) to assay electron flow as 02 uptake. 47, S. Izawa and N. E. Good, Vol. 23, p. 335.

426

METHODOLOGY

[3 9]

This problem can be circumvented by adding excess superoxide dismutase to the reaction mixture (see discussion of PS-II electron donors) or by using NADP/ferredoxin as the electron acceptor couple. However, the NADP/ferredoxin couple tends to permit a cycling of electrons around PS-I when artificial electron donors are present. Since this hidden cyclic electron flow is usually coupled to phosphorylation, one must exert caution in interpretating phosphorylation data from donor reactions involving NADP/ferredoxin. Donors of Electrons to P700 (Region 4) Of known electron donors to PS-I, the reduced forms of PMS and of DCIP (DCIPH2) seem to have the easiest access to P700. This is suggested by (a) the relative insensitivity of PMS-mediated cyclic phosphorylation and DCIPH2-supported electron transport to KCN 1~ and (b) electron paramagnetic resonance (EPR) experiments which showed a rapid dark reduction of photooxidized P700 by reduced PMS and DCIPH2 in KCNblocked chloroplasts. 48 However, PMS-mediated cyclic photophosphorylation is largely sensitive to KCN when the PMS concentration is low (<50 /.~M). Furthermore, although DCIPH2-supported PS-I noncyclic electron flow is only partially (50%) inhibited, concurrent phosphorylation is completely blocked by KCN. 15 These results suggest that the primary site of electron donation by reduced PMS and DCIPH2 is not P700 but is probably in the cytochrome flplastocyanin region (see next section). Presumably, however, given at very high concentrations any PS-I electron donor will gain sufficient access to P700 to support measurable electron flow, as indicated by the fact that even ferrocyanide does so when given at 0.2 M. 49 The inaccessibility of P700 to low concentrations of ferricyanide has been noted by Kok. 5° Clearly, however, the accessibility of P700 to exogenous redox agents is a function of the membrane integrity. 5~ Methods. A typical reaction mixture designed for assay of KCNresistant DCIPH2 oxidation by PS-I (02 uptake) is 0.1 M sucrose, 40 mM HEPES-NaOH buffer (pH 7.5), 0.2 mM DCIP, 1 mM ascorbate, 2/~M DCMU, 0.1 m M methyl viologen, and KCN-treated chloroplasts 15"26 equivalent to 50 ~g chlorophyll/ml.

4s S. Izawa, R. Kraayenhof, E. K. Ruuge, and D. DeVault, Biochim. Biophys. Acta 314, 328 (1973). 49 D. Rosen, R. Barr, and F. L. Crane, Biochim. Biophys, Acta 408, 35 (1975). so B. Kok, Biochirn. Biophys. Acta 48, 527 (1961). 5~ j. M. Gould and S. Izawa, Biochim. Biophys. Acta 314, 211 (1973).

[39]

CHLOROPLASTeLECTRONT~NSPOaT

427

Donors of Electrons to Cytochrome f/Plastocyanin Region (Region 5) The best known of PS-I electron donors and mediators of PS-I-dependent cyclic photophosphorylation all belong to this category: DAD (E0' = +0.22 V), TMPD (E0' = +0.22 V), DCIPH2 (E0' = +0.22 V), PMS (E0' = +0.08 V), and pyocyanine (E0' = -0.04 V). Of these, PMS and pyocyanine are only useful as mediators of cyclic photophosphorylation. Reactions supported by these substances are all insensitive to DBMIB but are largely or almost completely inhibited by KCN, indicating a site (or sites) of electron donation in the cytochrome flplastocyanin region. There is spectroscopic evidence which suggests that the main site of electron donation by DCIPH2 may be cytochrome f or some component before the cytochrome. 52,53 Besides DAD and TMPD, various other C- or N-substituted phenylenediamines, ~4 3,3'-diaminobenzidine (a histochemical reductant), 5s and indamines (4,4'-diaminodiphenylamines) ~6 have been shown to be useful as electron donors of this category. Among these amines, completely N-substituted compounds such as TMPD, Nphenyl-N,N',N'-trimethyl-p-phenylenediamine, and pentamethylindamine, are unique in two ways: (a) PS-I noncyclic electron transport supported by them is not coupled to phosphorylation, and (b) their oxidation, which is a monovalent oxidation to relatively stable free radicals, does not release protons. This structure-function relation has been inHsC ~ - - ~

ca3

H3C ~

.CH~

HsC ~

CHa

HaC ~

CH s

-b

(TMPD)

e °

(Oxidized TMPD or "Wurster s blue")

terpreted as indicating the involvement of a chemiosmotic energy coupling mechanism in PS-I-mediated photophosphorylation. The highly hydrophilic sulfonated PMS, sulfonated pyocyanine, 57 and sulfonated DCIP(H2) 2a have been shown to be ineffective as mediators of PS-I5z S. Izawa, in "Comparative Biochemistry and Biophysics of Photosynthesis" (K. Shibata, A. Takamiya, A. T. Jagendoff, and R. C. Fuller, eds.), p. 140. Univ. Park Press, State College, Pennsylvania, 1968. ~a A. W. D. Larkum and W. D. Bonner, Biochim. Biophys. Acta 267, 149 (1973). ~4 G. Hauska, W. Oettmeier, S. Reimer, and A. Trebst, Z. Naturforsch., Teil C 30, 37 (1975). ~5 j. Goffer and J. Neumann, FEBS Lett. 36, 62 (1973). z8 W. Oettmeier, S. Reimer, ~nd A. Trebst, Plant Sci. Lett. 2, 267 (1974). ~7 G. Hauska, FEBS Lett. 28, 217 (1972).

428

METHODOLOGY

[3 9]

supported reactions. This suggest that the component or components of the electron transport chain which accept electrons from PS-I donors are located near or on the inner surface of the lipid membrane, z3"57 M e t h o d s . An example of reaction mixtures for measurement (as 02 consumption) of very fast rates of electron transfer from DAD to methyl viologen and associated phosphorylation is given in the legend for Fig. 3. The reaction mixture contained excess superoxide dismutase to suppress superoxide oxidation of ascorbate (see discussion of PS-II electron donors). Donors of Electrons to Plastoquinone (Region 6) Tetramethyl-p-hydroquinone (durohydroquinone; E0' = +0.06 V) has recently been shown to donate electrons preferentially to the plastoquinone region of the electron transport chain in the presence of a PS-II inhibitor, DCMU. Although this reductant is susceptible to air oxidation, below pH 8 the oxidation rate is sufficiently slow to allow measurement of electron flow as 02 uptake using methyl viologen as the electron acceptor. 58"58aNo secondary donor (electron reservoir) is needed in short term measurements (3-4 rain). In fact, no reducing agent presumably exists which may be useful as an electron reservoir for this system. The durohydroquinone-supported electron flow is quite fast and well coupled (P/e2 0.6). It is highly sensitive to DBMIB, indicating that electron donation takes place at or very close to the plastoquinone pool. Unfortunately durohydroquinone is sensitive to the superoxide radical and this necessitates the use of exogenous superoxide dismutase when the electron flow rate is to be determined critically5s (see also section below). Other electron donors of this category include reduced forms of: 9,10phenanthrenequinone, 2-hydroxy-l,4-naphthoquinone, 2-methyl-l,4naphthoquinone (menadione), 1,4-naphthoquinone and, interestingly, ferredoxin. 5sb To date, however, the ability of these latter substances to donate electrons to plastoquinone has only been shown in terms of DBMIB-sensitive, anaerobic cyclic photophosphorylation. M e t h o d s . Durohydroquinone solution can be prepared by adding approximately 2 mg of NaBH4 to 1 ml of ice-chilled alcoholic solution of duroquinone (20 mM) and subsequently acidifying the solution with 10 /zl of 5 N HCI. 58 The rate-saturating concentration of durohydroquinone is about 1 mM, but in routine work 0.5 mM will suffice. A typical reaction mixture for phosphorylation experiments with durohydroquinone: 0.1 M 58S. Izawaand R. L. Pan, Biochem. Biophys. Res. Commun. 83, 1171 (1978). 5aaC. C. White, R. K. Chain and R. Malkin,Biochim. Biophys. Acta 502, 127 (1978). 5shG. Hauska, S. Reimer, and A. Trebst, Biochim. Biophys. Acta 357, 1 (1974).

[39]

CHLOROPLAST ELECTRON TRANSPORT m

4000

429

4000,

*

A

3 '~)r +gram.

i .gz o

+Pi

3000

'x: fi_

3000

t....

-Pi O

2000

2000

o

E

C:r m

tO00

0

I000

ATP y,,.

~

~

°

~ DAD(raM)

/f- 7 ATP / 9 / ~ ° - ° / /°

°--

6

pH

FIG. 3. Effects of DAD concentration and of pH on the rate of electron transport and phosphorylation in the ascorbate/DAD ~ PS-I ~ methyl viologen/O2 reaction. In the experiment of (A) the reaction mixture contained 0.1 M sucrose, 2 mM MgC12, 50 mM Tricine-NaOH buffer (pH 8.1), 0.75 mM ADP, 5 mM Na2Ha2PO4(Pl), 2.5 txM DCMU, 2.5 mM D-ascorbate, 0.1 mM methyl viologen, indicated concentrations of DAD, chloroplasts equivalent to 5 txg chlorophyll/ml and bovine eruthrocyte superoxide dismutase at 0.4 mR/ ml (approximately 1200 units/ml). When added, gramicidin (gram) was 4 ~g/ml. In the pH experiments of (B) the buffers used were the same as in Fig. 2B except that TAPS buffer was replaced by tricine buffer. In these experiments, rates of electron transport were calculated from 02 uptake rates based on the relation 02 = 2e-, which is valid in experiments such as this with excess superoxide dismutase. Although not shown in these figures, the 02 consumption rates were approximately 50% higher when the dismutase was omitted from the reaction mixture (see section for PS-II electron donors). The intensity of actinic light, 500 kergs sec -~ cm-2 (>600 nm). [Adapted from J. M. Gould, Biochim. Biophys. Acta 387, 135 (1975).] s u c r o s e , 40 m M t r i c i n e - N a O H b u f f e r ( p H 7.8), 3 m M MgClz, 5 m M NaH3zPO4, 0.8 m M A D P , 2 /~M D C M U , 0.1 m M m e t h y l v i o l o g e n , 0.5 m M d u r o h y d r o q u i n o n e a n d c h l o r o p l a s t s c o n t a i n i n g 12 /zg c h l o r o p h y l l .

D o n o r s o f E l e c t r o n s to P h o t o s y s t e m I I ( R e g i o n 7) D u r i n g the last s e v e r a l y e a r s a n u m b e r of n e w r e d u c t a n t s h a v e b e e n a d d e d to the list o f P S - I I e l e c t r o n d o n o r s g i v e n in the p r e v i o u s r e v i e w . 1

430

METHODOLOGY

[39]

Some of these new donors are now in frequent use for investigations of energy coupling at PS-II. Another recent development is the recognition of the problems of superoxide radical which are associated with the use of 02 as the terminal electron acceptor in the presence of certain reducing agents. This latter topic is discussed below because it is an important one, bearing upon the quantitation of donor-supported electron flow observed as 02 consumption. It should be noted that the use of ambient 02 as the terminal electron acceptor is a very convenient and the most widely applicable way of measuring both PS-I-mediated and PS-II-mediated electron-donor reactions.

Superoxide and the Stoichiometry of Electron Flow Assayed as 02 Uptake It is well known that addition of ascorbate greatly stimulates the 02 uptake by the Mehler reaction, i.e., the Hill reaction supported by an autoxidizable electron acceptor such as viologen and anthraquinone sulfonate. Since the enhanced 02 uptake is sensitive to PS-II inhibitors and is accompanied by an oxidation of ascorbate, the phenomenon has been interpreted as indicating that ascorbate contributes additional electrons to PS-II or replaces water as the electron donor. 1 However, it has been shown recently that the ascorbate-enhanced part of 02 uptake can be abolished by the addition of superoxide dismutase? 9-nl This demonstrates that ascorbate photooxidation by normal, water-oxidizing chloroplasts has nothing to do with electron donation to PS-II. It is a pure chemical oxidation by the superoxide radical O2-, the product of monovalent reduction of 02 by the photoreduced methyl viologen or anthraquinone. 02 uptake in the Mehler reaction is stimulated by ascorbate because O2-, which normally dismutates to H202 and 02, is mostly reduced to H202 by the ascorbate. Mn 2÷ also induces a similar "false" PS-II donor reaction. 5~ Some reductants, such as hydrazine, sulfite, dopamine, 62-64 are subject to chain oxidation when exposed to O2-, the result of which is a vast amplification of 02 uptake. Superoxide inevitably poses a problem when one is to deal with a donor-supported Mehler reaction, since it usually requires ascorbate (as an electron reservoir) which is one of the most O2--sensitive reductant. n9 B. L. Epel and J. Neumann, Biochim. Biophys. Acta 325, 520 (1973). 60 j. F. Allen and D. O. Hall, Biochem. Biophys. Res. Commun. 52, 856 (1973). 61 D. R. Ort and S. Izawa, Plant Physiol. 53, 370 0974). 62 K. E. Mantai and G. Hind, Plant Physiol. 48, 5 (1971). 63 K. Asada and K. Kiso, Eur. J. Biochem. 33, 253 (1973). 84 E. F. Elstner and A. Heupel, Z. Naturforsch., Teil C 29, 559 0974).

[39]

CHLOROPLASTELECTRONTRANSPORT

431

Some donors (e.g., catechol) are also 02- sensitive. In many cases the problem is a relatively simple one, ascorbate just scavenging all 02- and thereby doubling the rate of 02 uptake. However, as noted above, further amplification of 02 uptake can readily occur depending on the nature and the purity of the direct donor used. Thus, the only reliable solution to the problem is to add large amounts of superoxide dismutase to the reaction mixture to abolish whatever superoxide reactions that may be going on besides dismutation. It is also only under these conditions that the long-known and long-misused equation does apply: 2e-

= 02

that is, one molecules of 02 taken up for a pair of electrons transported through the photosynthetic chain (see formulae in footnote65). Inhibition o f Water Oxidation as Prerequisite to PS-H Donor Reactions

As has been noted by many workers, PS-II does not usually oxidize exogenous reductants unless forced to do so by inhibition of water oxidation. What appeared to be cases of normal chloroplasts preferentially oxidizing artificial reductants through PS-II (with O2 as acceptor) have turned out to be nonbiological superoxide oxidation (see section above). Selective blocking of water oxidation can be achieved in a variety of ways, 1,3 but the most often used are Tris treatment and hydroxylamine treatment. These two are also the only methods that have been shown to

65 The reaction sequence is as follows (symbols: A H - , ascorbate; A, dehydroascorbate; MV, methyl viologen): 2e - t r a n s p o r t

AH-

chloroplasts ) A + 2 e -

H + + (l)

MV

2 e - + 2 02 2 03- + A H - + 3 H +

..... ~ . 8

2e-

, 2 02-

(2)

, 2 I"I~) 2 + A

(3)

transport

2 A H - + 2 02 + 2 H +

~ 2 A + 2 H202

(02 = e - without excess dismutase)

chloroplasts

When excess superoxide is added to the reaction mixture, Eq. (3) will be replaced by dlsmutase

2 02- + 2 H +

(spontaneous)

~ H202 + 02

The overall reaction would then become 2e-

A H - + O2 + H +

transoort

chloroplasts

~ A + H202

(02 = 2 e - with excess dismutase)

432

METI'IODOLOGY

[39]

c~ b

0

6 ~.~

•.: -~

Z c5 Zc~

X .N

©

Z

~

=o,~

Z

Z

7o

~

~'.=

g~,z ga:, -," •~ ' ~

A

+

z

~",I c q

+

+

,~- ¢-1

I¢~ +

+

i

, ~ "~ ~ • ~

.,,a . " ~ ..

i

~

,.. c a .

,a

• R ~

M

z

~1

~

I~ I~

~

0

0

~

~=~'~.~

% •~

.~

~ =~ .~

"b

~ ' ~:B

e~

•

I

.

~o ~

[39]

CHLOROPLAST ELECTRON TRANSPORT

433

abolish the water oxidation reaction without appreciably impairing the energy-coupling efficiency of the membrane. 66,nr

Donors of Electrons to PS-II Of the various PS-II electron donors listed in the previous review, 1 1,5-diphenylcarbazide (DPC) 68 now seems to have established itself as the standard PS-II donor. It is routinely used to test the photochemical activity of PS-II and to locate the site of action of PS-II inhibitors. The main reason for the popularity of this donor appears to be the applicability of DCIP as an electron acceptor, and the relatively fast rate of electron transport it supports. The behavior of various carbazides and azobenzenes as PS-II donors has been studied in some detail. The oxidation of hydrazobenzene by "PS-II particles" is reported to be rather insensitive to DCMU. n9 Unique among the newly introduced PS-II electron donors are ferrocyanide (E0' = +0.42V), iodide ion (E0' = +0.56 V) TM and N,N,N',N'-tetramethylbenzidine (E0' = +0.5 V). 7x Electron transport from these non-proton-producing donors to PS-I acceptors (methyl viologen, NADP) generates ATP with only half the efficiency of electron transport from proton-releasing donors, such as hydroquinone, p-aminophenol, benzidine. Like the case of TMPD for PS-I, the result strongly points to a chemiosmotic coupling mechanism associated with PS-II. 7°'71 Another notable new PS-II electron donor is hydrogen peroxide, TM the well known end product of electron transport reaction in which Oz is utilized as the terminal electron acceptor. Given at high concentrations (>5 mM), H202 is rapidly oxidized to Oz in the presence of standard electron acceptors, such as ferricyanide, DCIP, and quinones. Furthermore, dimethylquinone reduction supported by H202 is coupled to phosphorylation with a near-normal efficiency (P/ez = 0.3). TM Thus, there are intriguing similarities between H20 and H202 as electron donors to PSII. Methods. Some technical data pertaining to selected new PS-II electron donors are summarized in Table I.

66 T. Y a m a s h i t a and W, L. Butler, Plant Physiol. 44, 435 (1969); see also Ibid. 43, 1978 (1968). 6r D. R. Ort and S. Izawa, Plant Physiol. 52, 595 (1973); see also Ort and Izawa. 6~ 6s L. P. V e r n o n and E. R. Shaw, Plant Physiol. 44, 1645 (1969). 69 j. H a v e m a n and M. D o n z , Proc. Int, Congr. Photosynth. Res., 2nd, 1971 p. 81 (1972). 70 S. Izawa and D. R. Ort, Biochim. Biophys. Acta 357, 127 (1974). 7~ E. Harth, W. Oettmeier, and A. Trebst, FEBS Lett. 43, 231 (1974). Tz H. Inoue and M. N i s h i m u r a , Plant Cell Physiol. 12, 739 (1971). 73 R. L. Pan and S. Izawa, Biochim. Biophys. Acta. 547, 311 (1979)

434

METHODOLOGY

1"40]

Acknowledgments The author wishes to thank Patrick M. Kelley for reading through the manuscript and for his assistance in art work. This work was supported by a grant (PCM76-19887) from the National Science Foundation.

[40] E l e c t r o p h o r e t i c A n a l y s i s o f C h l o r o p l a s t P r o t e i n s

By NAM-HA1 CHUA Electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gels has become a standard technique for structural and biosynthetic studies of chloroplast proteins. A number of electrophoretic systems with different degrees of resolution have been employed in such investigations. 1-12 This article describes a method 13-15 which combines the alkaline SDSdiscontinuous buffer system of Neville le,lr and, in the resolving gel, a linear acrylamide concentration gradient.IS The discontinuous buffer system is capable of stacking SDS-protein complexes over a wide range of molecular weights, thus providing very sharp bands, while the linear pore gradient ls,la allows the separation and resolution of polypeptides with widely different molecular weights. The combination of these two meth-

1j. K. Hoober, J. Biol. Chem. 245, 4327 (1970). 2 R. P. Levine, W. G. Burton, and H. A. Durham, Nature (London) 237, 176 (1972). 3 S. M. Klein and L. P. Vernon, Photochem. Photobiol. 19, 43 (1974). a j. p. Thornber and H. R. Highkin, Eur. J. Biochem. 41,109 (1974). 5 A. R. J. Eaglesham and R. J. Ellis, Biochim. Biophys. Acta 335, 396 (1974). 6 W. Bottomley, D. S. Spencer, and P. R. Whitfeld, Arch. Biochem. Biophys. 164, 106 (1974). 7 0 . Machold, Biochim. Biophys. Acta 382, 494 (1975). s W. G. Nolan and R. B. Park, Biochim. Biophys. Acta 375, 406 (1975). a K. Apel, L. Bogorad, and C. L. F. Woodcock, Biochim. Biophys. Acta 387, 568 (1975). 10 j. j, Morgenthaler and L. Mendiola-Morgenthaler, Arch. Biochem. Biophys. 172, 51 (1976). H A. R. Cashmore, J. Biol. Chem. 251, 2848 (1976). lz S. Bar-Nun, R. Schantz, and I. Ohad, Biochim. Biophys. Acta 459, 451 (1977). 13 N.-H. Chua and P. Bennoun, Proc. Natl. Acad. Sci. U.S.A. 72, 2175 (1975). 14 N.-H. Chua, K. Matlin, and P. Bennoun, J. Cell Biol. 67, 361 (1975). 15 L. Y. W. Bourguignon and G. E. Palade, J. Cell Biol. 69, 327 (1976). t~ D. M. Neville, Jr., J. Biol. Chem. 246, 6328 (1971). z7 D. M. Neville, Jr. and H. Glossmann, Vol. 32 p. 92. is j. Margolis and K. G. Kendrick, Anal. Biochem. 25, 347 (1968). 19 D. Rodbard, G. Kapadia, and A. Chrambach, Anal. Biochem. 40, 135 (1971). Copyright© 1980by AcademicPress,Inc. METHODSIN ENZYMOLOGY;VOL,69

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