Richard K. Chain

On the interaction of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with bound electron carriers in spinach chloroplasts

Abstract 2,5-Dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), when added to chloroplasts as the sole electron donor, is an effective reducing agent. Low concentrations of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone reduce cytochrome f , plastocyanin, and P700 in the dark but do not reduce the high-potential form of cytochrome b 559 . 2,5-Dibromo-3-methyl-6-isopropylbenzoquinone appears to interact at or near the site of function of the “Rieske” iron-sulfur center, as evidenced by a shift in the g value of the electron paramagnetic resonance signal of the reduced center.

Involvement of plastoquinone and lipids in electron transport reactions mediated by the cytochrome b6-f complex isolated from spinach

The isolation of a cytochrome b 6‐f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol‐plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol‐plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.

Regulatory electron transport pathways in cyclic photophosphorylation: Reduction of C-550 and cytochrome b6 by ferredoxin in the dark

Recent work [l-4] has strengthened and extended in several important respects the concept that ferredoxin is the physiological catalyst of cyclic photophosphorylation in chloroplasts and that this type of phosphorylation is a source of ATP needed for COZ assimilation, protein synthesis and other energydependent reactions [S-IO]. As reviewed in [2,1 A 1, noncyclic photophosphorylation alone cannot fully meet the ATP requirements of CO, assimilation because of the marked excess of ATP over NADPH that is needed: Cq plants require 5 ATPand Cs plants require 3 ATP per 2 NADPH used in the assimilation of 1 COZ to the level of sugar phosphate (cf. [ 121). The recent findings pertaining to cyclic photophosphorylation in chloroplasts may be summarized as follows: (i) Cyclic photophosphorylation by chloroplasts proceeds optimally in the presence of air and is catalyzed by low concentrations of ferredoxin (10 PM), the same as those required for NADP’ reduction [ 1,2]; (ii) Pseudocyclic photophosphorylation, which depends on electron transport from photoreduced ferredoxin to oxygen [7], is of minor importance as a source of ATP in chloroplasts [ 1,4]; (iii) In the presence of NADP+, the ferredoxin-depen-

Role of oxygen in ferredoxin‐catalyzed cyclic photophosphorylations

The enzymatic reactions responsible for the photosynthetic conversion of COZ to glucose or its equivalent require an excess of ATP over NADPH. Specifically, for one mole of CO* the required molar ratio of ATP to NADPH is 1.5 for C3 plants [l] and 2.5 for C4 plants [2,3] . From early studies of the light reactions of chloroplasts it appeared that these two components of assimilatory power might be generated by two distincts and competitive reactions: ATP by cyclic photophosphorylation [4,5] and NADPH by photolysis of water [6] . This concept [7] was abandoned with the finding of noncyclic photophosphorylation, in which ATP and NADPH are formed in an equimolar ratio (P/e* l), not competitively but concomitantly and in a mutually enhancing manner [8] . Cyclic photophosphorylation now came to be regarded as a second source of ATP in chloroplasts, one that supplements the insufficient ATP generated for CO2 assimilation by noncyclic photophosphorylation and also serves as a source of ATP for processes other than COZ assimilation, for example, protein synthesis [8,9]. The assignment of this physiological role to cyclic photophosphorylation was strengthened by evidence that this process was catalyzed by ferredoxin, a native protein component of chloroplasts [lo131 . Some investigators sought to account for the extra ATP needed for CO2 assimilation by contending that the P/e, ratio of noncyclic photophosphorylation may equal 2 and that, therefore, no additional source of

THREE LIGHT REACTIONS AND THE TWO PHOTOSYSTEMS OF PLANT PHOTOSYNTHESIS

Abstract— Recent work in our laboratory yielded new evidence that noncyclic electron transport in chloroplasts from water to ferredoxin (Fd) and N ADP is carried out solely by System II which, unexpectedly, was found to include not one but two photoreactions (IIa and IIb). The evidence suggests that these operate in series, being joined together by a ‘dark’ chain of electron carriers that includes (but is not limited to) cytochrome b559 and plastocyanin (PC):

The relationship of the cyclic and non-cyclic electron transport pathways in chloroplasts

Light-induced redox changes of plastocyanin, the Rieske iron-sulfur center, and P-700 have been studied in situ in spinach chloroplasts. Plastocyanin and the Rieske center behaved in an analogous manner in that their steady states were fully oxidized in the light in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when an electron acceptor is present. After illumination under conditions of non-cyclic electron transfer from water to an electron acceptor, followed by a short-dark period, the steady state of both shifted to a more reduced level. A 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photo-reduction of the Rieske center was observed in ferricyanide-washed chloroplast fragments. With reduced ferredoxin as electron donor, it was possible to demonstrate a reduction in the dark of these electron carriers and of P-700; this reduction was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea but was inhibited by antimycin A. These findings are discussed in relation to a function for these electron carriers in the cyclic electron transport pathway in chloroplasts and to their function in the non-cyclic electron transport pathway.

Evidence for a reluctant-dependent oxidation of chloroplast cytochrome b-563

An electron carrier thought to be uniquely associated with the cyclic electron-transport pathway is cytochrome b-563 (bh) (reviewed in [ 11). This conclusion was based on the early observation that cytochrome b-563 underwent both a photosystem I-mediated oxidation and reduction. The function of cytochrome b-563 has been explained in terms of a mechanism in which ferredoxin, reduced by photo070system I, served as the electron donor for cytochrome b-563 and that the cytochrome then donated electrons into the plastoquinone pool [l-4]. However, recent findings suggest a reduction which appears to be dependent on the redox state of plastoquinone [3,5]. Such results, as well as those obtained in studies of flash-induced redox changes of the cytochrome [5-81 have led to a general model in which cytochrome b-563 functions in a ‘Q-cycle’ [5,8] similar to that proposed by Mitchell for the cytochrome b-cl region of the mitochondrial electron transport chain 191. 2.1. Chloroplast preparation Spinach chloroplast membranes were prepared from freshly harvested greenhouse-grown spinach [lo]. These chloroplast membrane fragments were substantially depleted of ferredoxin (some residual NADP+ photoreduction activity remained in the absence of added ferredoxin) and were capable of both cyclic and non-cyclic photophosphorylation with low quantum requirements [ 1 l] when used within 30-60 min of preparation and when supplemented with appropriate cofactors.

Oxidation-reduction reactions of cytochrome b6 in a liposome-incorporated cytochrome b6-f complex

The chloroplast cytochrome b6-f complex, incorporated into phospholipid vesicles, shows proton translocation with an observed H+/e- ratio of approximately 2. The oxidation-reduction behavior of cytochrome b6 during electron transport from duroquinol to plastocyanin is affected by incorporation. The most obvious effect of incorporation is an increase in the duration of a steady-state level of cytochrome b6 that persists during electron transport. Reagents that decrease activity increase the duration of the steady state while reagents that stimulate activity decrease this time. Uncoupling conditions yield cytochrome kinetics similar to those in the unincorporated complex. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone and 5-n-undecyl-4,7-dioxobenzothiazole inhibited reduction of cytochrome b6 in the incorporated complex, but this apparent inhibition was due to a rapid oxidation of the cytochrome by these compounds.

Circular dichroism studies of ferredoxin:Protein complexes

Abstract Complex formation between ferredoxin and ferredoxin:nitrite oxidoreductase (EC 1.7.7.1) or between ferredoxin and ferredoxin:NADP+ oxidoreductase (EC 1.6.7.1) is accompanied by alterations in the circular dichroism spectra of the proteins. These changes in CD spectra indicate both changes in the environment of the prosthetic groups of the proteins and increases in protein secondary structure content. Addition of substrate (NO2−) to nitrite reductase results in changes in the enzyme CD spectrum in the visible region but apparently did not affect the secondary structure, as indicated by the lack of changes in the CD spectrum between 200 and 250 nm.

The role of cytochrome f in ferredoxin-dependent electron-transport reactions in spinach chloroplasts

Wide acceptance of the hypothesis of two light reactions in photosynthesis [ 1,2] has led to attempts to determine what electron carriers are located between the two photosystems (photosystem II and photosystem I). Based on the antagonistic effect of red versus far-red illumination and on the effect of 3-(3’,4’-dichlorophenol)-1 ,I -dimethylurea (DCMU) [2], cytochromefhas been proposed as one such carrier. In experiments with spinach chloroplasts, the cytochrome was demonstrated to function between water and the physiological electron acceptor ferredoxin-NADP [3]. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) [4,5] was found to function in a manner similar to DCMU in that both inhibitors prevented the reduction of the cytochrome by water but not its oxidation by photosystem I. Although, based on data from kinetic experiments, others have suggested that cytochromefmay not be involved in the main noncyclic electrontransport chain [6], the data presented below will be interpreted in terms of cytochrome fbeing an obligate electron carrier between the two photosystems. As indicated above, the source of electrons for the reduction of cytochrome f under conditions of noncyclic electron transport can be either water (in the light) or (in the dark) more electronegative electron carriers, in the reduced form, between the primary electron acceptor for photosystem II and the cytochrome. In addition to this ferredoxin-dependent noncyclic electron-transport system, reduced ferredoxin functions as an electron carrier in a cyclic electrontransport reaction [7] that was thought to involve cytochrome f [8-l 11. Cyclic, in contrast to noncyclic electron transport, could be driven solely by photosystem I, and the associated ATP formation was inhibited (at low concentrations) by antimycin A [7] but not directly by DCMU. Cyclic photophosphorylation was also inhibited by DBMIB [7]. If cytochrome f is an electron carrier in both types of electron transport and inhibitors are used that are specific for the respective pathways (e.g., DCMU and antimycin A), it should be possible to demonstrate the reduction of the cytochrome from either water (as in noncyclic) or reduced ferredoxin (as in cyclic). (Cytochromes reviewed in [12,13].) This communication reports the ferredoxindependent reduction of cytochrome f and the inhibition of that reduction by antimycin A. The results presented are consistent with the hypothesis that cytochrome f functions as an electron carrier common to both cyclic and noncyclic electrontransport reactions.