Harry Y. Tsujimoto

Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin.

Abstract Nostoc muscorum (Strain 7119) cells were disrupted and the accessory pigment phycocyanin was removed from membrane fragments by digitonin treatment. The phycocyanin-depleted membrane fragments retained both Photosystem I and Photosystem II activity, as evidenced by high rates of NADP+ photoreduction either by water or by reduced 2,6-dichlorophenolindophenol, indicating that phycocyanin is not an essential component for electron transport activity. No separation of the two photosystems was effected by the digitonin treatment. Even drastic digitonin treatments failed to diminish significantly the remarkably stable electron transport from water to NADP+. Action spectra and relative quantum efficiency measurements demonstrated the existence of both Photosystem I and Photosystem II in membrane fragments which contained chlorophyll a as the only significant light-absorbing pigment.

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):

Effects of magnesium and chloride ions on light-induced electron transport in membrane fragments from a blue-green alga☆

The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP+. Magnesium ions were required not only for light-induced electron transport from water to NADP+ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg2+ lost the capacity to photoreduce NADP+ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP+ by DCIPH2 was independent of Mg2+ (or Cl-) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentration of MgGl2 produced a strong inhibition of NADP+ photoreduction with DCIPH2 without significantly affecting the rate of NADP+ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP+ with different electron donors are discussed.

Differential inhibition by plastoquinone analogues of photoreduction of cytochrome b-559 in chloroplasts

The high‐potential form of cytochrome b‐559 (b‐559 HP) is closely linked to the oxygenic photosystem (photosystem II) but its relation to other redox components of the photosynthetic apparatus, including plastoquinone, is still obscure. We investigated the photoreduction of cytochrome b‐559 HP by isolated chloroplasts in the presence of 3 antagonists of plastoquinone, of which, DBMIB (dibromothymoquinone) and DNP‐INT (dinitrophenyl ether of iodonitrothymol) are known to inhibit the oxidation of the plastoquinone pool (PQ) by the FeS‐cytochrome ƒ/b 6 complex and one, UHDBT (5‐n‐undecyl‐6‐hydroxy‐4,7‐dioxobenzothiazole) is known to inhibit the reduction of PQ by QB.QB is a protein‐bound plastoquinone that serves as a two‐electron gate for the reduction of PQ. We found that DBMIB and DNP‐INT did not inhibit but low concentrations of UHDBT severely inhibited the photoreduction of cytochrome b‐559 HP. These results suggest that the electron donor for the reduction of cytochrome b‐559 HP was either QB or a portion of the PQ pool that was oxidized by a new pathway free of binding sites for DBMIB and DNP‐INT.

Photoreduction of ferredoxin by chloroplasts with or without an accompanying photoreduction of the bound iron—sulfur centers: Contrasting effects of electron donors to photosystems I and II

This communication reports new evidence consonant with a recent finding [l] of ferredoxin photoreduction by water when the photoreduction of the bound iron-sulfur centers associated with photosystem I of chloroplasts [2-41 was strongly inhibited. Chloroplasts photoreduce ferredoxin by electrons that originate from water, via photosystem II, or by electrons supplied directly to photosystem I by artificial donors that bypass photosystem II [S]. Contrary to expectations, only with a direct donor to photosystem I (ascorbate/DCIP) was the photoreduction of ferredoxin by chloroplasts [l], like the photoreduction of ferredoxin by cyanobacterial membrane fragments [6], unequivocally associated with the photoreduction of the bound iron-sulfur centers. With water as donor, ferredoxin was found in [ 1 ] to be in a predominantly reduced steady state, even when the photoreduction of the bound iron-sulfur centers was inhibited by DBMIB (2,5-dibromo-3methyl&isopropyl-l ,Cbenzoquinone). DBMIB is an antagonist of plastoquinone, the chloroplast component deemed essential for electron transfer from photosystem II to photosystem I [7,8]. It appeared, therefore, that, with water as the electron donor, the photoreduction of the bound iron-sulfur centers by photosystem I was an accompaniment but not a prerequisite for the photoreduction of ferredoxin. Normally, electrons originating

Different roles of plastoquinone in the photoreduction of ferredoxin and of membrane-bound iron-sulfur centers of chloroplasts

Photosynthetic electron transport in chloroplasts was inhibited by the plastoquinone antagonist, dibromothymoquinone (DBMIB) in two steps. Lower concentrations of DBMIB inhibited the photoreduction of the bound iron-sulfur centers of photosystem I without inhibiting the photoreduction of ferredoxin. Higher concentrations of DBMIB did inhibit the oxygenic photoreduction (i.e., by water) of ferredoxin and NADP+, but their photoreduction was restored, wholly or partly, by each of four chemically diverse uncouplers, similar only in facilitating proton movement across membranes. By contrast, none of the uncouplers alleviated the DBMIB inhibition of the photoreduction of the bound Fe-S centers. These divergent responses to uncouplers are incompatible with the Z scheme but are consistent with the new concept of oxygenic and anoxygenic photosystems in plant photosynthesis (Proc. Natl. Acad. Sci. USA 78, 2942–2946, 1981).

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga☆

The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.

Oxygenic photoreduction of ferredoxin independently of the membrane-bound iron-sulfur centers of photosystem I

Abstract An investigation of the photoreduction of soluble ferredoxin and membrane-bound Fe-S centers of chloroplasts yielded results that are incompatible with some basic postulates of the now prevalent concept of photosynthetic electron transport (the “Z scheme”). In the Z scheme, plastquinone serves as an essential link in a linear electron transport chain from water via photosystem II to photosystem I and thence to the bound Fe-S centers, soluble ferredoxin and NADP + . In this formulation the oxygenic photoreduction of ferredoxin and of the Fe-S centers should have the same sensitivity to the plastoquinone inhibitors, dibromothymoquinone (DBMIB) and dinitrophenol ether of iodonitrothymol (DNP-INT). We found that the photoreduction of ferredoxin and the Fe-S centers exhibited differential sensitivity to these inhibitors. Ferredoxin was fully photoreduced by water at inhibitor concentrations that abolished the photoreduction of the Fe-S centers. These findings suggest that the oxygenic photoreduction of ferredoxin does not involve the participation of the Fe-S centers or other components of photosystem I. Only when an artificial, direct donor to photosystem I is used is the reduction of ferredoxin invariably preceded by the reduction of the Fe-S centers.

Affinity of ferredoxin for electrons from water and the regulation of cyclic photophosphorylation

New evidence is presented how, in chloroplasts, an auxiliary electron flow from water, induced by photosystem II, maintains the redox balance (poising) needed for the functioning of ferredoxin-catalyzed cyclic photophosphorylation, a process driven by photosystem I. Optimal poising of cyclic photophosphorylation was attained when ferredoxin was kept in a predominantly reduced state by electrons from water but only when the electron pressure from water was greatly attenuated to prevent overreduction. An unexpected finding was the unusually high affinity of ferredoxin for electrons from water. Ferredoxin was kept in a predominantly reduced state by the severely restricted electron flow from water that was generated by 715nm illumination, or under 554nm illumination in the presence of diuron.

Photoreduction of membrane-bound paramagnetic component X by water as electron donor

The use of electron paramagnetic resonance (EPR) spectrosopy for the study of photosynthetic electron transport has led in recent years to the identification in photosynthetic membranes of three bound paramagnetic components closely linked to the primary electron-transfer reactions of photosystem I. Two of the membrane-bound paramagnetic components, initially described as belonging to bound ferredoxins, are now generally known as iron-sulfur centers A and B [l-4]. The third, whose chemical identity is less certain, is known as component X [5-lo] and is thought to be identical with component AZ that was detected spectrophotometrically [ 11 ,121. Although the midpoint redox potential of component X has not been determined directly, the potential is believed to be lower than -700 mV [13,10]; the potential of center B is about -580 mV, and that of center A is about -530 mV [4,14]. In the reduced state, center A is characterized by resonances at g = 1.86,1.94 and 2.05 and center B, by resonances at g = 1.89,1.92 and 2.05 [l-4]; component X has a distinctive signal at g = 1.75-I .78 and less readily distinguishable signals atg= 1.88 and2.08 [5-lo]. Component X has been assigned the role of primary electron acceptor for electrons released by the photooxidation of P-700, the reaction-center chlorophyll of photosystem I [5-lo]. Component X has hitherto been detected only under extremely reducing conditions. Photoreduction was either reversible or irreversible at cryogenic temperatures, depending on the pretreatment of the sample prior