Photoreduction and Photophosphorylation with Tris-Washed Chloroplastst

The artificial electron donor compounds p-phenylenediamine (PD). N, N, N', N'tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 M tris (hydroxymethyl)aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 ,umi PD plus 330 rLNi ascorbate was largely inhibited by 1 aum 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) while that supported by 33 um TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 gm donor plus 330 5Lm ascorbate) photophosphorylation, which occurred with P/e. ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited. The demonstration that artificial electron donor systems such as DCPTP2 pllus ascorbate (1) or PMS plus ascorbate (2) couild restore the photoreducing power of chloroplasts which had lost their capacity to evolve oxygein opened utp a new method of exploring the photosynthetic electron transport system. A niumllber of artificial electron donor compounds have been studied, often with the goal of determining the site of electroni donationi relative to the site of coupling of phosphorylation ( 3, 4. 5,6). Any study of artificial electron donor systems for chloroplasts is firml/y bound to the metho(d of inhlihbiting nornmal electron trcansport from water. Mlost p)revious studies have used DCMU or slllmilar acting inhibitors which block electron transport just after PS2 and thus preclude the study of electron donors acting between water and PS2. Yamashita and Horio (7) inhibited the Hill reaction by washing chloroplasts with 0.8 M tris-HCl (pH 8.0). They found that photophosplhorylation and NADP photoreduction could be restored by adding DCPIP or TMPD with ascorbate. Cyclic photophosp)horylation (no requireThis work was supported by USPHS, National Institutes of Health Grant GM-15048 and a Charles F. Kettering Research Award. 2 Abbreviations: PD, p-phenylenediamine; TMIPD. N,N,N',N'-tetramethyl-p-phenylenediamine; DCPIP, 2,6dichlorophenol-indophenol; DCMU , 3(3,4-dichlorophenyl)-l,1-dimethylurea; PMS; phenazine methosulfate: PSI and PS2-photosystem I and photosystem II. ment for NADP) was obtained when the concenitrations of ascorbate and DCP'IP wNere about eqtlal but non-cyclic photophosphorvlation (strict requirement for NADP) predominated when an excess of ascorbate was present. The non-cyclic photophosphorvlation with DCPIP was inhibited 50 % by DCMUI or o-phenanthroline but that with TMPD was inhibited 100 %. 'The DCMTU inhibition of the TMPD photoreaction suggested that the tris-wash treatment inhibited before the DCMIU block and that TMPD could donate electrons prior to the DCMU block. Electron donation to the electroni transport systeml prior to the DCMIJ block has been suggested previously by several laboratories ('8, 9, 10) on the basis of experiments showing that the photooxi(lation of ascorbate by chloroplasts in the presence of auitooxidizable quinones was inhibited by DCMIU. More direct measurements of electron donation prior to the DCMIU block have been made recently. Katoh and San Pietro (11, 12) used ascorbate as anl electron donor for the photoreduction of NADP witl Euglena chloroplasts that had been inlhibited by a heat treatment. The ascorbate-supported photoreduction of N\ADP was inhibited by DCMUI. Yamashita and Bultler (13) showed that NADP photoreduction in spinlach chloroplasts inhibited with a tris-waslh treatment could be restored with PD plus ascorbate as an electron donor system and that the electron transport from PD was inhibited by DCMU. The present paper is an extension of the previous one with measuirements of both photoreduiction and