Photoreduction of the intermediary electron acceptor, pheophytin (Pheo), in photosystem II reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and redox potential Eh = -450 mV produces an EPR doublet centered at g = 2.00 with a splitting of 52 G at 7 K in addition to a narrow signal attributed to Pheo (g = 2.0033, AH -13 G). The doublet is eliminated after extraction of lyophilized TSF-II with hexane containing 0. Phototrapping of Pheo-in PS II at 295 K is accompanied by the appearance of an EPR signal with g k 2.0035 and AH 13 G (1 G = 10-4 tesla) (6, 7), similar to that of the monomeric The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §17734 solely to indicate this fact. 7227anion radical of Pheo in vitro (18). After phototrapping of Pheo at 220 K, however, an EPR "doublet" centered at g = 2.00 with a splitting of m52 G is also observed (7). In analogy with a similar observation in bacterial reaction centers (19)(20)(21)(22), it was suggested that the doublet is probably the result of interaction of Pheo' with Q, which includes Fe or some other transition metal (7). Here we report data supporting the idea that a PQ-Fe complex acts as the stable "primary" electron acceptor in PS II reaction centers and that an exchange interaction of its singly reduced form with Pheo-accounts for the split EPR signal. MATERIALS AND METHODSSpinach chloroplasts and subchloroplast particles (Tritonfractionated subchloroplast fragments, TSF-II and TSF-IIa), highly enriched in PS II reaction center components and free of P700, were isolated as described (23- Iron was extracted by incubating PS II preparations (Chl at 50 mg/ml) in 10 mM Tris-HCI (pH 8.0) containing 0.3-1 M LiCl04 with or without 2.5 mM o-phenanthroline, for 2 hr at 20C, followed by centrifugation (200,000 X g for 40 min) and dialysis (12 hr at 2°C) of the pellet against 10 mM Tris-HCl (pH 8.0)/50 mM NaCI/10 AM EDTA (David Knaff, personal communication; also ref. 27). In the reconstitution experiments the extracted material was additionally dialyzed for 12 hr at 20C under anaerobic conditions against 10 mM acetate buffer (pH 5.5) containing 0.2 M LiCl04 and 1 MM EDTA, with or without 0.2 mM Fe(NH4)2(SO4)2, MnSO4, or MgSO4.Nonheme iron content was determined by the bathophenanthroline method (28). Changes in absorbance and fluorescence yield, induced by actinic light (intensity t0.05 Jcm2-s-l) from a 1000-W incandescent lamp filtered by 5 cm of CuSO4 solution, were measured in a phosphoroscopic pho-

The use of diphenylcarbazide as an electron donor coupled to the photoreduction of 2,6-dichlorophenolindophenol by tris-washed chloroplasts or subchloroplast fragments provides a simple and sensitive assay for photosystem 2 of chloroplasts. By varying the concentration of tris buffer at pH 8.0 during an incubation period it is shown that the destruction of oxygen evolution activity is accompanied by a corresponding emergence of an ability to photooxidize diphenylcarbazide, as evidenced by absorbance changes due to diphenylcarbazide at 300 nm. The temperature-sensitive oxidation of diphenylcarbazide is inhibited by DCMU and by high ionic strengths. This activity appears to measure the primary photochemical reaction of photosystem 2.

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The properties and preparation by conventional methods of salts of 28 substituted phenylhydrazines, 3 substituted naphthylhydrazines, 2-hydrazinofluorene, 9-phenanthrylhydrazine, 3-hydrazinopyrene, and 6-quinolylhydrazine are described. Only low yields of the polycyclic arjdhydrazines were obtained. Hydrazines whose hydrochloride salts appeared unstable were converted usually to the more stable hydrogen oxalate, which sometimes changed to the neutral oxalate during recrystallization.In this laboratory during the past lour years about 120 hydrazine derivatives of diverse types were screened at a variety of concentrations in D-19 developer for ability to increase the speed of