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A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP+). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, I1/2, is defined as the actinic light intensity that, at a given concentration of electron donro, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, I1/2 is compared with another parameter, equivalent to I1/2, that is obtained independently of the reaciton-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow. The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP+ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.

Under aerobic conditions that are likely to prevail in chloroplasts in vivo, the optimal concentration of ferredoxin for cyclic photophosphorylation was found to be equal to that required for NADP reduction and about one-tenth of that needed for cyclic photophosphorylation under anaerobic conditions. In the presence of ferredoxin and NADP, cyclic photophosphorylation operated concurrently with noncyclic photophosphorylation, producing an ATP: NADPH ratio of about 1.5. The effective operation of ferredoxin-catalyzed cyclic photophosphorylation by itself required a curtailment of the electron flow from water which was accomplished experimentally by the use of either an inhibitor or far-red monochromatic light. An unexpected discovery was that the operation of cyclic photophosphorylation by itself was also regulated by a back reaction of NADPH and ferredoxin with two components of chloroplast membranes, component C550 and cytochrome b559. The significance of these findings to photosynthesis in vivo is discussed.

A new iron-sulfur protein, distinct from the soluble chloroplast ferredoxin, was isolated from chloroplast membranes. The isolated protein, purified to homogeneity, had a molecular weight of about 8000 and 4 atoms of iron and 4 inorganic sulfides per mole. Its absorption spectrum had a broad absorbance band in the 400 nm region, a shoulder at approximately 310 nm, and a peak around 280 nm. The absorbance ratio A(400) to A(280) was 0.55. The electron paramagnetic resonance spectrum (measured at 12 degrees K) of the reduced protein was similar to that of other reduced iron-sulfur proteins, showing a major resonance line at g = 1.94. The isolated protein, when photoreduced by spinach chloroplasts, can in turn transfer electrons to mammalian cytochrome c. However, the photoreduced protein cannot replace soluble ferredoxin in NADP(+) reduction because of its apparent inability to interact with the chloroplast enzyme, ferredoxin-NADP(+) reductase. The relation of the isolated iron-sulfur protein to the bound ferredoxin that acts as the primary electron acceptor in Photosystem I is discussed.

New evidence is presented in support of the concept that reducing power for photosynthesis is generated solely by photosystem II (the oxygenic photosystem) when it transfers electrons from water to ferredoxin without the collaboration of photosystem I, the anoxygenic photosystem responsible for cyclic photophosphorylation. Membrane vesicles of opposite sidedness were prepared from spinach chloroplasts by the two-phase partition method: inside-out-vesicles greatly enriched in photosystem II and right-side-out vesicles containing both photosystems and having the same sidedness orientation as unfractionated chloroplast membranes. In both types of vesicles, plastoquinone analogues were used to inhibit light-induced electron transport from water to ferredoxin and from water to native photosystem I acceptors, the membrane-bound iron-sulfur centers A and B. In right-side-out vesicles the photoreduction of iron-sulfur centers A and B was more sensitive to plastoquinone inhibitors than the photoreduction of ferredoxin, whereas the converse was found in inside-out vesicles in which a greatly enhanced sensitivity of ferredoxin reduction to plastoquinone inhibitors was detected: the photoreduction of ferredoxin was about 80% inhibited at low concentrations of plastoquinone inhibitors that had practically no effect on the photoreduction of iron-sulfur centers A and B. These findings appear to exclude the possibility that these photosystem I contaminants were involved in the photoreduction of ferredoxin by the PSII-enriched inside-out vesicles.

Spinach chloroplasts were found to contain three forms of cytochrome b(559) that have the same alpha-peak at 559 nm, but are distinguished from one another by their oxidation-reduction potentials. The high-potential (H) form (E(m) about 330-350 mV) is reducible by hydroquinone, the middle-potential (M) form (E(m) about 50-80 mV) is reducible by ascorbate but not by hydroquinone, and the low-potential (L) form is reducible by dithionite but not by ascorbate. The H form was the predominant one in freshly prepared chloroplasts and was strongly correlated with Photosystem II activity. Chloroplast treatments such as aging, sonication, and mild heating, singly or in combination, brought about a shift of the H to the M form. More drastic treatments resulted in shifts of the H and M forms to the L form. The role of cytochrome b(559) as an electron carrier in System II of plant photosynthesis is discussed.