Light-Dependent Oxygen Metabolism of Chloroplast Preparations. II. Stimulation by Manganous Ions.

The importance of manganese for photosynthesis was first suggested by McHargue (17) and Bishop (2). Pirson (21) found that photosynthesis in manganese-starved Chlorella cells was inhibited despite the absence of visible chlorosis; this inhibition could be relieved by adding manganese to the medium. Emerson and Lewis (6) observed that manganese deficiency affected the maximum quantum yield of photosynthesis. Gerretsen (7) found that manganese deficiency caused a reduction in the rate of photosynthesis in oats. He surmised that manganese is specifically involved in the oxygen-liberating stage of photosynthesis and went on to study the effect of manganese salts on the potentials of electrodes immersed in chloroplast suspensions (8). Working with chloroplasts in the absence of Hill reagents, Gerretsen found that the electrode potential was changed very markedly on addition of manganese and he suggested that peroxide is formed in the presence of manganese but not in its absence. More recently Kessler (15, 16), from his studies of photosynthesis and photoreduction in manganesedeficient algae, provided convincing proof that the role of manganese in photosynthesis is mainly, if not exclusively, concerned with oxygen evolution. Aside from the implication of manganese, we know very little about the process by which oxygen is evolved from the oxidized product of the splitting of water in photosynthesis. There is a comparable lack of experimental evidence concerning the role of manganese in photochemical oxygen production by isolated chloroplasts. Mehler (18) was the first to show that oxygen could function as a Hill reagent, i.e., as an oxidant which is effective in promoting photochemical oxygen production by chloroplast preparations. In the Mehler reaction there is a light-dependent consumption of oxygen which is a consequence of the simultaneous reduction of oxygen by a photochemically generated reductant and evolution of oxygen from the oxidized product of photolysis. Added ethanol and excess catalase act as a "sink" for the peroxide produced by the reduction of molecular oxygen (13). The stoichiometry of the over-all reaction is such that two molecules of oxygen are consumed for every one produced (Mehler and Brown, 20). More recently, Brown and Good (3) have shown that chloroplasts illuminated without addenda can produce and consume oxygen simultaneously. Both production and consumption are light dependent processes. The stoichiometry being 1: 1, the chloroplasts thus effect an exchange of the oxygen of vater for molecular oxygen dissolved in the suspending medium. In this exchange reaction (which differs from the Mehler reaction only in the method of removal of peroxide formed in the reduction of molecular oxygen), each mole of oxygen reduced by chloroplast preparations in the light is exactly balanced by the production of Y2 mole from the oxidized product of photolysis and X mole from the decomposition of peroxide by the endogenous catalase of the chloroplasts. Because there is no net change of oxygen tension in the exchange reaction, it can be followed only by tracer oxygen techniques. Mehler (19) tested the effect of added manganous ions on the Hill reaction and found no effect on the rate of oxygen evolution when quinone was used as the oxidant (whether or not catalase and ethanol were present). In the case of the Mehler reaction, however, he found that Mn+ + increased the rate of net oxygen consumption. The manganese-stimulated rates were approximately the same as those which Mehler (19) had observed following quinone reduction. This stimulatory effect of manganese was catalytic; marked stimulation was given by 10-4 M Mn+ f and the maximum effects were produced by a 10-3 M concentration. Andreae (1) has reported a flavin-sensitized photooxidation of manganese ions and has suggested that the cyclic oxidation-reduction of manganese might explain the effects of manganous ions on oxvgen uptake by illuminated chloroplast suspensions. Also, Kenten and Mann (14) have demonstrated a light-dependent oxidation of manganese in chloroplast preparations and have discussed its possible role in photosynthesis. Mehler's observation of the manganese stimulation has been confirmed by Good and Hill (9) and extended to preparations from chard and Chenopodium. 'Received July 16, 1959. 2 The contents of this paper are part of a thesis submitted to the graduate faculty of the University of Minnesota in partial fulfillment of the requirement for the Ph.D. degree. 3 Present address: Department of Biological Sciences, Goucher College, Towson, Baltimore 4, Md.