Stimulation of the Hill reaction by carbon dioxide

Hill reactions (i.e., ?2 evolution by illuminated chloroplast preparations) are generally assumed to represent the photochemical splitting of water. For about 20 years the view has prevailed that, in the course of a Hill reaction, photolysis generates an 02 precursor and a reductant which is capable of reacting directly or indirectly with any of a variety of oxidants. The metabolism of both photolysis products (oxidant & reductant) evidently is enzymatically controlled. The list of oxidizing agents effective in the Hill reaction began chronologically with ferric oxalate and now includes an impressive number of naturally-occurring as well as exotic substances. On the assumption that oxygen evolution and oxidant reduction are separate if not independent processes, there is no compelling a priori reason to believe CO2 is uniquely involved in the Hill reaction even in a catalytic capacity. However, because of the close analogy between photosynthesis and the various types of Hill reactions, the possible role of carbon dioxide in chloroplast reactions continues to be a subject of concern both theoretically and experimentally, especially so because several reports in the literature suggest that CO2 at least in some instances may have a catalytic effect on reaction sequences which involve water photolysis. Franck (7) found an influence of CO2 on the evolution of 02 by chloroplasts prepared from tobacco leaf. He studied a Molisch-type reaction in which no Hill oxidant was added to the preparations. Only small amounts of 02 were evolved but the rate was significantly higher when CO, was present. Brown and Franck (3) using C1402 found that carbon 14 was not taken up under the conditions of these experiments. They concluded that CO, could play only a catalytic role. Kessler (9, 10) observed that the rate of reduction of nitrite by illuminated Ankistrodesmus cells as measured by H2 uptake was increased in the presence of catalytic amounts of CO2. Experiments performed with C1402 showed that the reduction of nitrite with hydrogen was not accompanied by an appreciable fixation of carbon. Thus the effect of CO2 apparently was indirect. Boyle (2) reported manometric experiments from which he claimed at least small quantities of carbon dioxide were required for 02 evolution in the light using triturated spinach leaves. These experiments were repeated by Clendenning and Gorham (6) who found no differences in Hill reaction rate between vessels which contained carbon dioxide and those which did not. Because Boyle's results were not confirmed and besides were incompatible with prevailing theory they have generally been ignored. Warburg and Krippahl (11, 12) using kohlrabi chloroplast preparations with quinone as the oxidant reported that small amounts of CO. increased the rate of the Hill reaction. Similar effects were noted with frozen-dried Chlorella and with quinone-poisoned whole Chlorella cells. Such results are of course reminiscent of the earlier observations by Boyle, even though the experimental material and nmethods were different. It is patently desirable to verify the claim that CO2 can augment the Hill reaction using experimental methods which adhere closely to those of Boyle and of Warburg and Krippahl. We report here the results of such attempts at confirmation. It was found possible to obtain manometric measuremiients which were in all respects identical with those reported by Boyle although, as will be shown, these results do not support his interpretation that there was a CO2 effect on the Hill reaction. Manometric results also were obtained which were at least qualitatively similar to the findings of Warburg and Krippahl with kohlrabi chloroplasts. The reactions in which an apparently valid CO2 effect could be observed also were measured by mass spectrometry which has the important advantage over manometry of relatively unambiguous analytic specificity. Additional experiments were performed with tracer CO, and with tracer 02 which permitted the detection and measurement of simultaneous production and consumption of these gases thus providing detailed information of the nature of the CO, effect. This method could reveal the operation of a cyclic regeneration of CO. by illuminated kohlrabi chloroplast preparations.