Photosynthesis by Isolated Chloroplasts

Abstract

In 1943, Ruben (l), elaborating on earlier suggestions by Thimann (2) and Lipmann (3), proposed that sugar formation in photosynthesis is a completely dark, chemosynthetic process which depends on only two products formed by light reactions: reduced pyridine nucleotide and adenosine triphosphate. Ruben’s proposal distinguished two phases of sugar synthesis in the dark: a carboxylative phase, dependent on ATP only, in which COZ enters cellular metabolism by carboxylating an acceptor molecule, and a reductive phase, in which a carboxyl group is reduced by pyridine nucleotide with the aid of ATP. In the ensuing 15 years Ruben’s scheme received experimental support from several directions: (a) Calvin, Horecker, Ochoa, Racker and their associates (4-7) have identified the ATPdependent carboxylative phase in COZ assimilation. They have shown that the entry of COZ into the metabolism of photosynthetic cells depends on the phosphorylation of ribulose monophosphate by ATP to ribulose diphosphate, which is then carboxylated by COZ and cleaved to give 2 molecules of 3-phosphoglyceric acid. (b) The kinetic studies of’ Calvin’s group (8) suggested that the reductive phase of CO2 assimilation is the reduction of 3-phosphoglyceric acid to triosephosphate by a reversal of the well known glycolytic reactions, a mechanism that requires reduced pyridine nucleotide and ATP. (c) In addition to a carboxylative and a reductive phase, the findings in photosynthetic tissues of components of the pentose cycle (cf. 9) afforded a mechanism for the regeneration of the COZ acceptor in photosynthesis, in what might be designated as a third, regenerat,ive phase of CO2 assimilation. (d) Racker (10) obtained in the dark, a synthesis, driven by DPNH2’ and exogenous ATP, of hexose phosphate from C02, in a model multi-enzyme system consisting of glycolytic enzymes from rabbit muscle and yeast? and pentose cycle enzymes from spinach leaves. The carboxylative, reductive, and regenerative phases constitute a cyclic sequence of dark reactions which jointly might be termed a reductive carbohydrate cycle (cf. 7). Was this reduc* Aided by grants from the National Institutes of Health, United States Public Health Service, and the Office of Naval Research. 1 The abbreviations used are: FMN, flavin mononucleotide; 3-PGA, 3-phosphoglyceric acid; 1,3-diPGA, 1,3-diphosphoglyceric acid; TPNHz and DPNHZ, reduced tri- and diphosphopyridine nucleotide (these abbreviations will be used, in preference to the more common TPNH and DPNH; cf. Dixon, M., and Webb, E. C., Enzymes, p. 395. Longmans, Green and Co., London, 1958). (At the request of the authors, the Editors have permitted the use of this terminology in this instance, although they strongly prefer the symbols DPNH and TPNH.) 2 Racker, E., personal communication (1958). tive carbohydrate cycle then, although comprising exclusively dark reactions, a “photosynthetic” cycle for sugar formation, peculiar to chlorophyllous cells? This question was answered in the negative when the most distinctive enzyme of the reductive carbohydrate cycle, ribulose-di-P carboxylase, was found in such heterotrophs as Escherichia coli (9, 11) and when the cycle in its entirety was found in the nonphotosynthetic sulfur bacterium