Botany online 1996-2004. No further update, only historical document of botanical science!

The Dark Reactions of Photosynthesis, Assimilation of Carbon Dioxide And The CALVIN Cycle.

Due to the use of isotopes were M. CALVN and his collaborators at the University of California, Berkeley able to reveal completely the reactions taking place during the incorporation of carbon dioxide into carbohydrates in the relatively short period from 1946 - 1953. The quick success was based on the use of sensitive methods (two-dimensional paper chromatography, autoradiography), a suitable specimen and the rapid progresses of enzyme biochemistry. Cultures of the single-celled green alga Chlorella pyrenoidosa (that was introduced to photosynthetic studies in 1919 by O. WARBURG) were supplied with light and an even stream of air containing 12CO2.

At a given time (t= 0) was 14CO2 added to the stream of air for a short time. It was assumed that the labelled carbon dioxide molecules were successively incorporated into intermediates of the carbohydrate synthesis. After 3, 5 etc. seconds were the experiments stopped by adding boiling alcohol and the newly produced 14C-labelled intermediates were separated and identified by paper chromatography.

  1. The first stable compound that was labelled radioactively already after 3 seconds was 3-phosphoglycerate (3-PG), a substance we got to know previously as an intermediate of glycolysis. 14C is found in the carboxyl group of 3-PG. At first was it assumed that the molecule accepting the carbon dioxide would have to be a C2 unit. But after a futile search was finally ribulose diphosphate (RuDP), a C5 unit identified as the acceptor

    C5 + C1 = 2 C3

    This reaction is catalyzed by ribulose bisphosphate carboxylase (also called Rubisco or, formerly, fraction-1-protein), as far as quantity is concerned the most common protein of the world. The protein complex of green plants consists of eight times two subunits, eight large and eight small ones The picture to the right shows part of the enzyme together with ribulose phosphate , CO2 , and a magnesium ion (green ball) essential for the reaction. An interactive file demonstrates the single, subsequent reactions.

    After longer reaction periods (5 seconds, 10 seconds, etc.) were further labelled compounds found. CALVIN and BENSON determined the sequence of the incorporation and were able to unite the single steps to a pathway. Two results were especially interesting:

    • the resynthesis of ribulose diphosphate and
    • the production of the assimilate (the net product of the carbon dioxide assimilation).

    The production of ribulose diphosphate is best described by a cycle (the CALVIN cycle), while the assimilate production is a linear process. It is based on the fact that an intermediate of the CALVIN cycle is deducted from it.

  2. 3-phosphoglycerate is reduced to glycerinaldehyde-3-phosphate (GAP), the carboxyl group is transformed into an aldehyde group. The reaction consumes ATP and NADPH2. The reverse reaction occurs, too, in glycolysis though in photosynthesis NADP is needed instead of the NAD consumed during glycolysis. It is known today that the two reactions (and all others, too) are catalyzed by different enzymes and that the enzymes of photosynthesis use NADP (> NADPH2) as a cofactor.

    The CALVIN cycle has to be passed three times in order to produce one molecule of glycerinaldehyde-3-phosphate (a C unit) via photosynthesis since just one molecule of carbon dioxide is fixed in every round.

  3. Just as in glycolysis is part of the glycerinaldehyde-3-phosphate converted into dihydroxyacetonephosphate (DAP) by epimerization.

  4. Fructose-1,6-diphosphate (F-1,6-P) is formed by addition of one molecule glycerinaldehyde-3-phosphate and one molecule dihydroxyacetonephosphate.

  5. Fructose-1,6-diphosphate is converted into fructose-6-phosphate (F-6-P) by splitting off Pi. The F-6-P has two alternative fates:

  6. The E-4-P is coupled to one molecule of dihydroxyacetonephosphate (DAP). The result is a molecule of sedoheptulose-1,7-diphosphate (SDP), a C7 unit.

  7. After splitting off one of the two phosphate residues reacts the sedoheptulose-7-phosphate (S-7-P) with glycerinaldehydephosphate. Two C5 units are the result: ribulose-5-phosphate (Ru-5-P) and xylulose-5-phosphate (X-5-P).

  8. Ribulose-5-phosphate is phosphorylated to ribulose-1,5-diphosphate and starts a new round of the CALVIN cycle.

In summary, one can describe the end result of the dark reactions as follows:
6 RuDP + 6 CO2 > 12 3-PG
12 3-PG + 12 NADPH2 + 12 ATP > 12 GAP + 12 ADP + 12 Pi + 12 NADP
12 GAP > 1 glucose (net synthesis product of carbon dioxide assimilation) + 10 GAP
10 GAP + 6 ATP > 6 RuDP

NADPH2 and ATP stem, as we will see, from the light reactions of photosynthesis in which the light energy is converted into chemical energy.

After understanding the pathway in Chlorella pyrenoidosa arose the question whether it occurs in all other green plants, too. It could be shown to be an important pathway of all green plants. Even isolated chloroplasts (from spinach, for example) are still fully active and all reactions of the CALVIN cycle take part within them.

© Peter v. Sengbusch - Impressum