Photosynthesis is conversion of light energy into chemical energy utilizing Carbon di oxide, light energy, chlorophyll ( LHC II. PS II and LHC I PS I and electron transport chain ) along with water and generating ATP, reducing NADP+ to NADPH + H+ and photolysis of water with release of Oxygen as by product. This reaction takes place in light and is termed as “Light reaction”. C3 reduction or Calvin cycle CO2 → C3 → C6 (Melvin Calvin 1950s, Nobel prize in 1961) was termed “dark reaction”.
Although Calvin Cycle’ or ‘Carbon Reactions Pathway do not Require Light energy to occur but they do require energy captured by light reactions and they occur at same time as light reactions.
Enzymes&intermediates of the Calvin Cycle are located in the chloroplast stroma, a compartment somewhat analogous to the mitochondrial matrix. The Calvin Cycle, earlier designated as the photosynthetic "dark reactions," is now called the carbon reactions pathway Carbon fixation. I propose to call it “light dependent carbon fixation” as this takes place during light only.
Light-activated e- transfer is linked to pumping of H+ into thylakoid disks. This leads to increase of pH in the stroma to about 8. This pH range ( 7.8 to pH 8.0 ) is required for activity of RuBisco to take place effectively. Alkaline pH activates stromal Calvin Cycle enzymes RuBP Carboxylase, Fructose-1,6-Bisphosphatase&Sedoheptulose Bisphosphatase. The light-activated H+ shift is countered by Mg++ release from thylakoids to stroma. RuBP Carboxylase (in stroma) requires Mg++ binding to carbamate at the active site. Some plants synthesize a transition-state inhibitor, carboxyarabinitol-1-phosphate (CA1P), in the dark. RuBP Carboxylase Activase facilitates release of CA1P from RuBP Carboxylase, when it is activated under conditions of light by thioredoxin. The activase is a member of the AAA family of ATPases, many of which have chaperone-like roles. RuBP Carboxylase Activase is a large multimeric protein complex that may surround RuBisCO while inducing the conformational change to the open state. Activation of RuBisco activase and coversion of inactive form of RuBisco require electron transport and it can only take place in presence of light. Thioredoxin is a small protein with a disulfide that is reduced in chloroplasts via light-activated electron transfer. During illumination, the thioredoxin disulfide is reduced to a dithiol by ferredoxin, a constituent of the photosynthetic light reaction pathway, via an enzyme Ferredoxin-Thioredoxin Reductase. Reduced thioredoxin activates several Calvin Cycle enzymes, including Fructose-1,6-bisphosphatase, Sedoheptulose-1,7-bisphosphatase, and RuBP Carboxylase Activase, by reducing disulfides in those enzymes to thiols. RuBP Carboxylase Activase is an ATP hydrolyzing (ATPase) enzyme that causes a conformational change in RuBP Carboxylase from a closed to an open state. This allows release of tightly bound RuBP or other sugar phosphate from the active site, and carbamate formation.There can be several modes of carbon fixation in heterotrophs and CAM plants which are not light dependent and involve PEPcarboxylase enzyme. Anaplerotic CO2 fixation has been reported in actively growing regions of plants as they require carbon skeleton due to rapid synthesis of amino acids for protein biosynthesis. Callus cultures of C3 have been shown to fix CO2 utilizing PEPcarboxylase in photosynthetic pathway and path of Carbon has been traced and published ( see Neumann, Kumar, Imani, 2010). This will be discussed separately. Regulation prevents the Calvin Cycle from being active in the dark, when it might function in a futile cycle with Glycolysis&Pentose Phosphate Pathway, wasting ATP & NADPH. Light activates, or dark inhibits, the Calvin Cycle (previously called the “dark reaction”) in several ways. Since photosynthetic light reactions produce ATP, the ATP dependence of RuBisCO activation provides a mechanism for light-dependent activation of the enzyme.
Ribulose Bisphosphate Carboxylase (RuBP Carboxylase), catalyzes CO2 fixation:
ribulose-1,5-bisphosphate + CO2 2 3-phosphoglycerate
Because it can alternatively catalyze an oxygenase reaction, the enzyme is also called RuBP Carboxylase/Oxygenase (RuBisCO). It is the most abundant enzyme on earth.
Extraction of H+ from C3 of ribulose-1,5-bisphosphate promotes formation of an enediolate intermediate. Nucleophilic attack on CO2 leads to formation of a b-keto acid intermediate, that reacts with water and cleaves to form 2 molecules of 3-phosphoglycerate. Transition state analogs of the postulated b-keto acid intermediate bind tightly to RuBP Carboxylase and inhibit its activity. "Active" RuBP Carboxylase has a carbamate that binds an essential Mg++ at the active site. The carbamate forms by reaction of HCO3- with the e-amino group of a lysine residue, in the presence of Mg++. HCO3- that reacts to form carbamate is distinct from CO2 that binds to RuBP Carboxylase as substrate. Mg++ bridges between oxygen atoms of the carbamate & substrate CO2. Binding of either RuBP or a transition state analog to RuBP Carboxylase causes a conformational change to a "closed" conformation in which access of solvent water to the active site is blocked. RuBP Carboxylase (RuBisCO) can spontaneously deactivate by decarbamylation. In the absence of the carbamate group, RuBisCO tightly binds ribulose bisphosphate (RuBP) at the active site as a “dead end” complex, with the closed conformation, and is inactive in catalysis. In order for the carbamate to reform, the enzyme must undergo transition to the open conformation. Reference:
Neumann, Karl-Hermann, Ashwani Kumar and Jafargholi Imani (2010) Plant cell and tissue culture – A tool in Biotechnology. Springer, Germany pp 333.
