Q.Are the enzymes that catalyse the dark reactions of carbon fixation located inside the thylakoids or outside the thylakoids?

The dark reaction (carbon fixation) occurs in the chloroplast's stroma, not inside the thylakoids — its enzymes are dissolved in the stroma, physically separate from the thylakoid membranes where the light reaction's pigments and electron carriers sit, even though the dark reaction depends on the ATP and NADPH2 that diffuse out of the thylakoids into the stroma. The full route from CO2 to sugar, the Calvin cycle (or C3 pathway), was worked out by Calvin, Benson and co-workers using radioactively labelled 14CO2 fed to the alga Chlorella (Calvin received the 1961 Nobel Prize for this work), and it proceeds through three distinct phases. In carboxylation, the CO2-acceptor molecule, the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP), reacts with CO2 (catalysed by the enzyme RuBP carboxylase, Rubisco — about 16% of chloroplast protein) to form an unstable 6-carbon compound that immediately splits into two molecules of the 3-carbon 3-phosphoglyceric acid (3-PGA); this step's significance is that it is the actual point where atmospheric carbon enters the organic world, and its 3-carbon product is what gives the whole pathway its name, the C3 pathway. In reduction (glycolytic reversal), 3-PGA is converted, at the cost of ATP, into 1,3-diphosphoglyceric acid, which is then reduced, using NADPH2 from the light reaction, into glyceraldehyde-3-phosphate (3-PGAL); this is the step that actually converts the fixed carbon into a reduced, energy-rich sugar-phosphate, and it is where the light reaction's two products (ATP and NADPH2) are put to direct chemical use. In regeneration, of every 12 molecules of 3-PGAL formed (from fixing 6 CO2), only 2 are exported to build one glucose molecule, while the remaining 10 are used, consuming 6 further ATP, to regenerate the 6 molecules of RuBP the cycle needs to keep running …