carbon fixation the light independent reaction. enzyme controlled (q 10 2) located in the stroma of...

CARBON FIXATION

THE LIGHT INDEPENDENT REACTION

THE LIGHT INDEPENDENT REACTION

Enzyme controlled (Q10 2) Located in the stroma of the chloroplast Occurs simultaneously with the light

dependent reaction It can continue in the dark provided the

necessary raw materials are available (CO2, NADPH + H+ and ATP)

© 2010 Paul Billiet ODWS

A B C D E F

Enzyme controlled reaction pathways

Ea Ea Ec Ed Ee

X = Precursor (raw material) added here


Enzyme controlled reaction pathways

To find out the sequence of the reactions and the point at which X is added in, two approaches can be used:

1. Label and trace the products formed through time

2. Cut the supply of X and observe what happens to the intermediates in the pathway

e.g. in studying photosynthesis, cut the CO2 supply or switch off the lightso cutting the supply of ATP and NADPH+H+


Calvin and Benson 1946 to 1953

Used 14C radioisotope for labelling

Unicellular algae: Chlorella and Scenedesmus

Simple plants which respond quickly to changes in the environment

So little time lagImage Credit Scenedesmus


The “Lollipop” vesselA flat-sided, round flask containing the culture of algaeThis shape:- provided even illumination of all the cells- permitted careful control of environmental conditions (e.g. pH, temperature)- permitted rapid mixing of contents- precise sampling time


Labelling and tracing carbon using 14C

Add NaH14CO3 solution At timed intervals the algae are sampled and

killed by dropping in hot methanol Two-way (2-dimensional) chromatography

used to separate the compounds Identify radioactively labelled compounds by

autoradiography


Two-way (2-dimensional) chromatography

A. Mixture placed at the origin

B.1st run

D. 2nd run E. Autoradiograph reveals the compound/s which are labelled with 14C

C. Rotate the paper 90°


Results

2sSalisbury & Ross (1969) Plant Physiology Wadsworth

Result

The compound that became labelled first was Phosphoglyceric Acid (or Phosphoglycerate) PGA

PGA is a 3 carbon sugar Therefore: Compound B + CO2 PGA

What is compound B?!!!


A B PGA D E F

Light independent pathway

Ea Ea Ec Ed Ee

CO2

What is compound B?!!!


Searching for the compound that combines with carbon dioxide

Feed the algae NaH14CO3 for over 15 min All the compounds involved in photosynthesis

are labelled Then reduce the level of NaH14CO3 rapidly

Prediction PGA levels should fall rapidly The compound that fixes CO2 should rise

sharply


Salisbury & Ross (1969) Plant Physiology Wadsworth

Result This compound was found to be Ribulose

Bisphosphate (RBP) RBP is a 5 carbon sugarTherefore

RBP + CO2 2PGA5C + 1C = 2x3C

The enzyme controlling this important step is Ribulose Bisphosphate Carboxylase (Rubisco)


A RBP PGA D E F


Ea RUBISCO Ec Ed Ee

CO2


RUBISCO

A large enzyme 500 000 DaltonsInhibited by oxygenA relatively slow enzyme50% of chloroplast proteinProbably the most abundant protein on EarthConsists of two subunits (1 large + 1 small)The large subunit is coded for by a gene on chlDNA Image Credit: Rubisco stick model


Carbon fixation and light

The light dependent pathways produce ATP and NADPH+H+ to be used in the light independent processes

Illuminate the algae for 15 min then cut the light

Result PGA levels rise Other compounds, after

PGA in the pathway, fall


Carbon fixation and light

The compound requiring the NADPH+H+ and ATP was found to be Glycerate 3-phosphate (GP)

GP is converted to various other three carbon sugars or Triose Phosphates (TP) some are used to produce hexoses such as fructose phosphate and glucose phosphate


A RBP PGA GP EHexoses


Ea RUBISCO Ec Ed Ee

CO2

12 ATP12 NADPH + H+


A curious turn When the light is cut

interestingly RBP levels also fall

This is for two reasons

RBP requires ATP in its synthesis

RBP is formed from some of the GP

In other words the reaction pathway is a CYCLE

Image Credit: Salisbury & Ross (1969) Plant Physiology Wadsworth


Squaring the circle! The Calvin-Benson Cycle

6 ATP 6CO2

(1C)

6 RBP (5C)

12 PGA (3C)

12 ATP12 NADPH + H+

12 GP (3C)

12 TP (3C)

1 Hexose(6C)

Intermediates

PHOTOSYNTHESIS OVERVIEW

CHLOROPLAST CYTOSOL

H2O O2Light dependent

reaction

Light independent reaction

CO2

Starch

Sugars

Fatty acids

Amino acids

ATP NADPH+H+

Triose phospates


carbon fixation the light independent reaction. enzyme controlled (q 10 2) located in the stroma of...

Documents

paul billiet odwsodws

light independent processes

light result pga levels

light dependent pathways

c intermediates

c radioisotope

phosphoglycerate pga

carbon fixation