10 April 2008 Dark reactions of Photosynthesis Andy Howard Introductory Biochemistry 10 April 2008

10 April 2008

Dark reactions of Photosynthesis

Andy HowardIntroductory Biochemistry

10 April 2008

10 April 2008 Dark Reactions p. 2 of 47

Dark reactions matter!

Not all of these reactions really take place in the dark; but some do, and even the ones that take place in daylight are not directly dependent on photon absorption


What we’ll discuss Dark reactions of

Photosynthesis RuBisCO Calvin Cycle

overview C5 to C3 to C6 Regenerating C5’s Energy bookkeeping

Sucrose & Starch Other C-fixation

paths


Dark reactions

Series of ordinary chemical reactions Powered by reducing power in NADPH Anabolic Some common features with pentose

phosphate pathway


Dark reactions: overview

RuBisCO fixes atmospheric CO2 into carbon skeletons

Reductions of 3-phosphoglycerate build up carbohydrate

Pathway is cyclic in that RuBP is regenerated for additional reactions


RuBisCO reaction Condensation of ribulose

1,5-bisphosphate (RuBP) with CO2 to produce two molecules of 3-phosphoglycerate

Enzyme is ribulose1,5-bisphosphate carboxylase / oxygenase(RuBisCO)

RuBP

3-phosphoglycerate


The unwanted (?) side-reaction of RuBisCO

Secondary reaction isribulose 1,5-bisphosphate+ O2 3-phosphoglycerate +2-phosphoglycolate

Uses up oxygen rather than CO2

No net carbon incorporation into organic molecules

2-phospho-glycolate


RuBisCO structure L8S8 stoichiometry

in higher plants:Mol.Wt. L=55kDa;Mol. Wt. S=12 kDa

TIM barrels in both All (?) catalytic activity in L

(large) subunit L coded for by chloroplast gene S by nuclear genome Does S play a controlling role?

PDB 1WDDOctamer of L8S8 unitsL2S2 shownfrom rice(cf. fig. 15.21)


RuBisCO regulation Plant growth closely associated with

carboxylation / oxygenation ratio:Carboxylation high means fast growth

Easy way to alter that: grow plants in high CO2

Difficult to do that without animal toxicity! Expensive to put your cornfield in a plastic

bubble (but not impossible)


Could you win genetically? Attempts to engineer proteins that

don’t do oxygenation(or even that have improved CO2/O2 activity ratios) have failed

There are some plants whose RuBisCO has a better SC/O than that of others

Maybe O2 and CO2 bind in precisely the same way!


Subsequent dark reactions, I Pair of 3-phosphoglycerate

molecules enter reductive pathway toward bigger sugars

Note that this reaction appears in glycolysis (in reverse) and in gluconeogenesis

Phosphoglycerate kinase activation:3-P-glycerate + ATP 1,3-bisP-glycerate + ADP PDB 1PHP

43 kDa monomerBacillus stearothermophilus(unfortunately!)


Subsequent dark reactions, II (cf. fig. 15.18)

Three glycolysis / gluconeogenesis rxns: GAPDH reaction:

1,3-bisP-glycerate + NADPH + H+glyceraldehyde-3-phosphate + NADP + Pi

TIM required to convert G3P to DHAP Aldolase makes fructose 1,6-bisphosphate

Some RuBP is recycled back in to provide input to subsequent condensations with CO2


RuBisCO, revisited 2-phosphoglycolate is the product

of the oxygenation reaction 2-P-glycolate is decarboxylated:

2 2-P-glycolate CO2 + 3-P-glycerate +Pi

The 3-P-glycerate can re-enter the Calvin cycle, but at the cost of some carbon

This lossy pathway is known as photorespiration


Be careful how you describe transketolase and transaldolase

A few days ago we said (in lecture) that the transketolase reaction wasKn + Am Kn-2 + Am+2

That’s wrong: we do donate two carbons from the ketose to the aldose, but they swap carbonyl positions when you do, so the reaction is really Kn + Am An-2 + Km+2

The notes have already been corrected!


Calvin cycle: first reaction

Begins with ATP-dependent phosphorylation of 3-phosphoglycerate to make 1,3-bisphosphoglycerate via phophosphoglycerate kinase

Same reaction found in gluconeogenesis; reverse of glycolytic step

Enzyme is 3-layer sandwich

PDB 1V6S86 kDa dimerThermus thermophilusMonomer shown


2nd Calvin-cyclereaction: GAPDH

NADPH-dependent reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate

As in gluconeogenesis, reverse of glycolytic reaction

GAPDH: typical NAD(P) dependent oxidoreductase

PDB 1RM4297 kDa octamerdimer + monomer shownspinach


The fates of glyceraldehyde-3-phosphate The pathway divides three ways at this

metabolite One equivalent toward fructose 1,6-

bisphosphate and gluconeogenesis Two head toward pentose phosphate

pathway, where a second bifurcation happens


C3 to C6 TIM converts one molecule of

glyceraldehyde 3-phosphate to dihydroxyacetone phosphate

Glyc-3-P and DHAP condense to form fructose 1,6-bisphosphate in standard aldolase reaction

Fructose 1,6-bisphosphatase removes the 1-phosphate to make fructose 6-phosphate

All of this happens in gluconeogenesis


Transketolase As we saw in the PPP,

fructose-6-P can react with glyceraldehyde-3-P in a transketolase reaction to form xylulose-5-phosphate and erythrose-4-phosphate

K6 + A3 A4 + K5 Typical TPP binding

structure

PDB 1ITZ297 kDa octamerdimer+monomer shownmaize


Fates of DHAP Can participate in F-6-P production Can condense with erythrose-4-P in an

aldolase reaction to form sedoheptulose 1,7-bisphosphate (K3 + A4 K7)

This can be dephosphorylated at the 1-position to form sedoheptulose 7-P via sedoheptulose 1,7-bisphosphatase


The final Glyc3-P

It can condense with sedoheptulose 7-phosphate in another transketolase reaction to form xylulose-5-phosphate and ribose-5-phosphate:K7 + A3 A5 + K5 (fig. 15.19)

The ribose-5-phosphate is an endpoint but it can also be isomerized to ribulose-5-phosphate

Xylulose-5-phosphate can be epimerized to form ribulose-5-phosphate too


Activation ofribulose-5-phosphate

Phosphoribulokinase uses ATP as a phosphate source to convert ribulose-5-phosphate to RuBP

Enzyme is similar to adenylate kinase

PDB 1A7J32 kDa monomerRhodobacter sphaeroides


What is unique here?

Not much Last reaction is specific to Calvin cycle Others are found in gluconeogenesis or

pentose phosphate pathway or both In this direction these reactions require

the NADPH and ATP derived from the light reactions of photosynthesis


Bookkeeping for dark reactions

Numbers given on fig.15.19 presuppose 3 input RuBP molecules per run of the cycle

This makes it easy to divide up the Glyceraldehyde 3-P later

Net reaction is:3 CO2 + 9ATP + 6 NADPH + 5 H2O glyceraldehyde 3-P + 9ADP +8 Pi + 6 NADP+


Cost of making Acetyl CoA• We get back 2 NADH, 2 ATP when we

convert glyceraldehyde 3-P to acetyl CoA• Therefore acetyl CoA costs 9-2 = 7 ATP

and 6-2=4 NAD(P)H• At 2.5 ATP per NAD, that total is 7 + 2.5 *

4 = 17 ATP required per acetyl CoA• When we oxidize acetyl CoA we get 10

ATP (see TCA-cycle lecture),so we’re 10/17 = 59% efficient


Carbohydrate storage in plants Glyc3P is converted to glucose-6-P or

glucose by gluconeogenesis Glycogen is storage polysaccharide in

bacteria, algae, some plants Other plants make starch (amylose or

amylopectin) from glucose-6-P Pathway begins with conversion of

glucose-6-P to glucose-1-P, catalyzed by phosphoglucomutase


Starch synthesis Glucose 1-P activated with ATP, not UDP

-D-glucose 1-P + ATP ADP-glucose + PPi

Reaction driven to the right by hydrolysis of PP i

ADP glucose is added to growing starch molecule with release of ADP:ADP-glucose + (Starch)n ADP + (Starch)n+1

Branching in amylopectin accomplished as in glycogen(Yao et al (2004) Plant Physiol. 136:3515)


Diurnal variations in starch

Starch synthesis in daylight:ATP is readily available

Starch degradation at night Starch phosphorylase cleaves

starch to produce glucose-1-phosphate;glucose-1-P to triose phosphates by glycolysis

Enzyme is similar to glycogen phosphorylase

PLP-dependent

PDB 2C4M350 kDa tetramerCorynebacterium callunae


Alternative path for night-time starch degradation

Starch to dextrins via amylase Dextrins are oligosaccharides

beginning with a -1,6 link Dextrins eventually degraded

to glucose Glucose is phosphorylated by

hexokinase Enzyme:

sheet domain + TIM barrel

PDB 1HT645 kDa monomerbarley


Sucrose: mobile carbohydrate

Synthesized in chloroplast-containing cells; exported tovascular system so otherplant parts can use it

Two fructose 6-phosphatemolecules are starting points(fig 15.25)

One is converted to Glucose-1-P (via glucose 6-P) and thence to UDP-glucose

That condenses with the other Fructose-6-P with the help of sucrose 6-P synthase to form sucrose 6-P

That gets dephosphorylated to make sucrose


Enzymes in sucrose synthesisEnzyme Reactant Product Glucose 6-phosphate

isomerase F-6-P G-6-P Phosphoglucomutase G-6-P G-1-P UDP-glucose G-1-P + UDP-

glucosepyrophosphorylase UTP + PPi

Sucrose 6-phosphate F-6-P + Sucrose-6-Psynthase UDP-glucose

Sucrose phosphate Suc-6-P Sucrosephosphatase + H2O + Pi


UDP-glucose pyrophosphorylase

Catalyzesglucose-1-P + UTP UDP-glucose + PPi PDB 2ICY

103 kDa dimerArabidopsis


Sucrose 6-phosphate phosphatase

Contains “tongs” that release free sucrose into the cell:Fieulaine et al, Plant Cell 17: 2049-2058

Rossmann fold + complex

PDB 1TJ527 kDa monomerSynechocystis


How sucrose is used Sucrose taken up by non-photosynthetic

cells Broken down to glucose and fructose

supplies energy by glycolysis and TCA Glucose and fructose can be built back up to

starch in storage tissues:Amyloplasts (modified chloroplasts with no photosynthetic mechanisms) in root cells do this


Other carbon-fixation pathways Purpose: increase local [CO2] / [O2] to

improve performance of RuBisCO C4 pathway (high temp, lots of light) Crassulacean acid metabolism (high

temp, limited water)


C4 pathways Common in maize, sorghum, sugarcane,

weeds Needed at high temp because

rate(oxidation)/rate(carboxylation) increases with temperature

External CO2 acceptor is PEP via PEP carboxylase; product is oxaloacetate

This occurs in mesophylls; bundle sheath cells continue to do ordinary RuBisCO-based carbon fixation using CO2 released from metabolites


PEP Carboxylase

PEP + HCO3-

oxaloacetate + Pi

Occurs outside C4 metabolism too

One TIM barrel per monomer

PDB 1JQO427 kDa tetramermaize


C4 interplay

Diagram courtesyMIT: ESG Biology program


Crassulacean acid metabolism

Leaf cells open to CO2 uptake lose a lot of water during the day(high evaporation rate)

Solution: assimilate carbon at night Reactions are as in C4 pathway;

cellular specialization and enzyme regulation are different


Stomata and vacuoles Stomata (spaces between cells that

can open to allow access for respiration) near mesophylls open only at night, enabling PEP carboxylation to oxalacetate and then reduction to malate

Malate stored in central vacuole, then released during the day when the stomata are closed


CAM: day and night

University of Newcastle, Plant Physiology program


iClicker quiz question 1 Oxidation of a 2n-

carbon fatty acid yields (n-1) QH2,(n-1) NADH, and n acetyl CoA. Initiating the process costs 2 ATPs. Assume we can get 10 ATP per acetyl CoA. How much ATP can we get from oxidizing palmitate?

(a) 104 ATP (b) 106 ATP (c ) 108 ATP (d) 112 ATP (e) Undeterminable

given the data supplied


Answer to 1st question Palmitate is a C16 carboxylic acid.

Therefore in the conditions of the problem, 2n = 16, n = 8, n-1 = 7.

Thus we get 7 QH2, 7 NADH,8 acetyl CoA produced by its oxidation

Thus we get 7*2.5 + 7 * 1.5 + 8 * 10 = 17.5 + 10.5 + 80 = 108 ATP produced

Starting the process costs 2 ATP, so the net result is 106 ATP gained


iClicker quiz question 2Why would you not expect to find crassulacean

acid metabolism in tropical plants? (a) Tropical plants do not photosynthesize. (b) Tropical plants cannot develop the stomata

that close off the chloroplast-containing cavities (c) Water conservation is less critical in areas of

high rainfall (d) The waxy coating required to close off the

leaves’ access to O2 would dissolve in the high humidity and high temperature of the tropics

(e) None of the above


Answer: (c)

The primary significance of CAM is conservation of water in regions of low humidity, where evaporation rates are high and water is scarce. Neither of these conditions pertains in the tropics.


Control of CAM PEP carboxylase inhibited by

malate and low pH That prevents activity during

daylight, which would lead to futile cycling and competition for CO2 between PEP carboxylase and RuBisCO


Compartmentation in bacteria In photosynthetic bacteria,

RuBisCO is concentrated in protein microcompartment called a carboxysome

Active carbonic anhydrase there: catalyzes HCO3

- OH- + CO2

That tends to keep the CO2 / O2 ratio high

Documents

10 April 2008 Dark reactions of Photosynthesis Andy Howard Introductory Biochemistry 10 April 2008