Rubisco Step 1 First Step: CO Adds to Enediol Form of Rubpi

Home , Carbon fixation, RuBisCO, Stroma (fluid)

The “Dark” Reaction: Carbon Fixation Requires Rubisco: Ribulose-1,5-bisphosphate carboxylase

RuBPi + CO2 6 2 x 3PG MW = 550, 000 g/mol Subunits eight small (14,000 g/mol) eight large (53,000 g/mol) 4 mM in stroma (250 mg/mL) Mg2+ required in active and allosteric sites

“dark” means Allosteric site lysine is bound to CO2 Mg2+ lysine-NH2 + CO2 light-indendent! - + à lysine-NH-CO2 + H (carbamate – nonsubstrate CO2)

Mechanism of CO2 Fixation: Rubisco Step 1

Ribulose 1,5-Bisphosphate Carboxylase “rubisco”

Fig. 20-23

First Step: CO2 adds to enediol form of RuBPi - - (not HCO2 or lysine-NH-CO2 !)

1 Mechanism of CO2 Fixation: Rubisco Step 2

H2O adds to b-keto intermediate

Fig. 20-23

Mechanism of CO2 Fixation: Rubisco Step 3

Fig. 20-23 Hydrated intermediate breaks down to 2 x 3PG

**C3 plants** (nontropical)

All steps catalyzed by rubisco! Enzyme makes up ~50% by wt total chloroplast protein Most abundant protein in biosphere

11 10 tons CO2 fixed per year!

2 Why is rubisco reaction energetically favorable?

Fig. 20-23

o’ RuBPi + CO2 6 2 x 3PG DG = -35 kJ/mol Step 1: highly unfavorable Step 3: highly favorable

Regulation of Enzymatic Activity of Rubisco (stroma)

+ Rubisco pHmax ~ 8 (H pumped into lumen from stroma) With hv, as H+ pumped into lumen, Mg2+ pumped out into stroma As hv 8, NADPH 8, ATP 8, [Mg2+ 8 H+ 9], Rubisco activity 8

CO2 fixation does not take place in the dark! At night, glycolysis and oxidative phosphorylation meet energy needs of the plant. Rubisco activity is controlled by a redox system that is dependent upon [NADPH]

3 Fig. 20-36 Redox regulation of Rubisco ox active Rubisco (red)

NADPH

NADP+ red inactive Rubisco (ox) Where is this protein found? What controls reduction of Fd? SUMMA RY O F ELECTRON TRA NSF ER IN PS I TO NADP+ hv + PSI 4 Cu+ +. -. 4 Fe 2+ 4 Fe 3+ 2 NADPH Chla Chla 4 Chla Ao plastocyanin 4 hnn Fe4S4 Fd What glycolytic enzymes must be +2 4 PQ A1 3+ 2+ + + 4 Cu Chla Chla 4 Fe 4 Fe 2 NADP + H RXN CTR activated? deactivated? FBPi phosphatase 8 , PFK9

Allosteric regulation of Rubisco

At high CO , no ATP or activase is allosteric subunit 2 required.

RuBPi At low CO2, ATP required to help displace a RuBPi which blocks the lys and prevents carbamylation

+ RuBPi allosteric subunit

RuBPi binds in both the active site and the allosteric site?

Fig. 20-33

4 From where comes the ribulose 1,5-bisphosphate (RuBPi)?

Understanding of C5 sugar synthesis requires a knowledge of how the pentose phosphate pathway works for carbon fixation!

Pentose-Pi pathway: Pentose Pi “Shunt”

Functions: oxidative portion Produces NADPH for biosynthesis (fatty acids, steroids).

Produces Ribose (C5) for RNA and cofactors (ATP, NAD, FAD, etc.).

Provides a breakdown pathway for C5 sugars.

Provides a pathway to regenerate Ru-5-Pi (C5) for carbon fixation.

regenerative (nonoxidative) portions

Ribulose 5-phosphate is produced in 3 steps! Ribulose 1,5-bisphosphate is synthesized how?

Ru-5-Pi + ATP º Ru-1,5-BPi + ADP

5 Pentose-Pi pathway (oxidative): Step 1

glucos e-6-Pi-de hydrogenase PiO NADP+ NADPH + H+ PiO O O H OH 1 OH O HO OH HO OH OH

glucos e-6-Pi 6-Pi- glucono- d-lactone C6 C6

Typical hydride transfer mechanism to NADP+ What “H-” is transferred? What is the source of G-6-Pi? Comes from phosphorylation of glucose and from G-1-Pi from glycogen phosphorylase breakdown of glycogen using Pi

Pentose-Pi pathway (oxidative): Step 2 lactonase

H+ PiO H2O PiO O - 2 OH O OH O OH O HO HO OH OH

6-Pi-glucono-d-lactone 6-Pi-gluconate C 6 C6 Typical hydrolysis of an ester bond. Where is the ester?

6 Pentose-Pi pathway (oxidative): Step 3 6-phosphogluconate dehydrogenase

NADP+ PiO PiO OH - OH O- O OH 3 O O HO HO O OH H OH 6-Pi-gluconate unstable bb-keto acid C 6 + Typical hydride transfer mechanism to NADP+ 3 NADPH + H H+ What “H-” is transferred? CO2

b In what other pathway was a -keto PiO acid formed? Name of enzyme? OH CAC: OH isocitrate dehydrogenase! HO O ketose + ribulose-5-Pi isocit + NAD º a -KG + CO2 + NADH C5

Phosphorylation of Ribulose-5-Pi to Bisphosphate

PiO ribulose-5-Pi kinase PiO OH OH OH + ATP OPi + ADP HO HO O O ribulose-5-Pi ribulose-1,5-BPi C5 C5

7 A review of C6 synthesis: Start with ?

Using “Cx” notation, summarize the rubisco reaction:

C5 + C1 à 2 C3

How is C6 made from 2C3?

Reverse of glycolysis starting from 3-phosphoglycerate! O - 3,4 C O H 2,5C OH

1,6 CH2OPi 3-phosphoglycerate (3PG)

A review of C synthesis: Continue with ? O 6

3,4 CH H2 ,5C OH

1,6 CH2OPi glyceraldehyde-3-ph osphate (GAP) Pi + NADP+ 6 - glyceraldehyde-3-phosphate dehydrogenase 6 DD GBBNN = + 6.3 kJ/mol NADPH + H + O O - 3, 4 C O P O H 2 ,5 C OH O-

1 ,6 CH2OPi 1,3-bisph osp hoglycerate (1 ,3-B PG) ADP 7 - phosphoglycerate kinase BB 7 DDG NN = - 18.5 kJ/mol ATP O 1st energy conservation step - 3,4 C O 2,5 H C OH 7 Start here 1,6 CH2OPi 3-phosphoglycerate (3PG)

8 A review of C6 synthesis: End with ?

O C6 PiO 6 1 OPi OH 2 5 OH 4 - aldolase BB 4 DDG NN 3 = + 24.0 kJ/mol Yeah! OH fructose-1,6-bisphosphate Cleavage of C into 2 x C fragments (F6P) 6 3 4 5 - triosephosphate isomerase BB NN PiO 6 OH 1 OPi DDG = - 7.6 kJ/mol 5 OH 2 5 OH 4 Isomerization of C3 to interconvert forms 3 O glyceraldehyde-3-phosphate + dihydroxyacetone phosphate (GAP) (DHAP)

For 2 C3 à C6, how many ATP required? NADPH? Note: 1 ATP and 1 NADPH

3PG (C3)

How is C5 regenerated? Rubisco reaction:

C5 + C1 à 2 C3 C Multiply by 6 6 Oops!

6 C5 + 6 C1 à 12 C3 C5 consumed 30 6 36 10 2 ? 10 C 2 C3 30 3 6

C5 regenerated using:

1) C2 transfers (transketolase) C 2) Condensations (transaldolase)! 6

9 CO2 Fixation is called “Calvin Cycle”

6 C5 + 6 C1 à 12 C3 30 6 36 10 2

10 C3 2 C3 30 6

http://www.nobel.se/chemistry/laureates/1961/calvin-bio.html

Photorespiration: Oxygenase Activity of Rubisco

O2 competes with CO2 for enol form of RuBPi

Km O2 350 m M Km CO2 9 m M

Only 1 x 3PG formed

Other product, Pi-glycolate, is salvaged through a complex series of reactions involving both peroxizomes and mitochondria!

Fig. 20-38

10 Salvage Pathway of Pi-glycolate

Photorespiration is a wasteful process, and is especially problematic at higher temperatures (above 28°C) At 25°C, the carboxylase activity is four times the oxygenase activity)

Fig. 20-39

Function of Photorespiration? Oxygenase Activity of Rubisco?

Km CO2 9 m M Km O2 350 m M Rubisco…

…most likely evolved before atmosphere had O2!

…retains ability to react with O2? Why?

Photorespiration… …dominates if light levels are high à high O2 production …scavenges O2 to help prevent oxidative damage to cells under intense light conditions

11 CO2 Fixation by C4 Plants (Tropical)

Tropical grasses such as sugar cane use a different pathway for CO2 fixation 14 14 Short-time CO2 fixation with CO2 leds to C in C4 intermediates

CO2 is fixed as follows: PEP + CO2 à OXAL (PEP carboxylase) Good summaries: http://methanogens.pdx.edu/boone/courses/BI336/BI336Lectures/200201/BI336Lec16.html