Lecture 9 RUBISCO.pdf

The problem of oxygen – In a photosynthetic organism, what is level of respiration in the light? 2 CO2 exchange in leaf when placed in darkness Post-illumination burst (PIB)...

The problem of oxygen – In a photosynthetic organism, what is level of respiration in the light? 2 CO2 exchange in leaf when placed in darkness Post-illumination burst (PIB) PIB increases with O2 Not saturated until O2 100% 3  W. Ogren: Ribulose bisphosphate carboxylase = bi-functional enzyme – can use O2 as well as CO2 2(346) Ribulose bisphosphate carboxylase/oxygenase = RUBISCO 4 Rubisco, Calvin-Benson cycle and photorespiration Almost all inorganic carbon is fixed into Rubisco organic form by Rubisco CO2 or O2 Rubisco carboxylates RuBP, to produce two molecules of 3-PGA 3-PGA enters the Calvin-Benson cycle, energy energetically creation costly regenerating RuBP Rubisco also oxygenates RuBP, producing 3-PGA and 2-PG 2-PG is recycled into RuBP through energetically costly photorespiration 5 Glycine decarboxylase (GDC) and serine hydroxy methyl transferase (SHMT) SHMT 6 Mechanism of Glycine Decarboxylase Complex NHy Ch 200 - CH2-NHzt Chin W GDC reaction cycle THF = tetrahydrofolate 7  Serine hydroxymethyl transferase (SHMT) CH3- THF + Gly Ser + THF CHI + HF = + Gly One carbon methylene carried on tetrahydrofolate (THF) is transferred to a second glycine, producing serine. 8  Folate Structure Tripartite molecule C1 units at various 1 23 oxidation levels are attached to N5 and/or N10 Polyglutamylated in vivo Photolabile Is photorespiration essential? High CO2 Ambient CO2 Mutations in photorespiratory pathway are deleterious  Photorespiration Decreases plant productivity approx. 30% Consequence of oxygen in atmosphere Recovers 3 out of 4 C otherwise lost Provides turnover of reducing power and ATP (decreases photoinhibition – removes excess energy) Can it be avoided? Mutations or chemical inhibition in photorespiratory pathway are deleterious High CO2 C4 – photosynthesis (in 2 lectures time…) 11 Ribulose bisphosphate carboxylase/oxygenase - Rubisco is an enigma Most abundant enzyme on planet – 50% soluble leaf protein, 75% chloroplast stromal protein Very slow reaction rate kcat = 3 sec-1 most enzymes ca >106 sec-1 Bifunctional enzyme – acts as carboxylase and oxygenase Large subunits (LSU) 54kDa (chloroplast encoded) Small subunits (SSU) 15kDa (nuclear encoded) Higher plants, algae, cyanobacteria - L8S8 (550kDa) Photosynthetic Bacteria – L2 12  Subunit composition of Rubisco (8LSU, 8 SSU) 550 kDa zolsu SSU subunit encoded Jou b , by rbcS in nucleus (14kDa) 8 LSU subunit encoded by rbcL in chloroplast 35 (55 kDa) 13  Rubisco is found in a variety of forms Form I: Plants and most Form II: Some cyanobacteria have a SSU bacteria and some LSU hexadecameric form consisting dinoflagellate algae of 8 Large subunits and 8 have form II, made Small subunits (L8S8) of dimers of L, Only Form I has small subunits ranging from L2 to L8 Form IV: Rubisco-Like Proteins with no catalytic activity. Some bacteria. Found as L2 Form III: Some archaea. Form III is found as L2, (L2)4 or (L2)5. Form III also serves a non- photosynthetic function Whitney, S.M., Houtz, R.L. and Alonso, H. (2011). Advancing our understanding and capacity to engineer Nature’s CO 2-sequestering enzyme, Rubisco. Plant Physiol. 155: 27-35; See also Tabita, F.R., Satagopan, S., Hanson, T.E., Kreel, N.E. and Scott, S.S. (2008). Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. J. Exp. Bot. 59: 1515-1524. © 2016 American Society of Plant Biologists Ribulose bisphosphate carboxylase is a bi-functional enzyme – WHY does it use O2 as well as CO2? Ribulose bisphosphate carboxylase/oxygenase = RUBISCO 15  Activation of Rubisco by CO2 - carbamoylation of lysine in active site Inactive Active (l > to - be substituted w/Ht Mg2+ stabilizes carbamoylated Lys, facilitates interaction with ribulose 1,5-bisphosphate → forms enediolate intermediate Mg2+ necessary for activation but not carboxylation Second molecule of CO2 used in catalysis 16 Activated Rubisco: carboxylation reaction & - I I Carbamoylated Lys of Rubisco attracts proton from C3 of RuBP, carbonyl group → forms enediolate intermediate Addition of CO2 to enediol intermediate followed by hydration of 6-C unstable ketone 17  Activated Rubisco: oxygenase reaction = o ofo ↓ 2H2ecules j (2PG) H + o (PGA) c - - + 20 C = 0 the H2 0 -c =0 Carbamoylated Lys of Rubisco attracts proton from C3 of RuBP, carbonyl group → forms enediolate intermediate Addition of O2 to enediol intermediate, followed by hydration of 5-C unstable ketone, cleaves bond between C2 and C3 of intermediate 18 Changes in atmospheric oxygen Early forms of Rubisco – O2ase activity not invoked ie no disadvantage Dual specificity of Rubisco is an accident of evolution arising from success of oxygenic photosynthesis 19  Rubisco specificity factor Measure of tendency for oxygenation over carboxylation Higher values = favour photorespiration Implies evolution has “improved” photosynthetic activity, affinity for CO2 20  Higher temperatures favour Rubisco oxygenation reaction – differences in solubility of O2 and CO2 C’ase vs O2ase (reverse of previous slide) 21  Can Rubisco be “improved”? Mn2+ can be used instead of Mg2+ But………..favours oxygenation 22  Activation of Rubisco requires another enzyme - Rubisco activase (RCA) rca mutants in Arabidopsis inhibit PS, inactive Rubisco O ↳ 5-S retuct-active oxidized 2 Subunits, 43 and 46 kDa hetero-tetramer? RCA activated by light – thiol reduction by thioredoxin (non-cyclic ET) Activation of Rubisco requires ATP 23 Rubisco activase a dislodges RuBP , frees bindingsitefr 24  Rubisco is also inhibited by a “nocturnal” inhibitor 2-carboxyarabinitol 1-phosphate inhibits carbamoylated rubisco. – transition state analog of -keto acid intermediate – synthesized in the dark in some plants 25 Ca1P is transition state analogue t 2-carboxyarabinitol 1,5 bisphosphate 26 Rubisco is inhibited by Ca1P 27 Biosynthesis of Carboxyarabinitol 1-Phosphate Hamamelose – witch hazel (bark) 28 (Inh ). 29  Assembly of Rubisco Small subunit – SSU (14 kDa) nuclear encoded Large subunit – LSU (55 kDa) chloroplast encoded 30 31 from nurs Protein import translocon removes - 32 Rubisco: Bifunctional – carboxylase, oxygenase Affected by atmospheric conc. of gases Activated by CO2 + Mg2+ – distinct from catalysis Regulated by RCA - activated by ATP and light RCA removes Ca1P or RuBP from active site 2 subunits, nuclear and chloroplast encoded Correct assembly requires molecular chaperones (HSC60/HSC70) Most abundant enzyme on planet 33