ATP is the cell’s “energy” BUT –Cells also have….REDUCING POWER! Processes (such as photosynthesis) require NADPH as well as ATP NADH and NADPH are NOT interchangable Pentose Phosphate Pathway Hexose monophosphate (HMP) shunt / Phosphogluconate pathway.

NADH and NADPH are NOT interchangable NAD + participates in synthesis of ATP glycoloysis, oxidative phosphorylation NADPH is a reducing agent produced in light reactions and consumed in Calvin cycle of photosynthesis NADP + + 2H ---> NADPH + H + In the cell… [NAD+] ~ 1000 [NADP+] ~ 0.01 [NADH] [NADPH]

1C5 to 1C5 2C5 to 2C5 2C5 to 1C7 + 1C3 3C6 3C5 3C1 1C7 + 1C3 to 1C6 + 1C4 1C4 + 1C5 to 1C6 + 1C3

Summary of carbon skeleton rearrangements in the pentose phosphate pathway. 3C 6 ---> 3C 5 + 3C

3 ribulose-5-P ---> 2 xylulose-5-P + 1 ribose-5-P

Transketolase: catalyzes the transfer of C2 units

C3C3 C7C7 CH 3

Transaldolase: catalyzes the transfer of C3 units

C7C7 C4C4 C6C6 C3C3

C5C5 C3C3 C4C4 C6C6

Summary of the pentose phosphate pathway 3G6P + 6NADP + + 3H 2 O 6NADPH + 6H + + 3CO 2 + 2F6P + GAP Important intermediates Ribose-5-phosphate (nucleic acids, histidine) Erythrose-4-phosphate (aromatic amino acids)

What is the purpose of the pentose phosphate pathway? 1)Biosynthetic precursors 2)NADPH for biosynthesis 3)NADPH to keep cell reduced

O 2 + 2e - + 2H > H 2 O 2 H 2 O 2 + 2e - + 2H > 2H 2 O O 2 + 4H + + 4e > 2H 2 O Eº (V) vs. NHE Oxygen Biochemistry Reduction of O 2 or H 2 O 2 can be used as a thermodynamic driving force to drive oxidation of various molecules

O 2 + 2e - + 2H > H 2 O 2 S ----> S e - O 2 + 4e - + 2H > 2H 2 O 2S ----> 2S e - H 2 O 2 + 2e - + 2H > 2H 2 O S ----> S e - Oxidative Damage

Peptide and phosphodiester cleavage Iron-sulfur cluster disassembly

Oxygen Diradical OO 1s 2s 2p x 2p y 2p z 1s 2s 2p x 2p y 2p z  2s  2s*  1s  1s*  2p x  2p x *

3 O 2 (up/up) + 1 X (paired) ---> 1 XO 2 (paired) 1 O 2 (paired) + 1 X (paired) ---> 1 XO 2 (paired) 3 O 2 (up/up) + 3 X (up/up) ---> 1 XO 2 (paired) Need to alleviate spin restriction

O 2 + e > O 2 - O e - + 2H > H 2 O 2 H 2 O 2 + e - + H > H 2 O + OH OH + e - + H > H 2 O O 2 + 2e - + 2H > H 2 O 2 H 2 O 2 + 2e - + 2H > 2H 2 O O 2 + 4H + + 4e > 2H 2 O Eº (V) vs. NHE

Homolytic peroxide cleavage

Heterolytic peroxide cleavage: The Fenton Reaction Eº = V Catalyzed by metals like iron and copper

OH + RH ----> H 2 O + R R + O > ROO RH + ROO ----> R + ROOH

Antioxidants

Initiation X > 2X X + RH ----> XH + R Propagation R + O > ROO ROO + RH ----> ROOH + R Termination R + ROO ----> ROOR R + R ----> R 2 ROO + ROO ----> ROOOOR ----> O 2 + ROOR Free Radical Chain Reactions X = OH, O 2 -, O 2

If R = lipid The E/C couple Termination R + EH ----> RH + E ROO + EH ----> ROOH + E Recovery AH - + E ----> A - + EH A - + E ----> A + EH A + NADPH ----> AH - + NADP + 1/6Glucose + NADP > 1/3CO 2 + NADPH DHAR DHAR = dehydroascorbate reductase PPP = pentose phosphate pathway PPP or Photosynthesis

If R = soluble, C or GSH Termination R + AH > RH + A - ROO + AH > ROOH + A - 2A - + H > AH - + A Recovery A + NADPH ----> AH - + NADP + 1/6Glucose + NADP > 1/3CO 2 + NADPH Termination R + GSH ----> RH + GS ROO + GSH ----> ROOH + GS 2GS ----> GSSG Recovery GSSG + NADPH + H > 2GSH + NADP + 1/6Glucose + NADP > 1/3CO 2 + NADPH

Peroxide reduction Eº = V Can be used to extract hydrides from substrates

Oxygen reduction Eº = V Can be used to extract hydrides from substrates

Acetyl-CoA Some Bacteria/Plants CO 2 fixation Fungi/plants

Extant ways of fixing CO 2 Reductive TCA cycle Calvin Cycle Acetyl-CoA Synthase

Reversing the TCA Cycle Pyruvate ∆G ~ 0 ∆G <<< 0

How do you reverse KGDH? Ketoglutarate synthase 2-oxoglutarate:ferredoxin oxidoreductase Photosynthetic bacteria Anaerobic bacteria

What about isocitrate dehydrogenase? This step can be made reversible if you use a different source of electrons. Use NADPH instead of NADH.

Citrate lyase

Pyruvate synthase Acetyl-CoA + CO 2 ---> pyruvate Pyruvate:ferredoxin oxidoreductase Photosynthetic bacteria Anaerobic bacteria

Furdui, C. et al. J. Biol. Chem. 2000;275: Other bacteria

The Calvin cycle. 3CO > GAP 9 ATP and 6 NADPH

3C5 3C1 6C3 1C3 C6 C3+C3 C3+C4 C6+C3 C5 C4 C7+C3 C7 C5

Most important enzyme is Ribulose-5- phosphate carboxylase (Rubisco)

Transketolase: catalyzes the transfer of C2 units Aldolase: catalyzes the condensation of C3 ketoses with aldoses

C3 + C3 ---> C6 C3 + C6 ---> C4 + C5 C3 + C4 ---> C7 C3 + C7 ---> C5 + C5 Overal reaction = 5C3 ---> 3C5 1 GAP molecule is made from 3CO 2 3CO 2 + 9ATP + 6NADPH ---> GAP + 9ADP + 8P i + 6NADP + GAP is converted to glucose by gluconeogenesis

C3 + C3 = C6 Aldolase Reverse of the step in glycolysis

C3 + C6 = C4 + C5 Transketolase

Acetyl-CoA synthase The Wood-Ljungdahl Pathway 2 CO > Acetyl-CoA

Western Branch Eastern Branch

CH 3, CH 2 OH, and CHO transfer

Corrinoid CH 3 transfer

Formate dehydrogenase Can also use Mo H- from NADPH

CODH: carbon monoxide dehydrogenase

Acetyl-CoA synthase

Assimilating Acetyl-CoA:The glyoxalate cycle

Acetyl CoA Citrate Synthase Oxaloacetate Claisen condensation Ligase

Aconitase: lyase

Isocitrate lyase

Malate synthase

Malate Dehydrogenase

Acetyl-CoA Some Bacteria/Plants CO 2 fixation Fungi/plants

Pyruvate AA’s AA’s, Acetyl-CoA AA’s FA’s, AA’s

Malate Pyruvate Malate dehydrogenase/cytosolic Oxaloacetate Glycolytic intermediates Pyruvate carboxylase PEP Carboxykinase

∆G ~ 0 ∆G <<< 0 ∆G ~ 0 ∆G <<< 0

Pyruvate Carboxylase

Fructose-1,6-bisphosphate + H 2 O ---> fructose-6-phosphate + P i ∆G’º = kJ/mol Glucose-6-phosphate + H 2 O ---> glucose + P i ∆G’º = kJ/mol