Biochem midterm #3

evolution of photosynthesis

• Oxygenic photosynthesis generates O the atmosphere

• Plants MAKE oxygen which heterotrophs USE for cellular respiration

• Rise of aerobic respiration occurred after plants made O2

Autotroph - “Self-feeders”

Take simple molecules and make macromolecules to feed themselves and other organisms

Overview of photosynthesis + split into what?

• Convert light energy into chemical energy

• Plants take in CO2 and water, produce carbohydrates, and release O2

Split into

1. Light dependent reactions

   1. NADP+ → NADPH

2. Carbon-assimilation (carbon fixation) reactions

   1. ATP → ADP + Pi

   2. CO2 → Triose phosphates

Compare/contrast chloroplast structure to the mitochondria. How does compartmentalization drive function?

• Double mem

• Stroma - like the mitochondrial matrix

• Thylakoids - membrane-filled spaces (lumen)

• Proton Pumping Direction:

  • Mitochondria: Protons are pumped OUT of the matrix into the Intermembrane Space.

  • Chloroplast: Protons are pumped IN from the stroma into the Thylakoid Lumen

explain proton pumping direction for photosyn vs. cell resp

Mitochondria: Protons are pumped OUT of the matrix into the Intermembrane Space.

Chloroplast: Protons are pumped IN from the stroma into the Thylakoid Lumen.

properties of excited electrons

caused when a photon hits a ground state e-

• Increased potential energy

• Transfer energy (excitons)

• Easier to transfer (oxidation)

Things that can happen from excited→relaxed state

1. Heat

2. Fluorescence

3. Energy transfer to neighboring P molecule

4. Redox reactions

Pigments definition

Molecules that absorb specific wavelengths of light

chlorophyll structure

• contain a porphyrin ring with conjugated double bonds

  • Conjugated = alternating single and double bonds

  • Absorb photons within the visible spectrum

• Minimize energy loss through thermal vibration (de-excitation)

which wavelengths/colors do chlorophylls absorb well

Chlorophyll absorbs well in blue/red regions, reflects green

• 450 and 650 nm preferred

Accessory pigments help absorb the others

accessory pigments

extend the range of light absorption

absorption spectrum correlation with photosynthesis

Light Harvesting Complexes location

thylakoid membrane

pigments location

bound to Light Harvesting Complexes within the thylakoid membrane

Antenna complex

• provides a network of pigment molecules to capture and transfer energy.

• No matter where the light hits, the light can be transferred to neighboring molecules

• Will eventually hit the reaction center → like a funnel to the reaction complex

Reaction center

• pigments where absorbed light energy drives electron transfer

• Antenna complex leads to the reaction center

• Reaction center chlorophyll enables efficient redox reaction

• Light excites and relaxes e- in antenna molecules until it reaches the reaction center and excites it

Photosystems

• energy harvesting proteins that facilitate transfer of excited electrons

• multiple Light Harvesting Complexes (LHC) that direct energy to the core complex.

  • Separate subunits that contribute to the photosystem

• Energy is funneled to the chlorophyll special pair in the reaction center.

  • Middle of the photosystem has the reaction center

reaction center structure

Reaction center chlorophyll enables efficient redox reaction

Excitation causes a separation of charges in the reaction center and initiates a redox chain.

Photosystem II location

within thylakoid membrane

Photosystem II function

• first protein complex in light-dependent reactions of photosynthesis

• Reduce PQ

  • Special pair passes e- up to PQ

  • PQ = plastoquinone

• OEC - oxygen evolving complex

  • Take e- from H2O (oxidize) → produce oxygen

  • Replace the e- in the reaction center chlorophyll with the e- from H2O

Oxygen-evolving complex in photosystem II (OEC)

• Sends the e- from the H2O to the reaction center chlorophyll

  • Take e- from H2O (oxidize) → produce oxygen

  • Replace the e- in the reaction center chlorophyll with the e- from H2O

• manganese ions (Mn)

  • Mn complex oxidizes H2O

  • In lumen (inside of the thylakoid

• Every 4 flashes/excitations → produce 1 O2

  • Water re-reduces Mn atoms in OEC in a single step to produce O2

  • Mn center donates e-

  • Getting excited + pulling e- off of water

photosystem II importance/facts/structure

• first protein complex in light-dependent reactions of photosynthesis

• special pair = P680

• includes Phe and PQ

Cytochrome b6f complex

COPY AS COMPLEX III in ETC -

• moves H+ from stroma to lumen

• Q cycle type thing for PQ instead

Problem:

• Plastohydroquinone (PQH 2) transports 2 e-

• plastocyanin (PC) can only transport one e- at a time.

flow of e- in thylakoid membrane light reactions

1. H2O

2. photosystem II

3. cytochrome b6f complex

4. photosystem I

   1. Fd shuttle

5. NADP reductase - produce NADPH

Plastoquinone

Same as ubiquinone :D

Transports 2 e-

2-step PQ cycle - steps + results

• steps

  • first QH2 oxidized, Q- produced

  • second QH2 oxidized, regen another QH2 from the Q- radical

• Overall

  • Remove 2 H+ from the stroma

  • Pump 4 total H+ into the thylakoid lumen

  • Move both electrons from QH2

Photosystem I facts

• Steps

  • Accepts e- from PC

  • Excite e-

  • Transfer e- to Ferredoxin (Fd)

• Ferredoxin (Fd)

  • Shuttles e- NADP Reductase to produce NADPH.

  • Shuttle e- to Cyt b6f complex to produce ATP

Ferredoxin (Fd)

• shuttles between photosystem I to NADP reductase

  • producs NADPH

• ALSO - Shuttle e- to Cyt b6f complex - makes H+ gradient to produce ATP

Z-scheme in light reactions

• Tracking e-

  • Increase in E when e- are excited at the photosystems

  • Small drops in E when transferring carriers

• Photosystems don’t pump any H+, only cytochrome b6f complex

• Fd can go to FNR → NADPH

  • OR back to cytochrome b6f complex

what can pump H+ in thylakoid membrane light reactions

only cytochrome b6f complex

Phosphorylation step of thylakoid membrane light reactions

• light generates a proton gradient that drives ATP synthase

  • via cyt b6f complex

• ATP synthase SAME structure

  • H+ move from LUMEN to STROMA

  • 12 H+ drive full rotation

what direction do H+ move in thylakoid membrane light reactions?

cyt b6f - stroma to lumen

ATP synthase - lumen to stroma

linear flow of e- products (light reactions)

1 ATP and 1 NADPH produced

energy requirement of calvin cycle to build sugars?

9 ATP 6 NADPH - 3:2 ratio of ATP to NADPH

• Need a proton gradient to make ATP

  • Need more ATP than NADPH

• Solution: Fd → decision making point, it can transport e- to make NADPH or deliver it back to Cyt b6f

2 systems of e- transport for prep for Calvin Cycle

Plants utilize both to maintain 3:2 ATP:NADPH required for carbon fixation.

1. Noncyclic electron transport

   1. Photosystem II and Photosystem I are required

   2. results in production of NADPH and ATP

2. Cyclic electron transport

   1. Photosystem I

   2. results in production of ATP but NOT NADPH

Noncyclic electron transport

• Photosystem II and Photosystem I are required

• results in production of NADPH and ATP

• electrons flow from water —> PSII —> cyt b6f —> PS1 —> NADPH

Cyclic Electron Transport

• only Photosystem I required

• results in production of ATP but NOT NADPH

• Fd shuttles back to cyt b6f complex to pump H+

  • used by ATP synthase to make ATP

  • no NADPH made because doesn’t pass by PSII

how are the light reactions regulated?

appressed and nonappressed thylakoids

appressed thylakoids

• Stacked tight

  • Happens when they’re being hit by a LOT of light

  • Intense production of H+ gradient

• Packed with PSII and LHC (light harvesting complexes) to optimize light capture

• LHC mediates connection between membranes

nonappressed thylakoids

• Unstacked regions with access to stroma (ferredoxin, NADP+, ADP)

• PSI and ATP synthase

• More stroma access → more ATP synthase

how do the thylakoids stack/appress?

• dynamic in response to light

  • Controlled by covalent modifications - phosphorylation

• LHC mediates the connection between membranes

  • Span thylakoid membrane

  • Thr-OH residue anchors it

  • Phosphorylation of the Thr residue releases it to nonapressed state

• LHC are mobile and can help funnel energy to PSI when PSII is more active

Stoichiometry of CO2 assimilation in Calvin Cycle

• For every 3 CO 2 molecules fixed:

  • 9 ATP and 6 NADPH are consumed

  • 1 glyceraldehyde 3-phosphate is ‘produced’.

    • 6 are acutally made, but 5 are re-used for the cycle

• Remember: Plants balance cyclic and non-cyclic electron transport to maintain this 3:2 ATP:NADPH ratio.

stage 1 calvin cycle

CO2 fixation to 3-phosphoglycerate (3-PG)

3 R1,5BP + 3 CO2 —> 6 3PG

• uses RuBisCO - Ribulose 1,5-bisphosphate carboxylase/oxygenase

• Very slow: Large amounts are needed to achieve high carbon fixation rates.

• ~50% of soluble protein in chloroplasts

• Forms alternative products in the presence of oxygen

  • If O2 binds, there will be a reaction still

what is RuBisCO + what’s it used for

• Ribulose 1,5-bisphosphate carboxylase/oxygenase

• most abundant protein (enzyme) on earth

• fixates CO2 onto ribulose 1,5-bisphosphate —> 3PG

Carboxylase activity of RuBisCO

• CO2 is a very poor electrophile – R-1,5-BP needs to be activated to glycerate attack it.

• Enediolate intermediate → extremely reactive, used to activate R1,5BP

  • So that the reaction can more easily proceed

• Problem: once it is primed for attack, it cannot discriminate between CO2 and O2

  • Solution: plants have evolved different mechanisms to increase [CO2] in leaf tissue

stage 2 of calvin cycle

reduce 6 3-PG —> 6 G3P

• uses 6 ATP and 6 NADPH

• Note: we need 5 G3P to regenerate RuBP

  • 1 G3P is used to make other stuff like glucose, cell wall, etc

• Phosphoglycerate kinase

  • Uses ATP to convert 3PG → 1,3 BPG

Phosphoglycerate kinase

Uses ATP to convert 3PG → 1,3 BPG + NADPH —> G3P

• stage 2 of calvin cycle

stage 3 calvin cycle

regeneration of 3 ribulose-1,5-bisphosphate using 3 ATP

3 ATP + 5 G3P —> 3 R1,5BP

• Problem: RuBP is a pentose (5C), but G-3-P and DHAP are 3C.

  • Take home: Triose phosphates interconvert to form pentose phosphates.

• Note: Blue arrows are exergonic steps that make the entire process irreversible.

  • G3P and DHAP transfer C’s between each other to make a 5

• ribulose -5-phosphate kinase

  • Uses ATP to convert ribulose-5-phosphate → ribulose-1,5-bisphosphate

ribulose -5-phosphate kinase

used in generation of R1,5BP

• Uses ATP to convert ribulose-5-phosphate → ribulose-1,5-bisphosphate

how are Enzymes in Calvin Cycle are indirectly activated by light

Remember: the thylakoids shift between the appressed and nonappressed states

• Thylakoid lumen can regulate the amount of Mg

  • This changes the amount of RuBisCO active sites

• Increasing Mg2+ increases RuBisCO activation

• Fructose 1,6-bisphosphatase (Stage 3) is pH dependent and requires Mg2+.

  • In light conditions, its activity increases more than 100- fold.

When chloroplasts are illuminated:

1. Stromal [Mg2+] increases (2-3mM → 6-8mM)

2. Stromal pH increases (ph 7 → pH 8)

What happens when chloroplasts are illuminated? impact on calvin cycle?

• stromal Mg2+ and pH increase

  • activity of stage 3 enzyme fructose 1,6 bisphosphatase + RuBisCO INCREASE

what are the 4 fates of glucose?

1. synthesis of structural polymers - cell wall, matrix

2. storage - glycogen, starge, sucrose

3. PPP - form ribose 5-phosphate

4. glycolysis - pyruvate + reverse=gluconeogenesis

list of tissues diff function for glucose fate + takehome

1. brain - depends on blood sugar

2. liver - 90% of GNG

3. skeletal muscle - glycolysis

4. kidney - 10% of GNG

brain usage of glucose

• High-priority glucose user.

• No glycogen stores

• cannot do GNG.

• Depends on blood sugar.

liver glucose usage

• Stores glycogen to share and performs

• 90% of GNG to maintain blood sugar

• Provides the blood glucose for the brain and other parts of the body

skeletal muscle glucose usage

• Burns glucose for ATP.

• Stores glycogen but won’t export glucose (won’t share with anyone else, cannot give glucose to other organs like brain)

• Mostly performs glycolysis using the glycogen

• Will export lactate under anaerobic conditions.

kidney glucose usage

Performs about 10% of GNG, mostly during prolonged fasting.

Cori Cycle

• liver does GNG to produce blood sugar

  • blood sugar used by brain and muscles

• skeletal muscle uses the glucose in glycolysis

  • Uses O2 and glucose so quickly → switches to anaerobic respiration

  • Muscles export lactate into blood → liver uses it to make glucose

how are sugars catabolized?

• Glycolysis is central to all of metabolism

• Each mono or disaccharide does not have its own catabolic pathway

  • They feed into glycolysis in the shortest path possible

  • Digesting carbohydrates → try to get it into a form that is a glycosidic intermediate

  • If you convert into a glycosidic intermediate → can be digested via glycolysis and harvest E

• Polysaccharides are also broken down into monosaccharides that feed into glycolysis

marcomolecular structure of carb polymers

1. Macromolecular structure

   1. Cellulose - linear

   2. Starch - branched

   3. Glycogen - highly branched

2. Types of glycosidic linkages differ, as do the branching patterns

Glycogenesis overview

makes glycogen

• 20% liver, 80% muscle

• Solves a key osmotic problem for cells:

  • If stored as free glucose, the amount of water required would burst the cell!

  • Enables parallel processing:

  • thousands of non-reducing ends can be cleaved simultaneously when the cell needs glucose

glycogen

• produced in glycogenesis

• main storage polysaccharide in animal cells

• alpha 1-4 linked glucose subunits

• heavily BRANCHED

  • alpha 1-6 branches

• contains glycogenin protein seed to start polymers

glycogenin

protein core = “seed” in the center of glycogen that can help start polymers

how do we prime glucose for glycogenesis

activate it

1. Isomerize to glucose-1-phosphate Building a polymer of (α1→4)-linkage

   1. move phosphate from 6C to 1C for the a1—>4 linkages

2. add uracil phosphate —> UDPG

   1. adds another phosphate group to the C1 phosphate

why do we add P and UP to glucose?

we add phosphate and uracil phosphate —> UDPG

1. Binding Energy:

   1. the nucleotide group forms favorable interactions with enzymes, and the free energy of nucleotide group binding enhances catalytic activity.

2. Reactivity:

   1. the UDP is a good leaving group. The attached sugar carbon is activated for nucleophilic attack.

3. Separate Pools:

   1. tagging hexoses with nucleotides sets them aside for biosynthesis separate pools are available for energy production and storage.

what is the role of sugar nucleotides in biosynthesis

Making sugar nucleotides is a common substrate for polymerization

• Formation of sugar nucleotides is highly exergonic and essentially irreversible, driven by the large free energy of PPi hydrolysis.

  • Break off the PPi

  • Sugar phosphate attacks the NTP → removing the PPi

• PPi

  • Concentration of pyrophosphate very low

  • Very high energy

Glycogen synthesis (glycogenesis) steps by glycogenin

Glycogenin adds the first 7 glucose molecules

Remains covalently bound to the reducing end of the completed glycogen molecule

1. UDP-glucose

   1. Tyr on glycogenin attaches to C1 of UDP-glucose

2. Glucosyltransferase adds another UDP-glucose

3. C4 of the 1st UDP-glucose attaches to C1 of another one

4. repeats 6 times

glycogen synthase

• catalyzes alpha 1,4 linkages

• Glycogen synthase does NOT do BRANCHING, just lengthens the chain

Glycogen-branching enzyme

• Synthesizes alpha 1-6 linkages → BRANCHING

• Glycogen-branching enzyme catalyzes:

  • transfer of a terminal fragment (6 or 7 residues long) from the nonreducing end of a branch to the C-6 hydroxyl group of a glucose residue on the same chain or another chain

  • creating a branch with an (α1→6) linkage

  • Cut alpha 1-4 link and make it alpha 1-6

• Have 2 nonreducing ends → can add more reducing glucose to these ends

overall steps of glycogenesis

1. take glucose 6-phosphate —> glucose 1-phosphate

2. glucose 1-phosphate + uracil-phosphate —> UDPG

3. 7 UDPG connect using glycogenin

4. Glycogen synthase extends linearly a1-4

5. glycogen branching enzyme adds branches a1-6

Phosphorolysis overview

• degrade glycogen

• Formation of a phosphate ester preserves some bond energy

• uses 3 enzymes

  • glycogen phosphorylase

  • transferase

  • glucosidace

glycogen phosphorylase

• degrades glycogen

  • processive enzyme - catalyzes consecutive reactions without releasing its polymeric substrate

• Sequentially removes terminal residues → G1P

• Stops 4 residues from branch point

processive enzyme

catalyzes consecutive reactions without releasing its polymeric substrate

Transferase

• glycogen breakdown

• debranching enzyme

• shifts a block of 3 glucose residues to nonreducing end of the same or a different glycogen molecule

  • leaves 1 glucose behind

glucosidase

• glycogen breakdown

• debranching enzyme that resolves

  • a(α1→6) linkage → glucose (not G-1-P)

reciprocal regulation of glycogen metabolism

Glycogen phosphorylase (breakdown) and glycogen synthase (production)

• Opposing pathways → if one is activated, the other is inhibited

  • Glucagon = low energy, need to increase blood sugar

  • Glucose = high energy

  • The conditions that activate glycogen synthesis deactivate glycogen breakdown

• phosphorylase

  • inhibited by glucose, insulin(high energy, need less glucose)

  • activated by glucagon (low energy, need more glucose)

• synthase

  • inhibited by glucagon (low energy, need to store less)

  • activated by glucose, insulin (high energy, store and make glycogen)

why can’t we just reverse glycolysis to make glucose?

• In the cell, steps that are close to equilibrium are reversible

• Highly exergonic steps are irreversible (1, 3, 10)

bypass reactions

The three irreversible reactions of glycolysis are bypassed to synthesize glucose from pyruvate in gluconeogenesis

• Use a different set of enzymes

• Are exergonic and effectively irreversible

• Are reciprocally regulated

3 bypass reactions:

1. First bypass: (step 10 glycolysis)

   1. Pyruvate → phosphoenolpyruvate

2. Second bypass: (step 3 glycolysis)

   1. Fructose 1,6-bis-P → fructose 6-phosphate

3. Third bypass: (step 1 glycolysis)

   1. Glucose 6-phosphate → glucose

First Bypass overall reaction and 2 pathways

Pyruvate → phosphoenolpyruvate

1. Pyruvate into matrix → oxaloacetate → malate → export out into cytoplasm using malate-aspartate shuttle → oxaloacetate

   1. Have to move the reducing equivalents from matrix into cytoplasm

   2. NADH

2. Lactate in cytoplasm → oxidize into pyruvate → export into matrix

   1. Use NAD+ to lactate → pyruvate + NADH (in cytoplasm)

   2. Lactate better because NADH generated DIRECTLY in cytosol

   3. Cori cycle → USED BY LIVER

3. Both pathways are required to balance redox equivalents and PEP to start gluconeogenesis

   1. Overall: energy expensive

      Requires 2 ATP to synthesize 1 PEP

      In glycolysis, we gain 1 ATP from 1 PEP

problem about the first bypass

• Pyruvate is imported into the mitochondrial matrix, but the rest of our pathway is in the cytoplasm.

• Pyruvate carboxylase produces OAA that is available for anabolic reactions.

  • Produced in anaplerotic reaction

  • Other OAA is consumed in CAC

  • Also part of the shuttling system → malate-aspartate shuttle

• Malate-aspartate shuttle → moves the malate + NADH out of the mitochondria

• requires NADH - which has a low concentration in cytosol

Step 1: Pyruvate → oxaloacetate for 1st bypass in GNG

1. First, pyruvate is transported into the mitochondria

2. Pyruvate carboxylase is the first regulatory enzyme in these pathways and is stimulated by acetyl-CoA. Why?

   1. High acetyl-CoA = energy rich, CAC not running = ready for anabolism to store energy

   2. Pyruvate oxidation = makes acetyl CoA

3. Biotin - swings between 2 active sites to place the CO2

4. Add carboxylic acid to pyruvate = oxaloacetate

   1. Costs 1 ATP

Step 2: oxaloacetate (OAA) → PEP in GNG

• Happens either in cytosol or in mitochondria

  • Longer pathway in cytosol

  • Shorter pathway in mitochondria

• PEP is the highest-energy phosphorylated intermediate in the cell

  • requires two highly exergonic steps to drive its synthesis.

  • Use 1 GTP

first bypass is highly regulated