Exam 2 biochemistry citric acid/ETC/Proton motive

Citric acid cycle enzymes

Amphibolic: both anabolism (build) and catabolism (break

Enzymes: officer can i keep selling seashells for money

• Oxaloacetate, citrate, isocitrate, a-ketoglu.,succinyl CoA, fumarate, malate

Purpose of CAC

Generate NADH and FADH2

• Stage 1: 2 carbons in by oxaloacetate then citrate forms to generate 2 CO2

• Stage 2: Oxaloacetate regenerated

Net products of catabolism of 1 acetyl CoA

3 nadh out

1 atp

1 fadh2

2 co2

Physical link between citric acid cycle and ETC

2 carbon acetyl unit from acetyl coa condense with oxaloacetate to form citrate

then ox to form NADH

• high energy electrons used later in ETC to reduce O2 (final e- acceptor) for h2o and ATP

Why oxygen required for CAC to function

O2 electronegative, e- acceptor

• Glycolysis is anaerobic (fermentation) for NAD+

• CAC proceed only when enough O2 for ETC proceed using NADH and FADH2 and releasing ox form (nad+ fad) of them

Citric acid cycle picture

All the citric acid cycle steps

Step 1: Citrate synthase (Ligase) - Irreversible

Step 2: Aconitase (Lyase) - Reversible

Step 3: Isocitrate Dehydrogenase (Oxidoreductase) - Irreversible

Step 4: α-ketoglutarate dehydrogenase complex (Oxidoreductase) -

Irreversible

Step 5: Succinyl CoA synthetase (Ligase) - Reversible

Step 6: Succinate dehydrogenase (Oxidoreductase) - Reversible

Step 7: Fumarase (Lyase) - Reversible

Step 8: Malate Dehydrogenase (Oxidoreductase) - Reversible

ATP equivalent from complex oxidation of carbohydrates

Monosaccharides same amt ATP can glucose (30-32)
Glycerol to G3P (16.5-17.5)
Alanine to pyruvate (enter cycle) (12.5)
Lactate to pyruvate (produce ATP) (12.5)

CAC irreversible step

Citrate synthase- irreversible
Isocitrate dehydrogenase- irreversible
a-ketoglu. dehydrogenase complex-  irreversible

• PDH controls glycose acyl coa entrance 

• Production NADH, ATP, succinyl: inhibit CAC

Positive inhibitors CAC

Isocitrate: ADP and CA2+
a-Ketoglu.": Ca2+

Negative inhibitors CAC

• the main electron carrier is inhibitor

Acetyl coa; NADH, succinyl CoA
Isocitrate: NADH, ATP
a-ketoglut: NADH, succinyl CoA

Intermediates in CAC anabolic biosynthesis

• A-ketoglu: amino acid synthesis

• Succinyl coa: heme synthesis

• Oxaloacetate: GNG and amino acid synthesis

CAC intermediates replenished

• Anaplerotic reactions: replenish

• precursors for biosynthesis(anabolism) and energy status

Pyruvate Carboxylase: synthesize oxaloacetate

Malic Enzyme: produce malate from amino acids

Glyoxylate cycle

Bypasses 2 decarboxylation steps, synthesize carbohydrate from fat
succinate convert to oxaloacetate then glucose

• Oil rich seeds (plants) organelles not in animals 

Glyoxylate cycle enzymes

Isocitrate lyase- reversible 

• split isocitrate to glyoxylate and succinate

Malate synthesis- irreversible

• condense glyoxylate with acetyl CoA form malate 

Electron Transport Chain and Ox phospho. occurs

Inner mitochondrial membrane

• ETC and ATP synthesis 

Redox potential

Molecule tendacy to donate or accept electrons

• strong reducing agent: donate electron

  • NADH, negative value

• strong oxidizing agent: accept electrons 

  • NAD+ and FAD, positive value

Electron carrier ETC

NADH: 2 e-

FADH2: 2 e-

Flavin mononucleotide: 2 e-

Iron sulfur protein: 1e-

Cytochrome C: 1 e-

Cytochrome Q/QH2: 1-2 E (split 2 e-)

Reduced vs oxidized electron carrier: ETC

Fe 2+ reduced
Fe 3+ oxidized
Coenzyme Q: Reduced has R
Coenzyme Q: Oxidized has double bond/Ch3/H
Flavin reduced: R and 2 H
Flavin oxidized: ch2-h-oh-h-oh-h-oh-ch2po3 2-

All 4 enzyme complex in ETC

1-NADH-Q Oxidoreductase

2-Succinate Q Reductase (dehydrogenase)

3-Q-Cytochrome c oxidoreductase

4-Cytochrome c Oxidase 

Complex 1-NADH-Q Oxidoreductase

• transfer electrons from NADH to Q

• Electron carriers FMN and Fe-S

• Proton pump: 4 out of matrix

Complex 2-Succinate Q Reductase (dehydrogenase)

• FADH2 reduce to Q and QH2 into Q

• Electron carriers FAD and Fe-S

• No proton pump

Complex 3-Q-Cytochrome c oxidoreductase

• Coenzyme Q binds to protons/electrons: entry point

• Allows electron to flow from Q to C

• Electron carriers Heme (3) and Fe-A

• Proton pump: 4 into intermembrane space

Complex 4-Cytochrome c Oxidase 

• Catalyze reduction of O2 to H2O

• Electron carriers: Heme (2) and Cu (2)

• Proton pump: 4 from cytochrome C

Flow of electrons from NADH vs FADH2 to O2

NADH to O2: More proton across membrane because electrons enters at complex 1

FADH2 to O2: less protons pumped across membrane because electrons enter at complex 2

Q cycle overview

Occurs in complex 3 (cytochrome complex) of ETC
funnels 2 electrons from Qh2 to 1 electron cytochrome c
proton motive force pump proton inner mitochondrial membrane 

Q cycle funnel electrons step 1

QH2 donate 2e and 2H

• 1e: Fe-S: Cyt c1: Cyt c

• 1e: cyt b: Q at Qi site

2H release to intermembrane space 

Q cycle funnel electrons step 2

QH2 donate 2e and 2H

• 1 e-: Cyt c

• 1e-: cyt b: cyt b: reduce Q-: QH2

2H+ released to intermembrane space and 2H matrix 

Coenzyme Q vs Cytochrome C

Coupling electron transfer from QH2 to cytochrome c
Electron accepter to catalyze reduction of O2 to 2 H2O

Advantages of Respirasome (etc in membrane)

Electrons transfer between non-contacting group

• Large enzymatic complex enhance enzyme

• proteins improve electron transfer

Disadvantages of Respirasome (etc in membrane)

Reactive Oxygen Species formed

• Reduction of O2 gives Superoxide ion, peroxide ion, hyroxyl ion

ETC in membrane

Exergonic reactions coupled to pumping protons in matix

• proton gradient built up and used to produce ATP 

Toxic oxygen species produced

Superoxide dismutase enzyme catalyze conversion of 2 O2 and 2H+ into H2O2 and O2

Peroxides produced by oxidases/Catalase heme protein catalyze H2O2 into h2o and o2

Goal of ETC

Transfer E from NADH/FADH to O2, creating proton gradient
certain toxins block e flow, stopping ATP and causing cellular hypoxia

Toxins inhibiting complex 1 ONLY

- Rotenone: Blocks e transfer from Fe-S centers

• NADH cannot donate e and ATP goes down

-Piericidine A: Competes with CoQ for binding site

• NADH cannot donate e and ATP goes down

-Amytal: inhibit NADH-CoQ reductae

• stops NADH oxidation early in ETC

Inhibitors complex 1,3,4

Complex 1: prevent NADH reoxidized, not succinate

Complex 3: interfere e flow and cyt bH

Complex 4: prevent e transport to O2 (most toxic)

Proton Motive force

Electrochemical gradient of protons across membrane

• delta H: chemical gradient (proton difference)

• delta y: electrical potential (charge difference)

Drives ATP synthesis by ATP synthase

Chemiosmotic Hypothesis

Energy from etc pump protons across inner mitochondrial membrane
Provides energy for ATP synthesis
Reaction coupling: E flow: H+ pumping: proton gradient: ATP synthesis

PMF experiment: Chemiosmotic hypothesis 

Artificial vesicles (liposomes) contained

• Bacteriorhodopsin: light driven proton pump

• ATP synthase: synthesis ATP

Observation

• Bact. did pump H into vesicle

• ATP synthase synthesized ATP without ETC 

SO: proton gradient drive ATP synthesis without ETC 

ATP Synthase function

Converts proton gradient energy into ATP
acts as rotary motor enzyme

• F0 complex membrane: proton channel

• F1 complex matrix: catalytic piece synthesize ATP 

Proton flow to c Ring rotation (ATP synthase)

Proton enter F0 through subunit A
Then bind to c-subunit in membranse
then c-ring rotate
then other channel releases proton into matrix

Rotation to change to ATP release (ATP synthesis)

Motion drives catalysis

• y shaft rotates in a3b3 catalytic headpiece

• changes B subunits in 3 states

  • Loose: binds ADP and Pi

  • Tight: Catalyze formation of ATP

  • Open: releases ATP 

Proton flow 

Mitochondrial shuttles

Mitochondrial pyruvate carrier

Malate transporter

Glycerol 3 phosphate shuttle

Malate aspartate shuttle

ATP-ADP translocase

Phosphate Translocase 

Mitochondrial shuttle: Mitochondrial pyruvate carrier

Takes pyruvate from glycolysis and move to mitochondria

Mitochondrial shuttle: Malate transporter

leaves mitochondrial matrix to generate oxaloacetate for GNG

Mitochondrial shuttle: Glycerol 3 Phosphate

carries electrons from NADH across mitochondrial membrane in tissue 

Mitochondrial shuttle: Malate Asparatate

carries electrons from NADH across mitochondrial membrane in heart and liver

Mitochondrial shuttle: ATP to ADP translocase

Exchange cytoplasmic ADP for mitochondrial ATP, energetically expensive

Mitochondrial shuttle: Phosphate Translocase

provide substrate for ATP synthesis in matrix and remove synthesized ATP

ATP net yield of mitochondrial shuttle

Glycerol 3 phosphate: 30 ATP
Malate aspartate: 32 ATP 

Oxidative phosphorylation ad oxygen consumption

Depends on substrates for ETC and ATP synthesis
Respiratory control: Electrons do not flow through ETC unless ADP convert to ATP

Succinate

Nonshivering Thermogenesis

ETC uncoupled from ATP synthesis, heat generated
Adults in brown adipose tissue

Thermogenin (uncoupling protein 1)

Protein in inner mitrochrondrial membrane facilitates uncoupling in regulated fashion

generate heat in PMF

Effect of enzyme in ETC and ATP synthase

ETC inhibit: prevent proton motive force
Uncoupling of ETC from ATP synthesis stopping
inhibition of ATP exporting 

Complex 3 inhibitor

-Antimycin A

• Block electron transfer from Cyt b to cyt c1