Electron Transport (oxidative phosphorylation)

Substrate level phosphorylation reaction: Glycolysis and TCA cycle

A ~ X + Pi + ADP → A + X + ATP

Energy rich intermediates donate P to ADP

Oxidative phosphorylation reaction - electron transport system

ATP Synthase: ADP + Pi → ATP

Photophosphorylation reaction: Photosynthesis

ATP synthase: ADP + Pi → ATP

Which type(s) of phosphorylation are based on the chemiosmotic theory?

Oxidative and photo

What tissues are rich in mitochondria

nerve, heart, and muscle cells, eye, and brown adipose tissue

Structural features of the mitochondria and their significance

Outer mitochondrial membrane

Intermembrane space

Inner mitochondrial membrane (highly selective and high surface area; rich in membrane proteins)

Mitochondrial matrix (rich in enzymes and other cofactors and host for the TCA cycle and other pathways

Contain genomic DNA and codes for 37 proteins

What are the 4 stages of cellular respiration and where do each of them take place?

Glycolysis (cytoplasm), PDH (mitochondrial matrix), citrate cycle (mitochondrial matrix), oxidative phosphorylation (inner mitochondrial membrane)

The chemiosmotic theory

H+ leaves matrix into intermembrane space through electron transport system; H+ enters matrix through ATP synthase complex to make ATP. Both embedded in inner membrane of the mitochondria. High H+ concentration in the intermembrane space.

What are the proteins of each complex?

I: NADH-ubiquinone oxidoreductase

II: Succinate dehydrogenase

III: ubiquinone-cytochrome c oxidoreducase

IV: cytochrome oxidase

V: ATP synthase complex

Electron donors

NADH and FADH2

Electron carriers

FMN/FMNH2-, ubiquinone (Q), Fe-S clusters, Cytochromes b, c, and a+a3

Electron acceptor

O2

Is electron transport exergonic or endergonic

Exergonic

Standard energy of change for electron transport equation

DG*’ = -nFDE*’

DE*” = E*’ e acceptor - E*’ e donor

Proton circuit analogy to electrical circuit

Battery: electron transport system

Capacitor: proton gradient

Resistor: ATP Synthase

Free energy from proton motive force reaction

DG = RTln(C2/C1) + ZFDfork

What is the P side and N side referring to in the complexes

P side is the intermembrane space side, N side is the mitochondrial matrix side

Complex I NADH-ubiquinone oxidoreductase net reaction

NADH + H+ + Q + 4H+N → NAD+ + QH2 + 4H+P

Complex I three steps

1. NADH transfers 2 e- to FMN

2. 2 e- transferred from carrier (FMN) to carrier (FE-S)

3. 2 e- + H+ bind Q, forming QH2

Complex II succinate dehydrogenase net reaction

FADH2 + Q → FAD + QH2

Succinate dehydrogenase function in TCA cycle and electron transport chain

second entry point for electrons; oxidation of succinate to fumarate and reduction of FAD to FADH2; donates electrons to coenzyme Q to QH2; only a complex that doesn’t pump protons

Coenzyme Q reduction

Ubiquinone (Q) is reduced to Ubiquinol (QH2)

Complex III Ubiquinone-cytochrome C oxidoreductase

Docking site for QH2 and Cyt C; contains binding sites for ubiquinone (Qp and QN); transfers e- through Fe-S cluster center onto cyt C; reduces cyt c while translocating 4 H+

Where does the Q cycle occur? What is reduced? What happens to cytochrome c? Where do electrons come from?

Occurs in complex III. Cytochrome c is reduced in the process, it transports 1 e- from complex III to IV. This converts 2e- transport process into two separate 1e- transfers. Electrons come from CoQ (two QH2 participate, one QH2 is oxidized and the other returns to the fully reduced form)

Where is cytochrome c located? What does it do?

Localized to the intermembrane space; highly conserved mobile electron carrier; transports 1 e- from III to IV. Involved in electron transport and apoptosis

Complex IV Cytochrome c oxidase net reaction

2 Cyt C (red) + 4H+(n) + 1/2O2 → 2 Cyt C (ox) + 2H+ (p) + H2O

How many electrons can cytochrome c oxidase accept at a time? What is oxidized and reduced?

1; cytochrome c is oxidized and oxygen is reduced to water

Memorize one inhibitor for each complex minimum

I: rotenone

II: none!

III: Antimycin

IV: Hydrogen Cyanide/Carbon monoxide

V: Oligomycin

What is Complex V consisted of (large structural components)

F0 - acts as a proton channel crossing the inner mitochondrial membrane

F1: encodes the catalytic activity and synthesizes ATP

What are the 3 detailed structures of ATP synthase complex and their subunits

Rotor: y, ring and e subuntis

Headpiece: a3b3 units, b3 units (catalytic sites for ATP synthesis)

Stator: stabilizing arm (immobile)

F1 conformational change forms

L→T→ O; loose → tight → Open

What are the ATP states during each of these conformations

L= b-ADP

T=b-ATP

O= b-empty

At what angle does the rotor rotate

120

How many ATP is synthesized for 4H+?

2.5 ATP

what is the primary shuttle in liver cells

MAS; malate aspartate shuttle

what is the primary shuttle in muscle cells

GPS; glycerol-3-phosphate shuttle

MAS transport electrons from Cytosolic NADH to ______

mitochondrial NAD+

GPS transports electrons from cytosolic NADH to ____ using ____

mitochondrial matrix; FAD

Why is the supply of NAD+ maintained?

Glycolysis

How many ATP are generated per glucose for MAS

32 in liver

How many ATP are generated per glucose for GPS

30 in muscle

How do electrons from NADH go into ETS in GPS?

Through coenzyme Q

GPS contains 2 isozymes of glycerol-3-phosphate dehydrogenase, what kinds of isozymes are they?

Cytosolic and mitochondrial

Between muscle and liver cells, which process yields more ATP per glucose? Why? In which step does it happen?

Liver cells, because of MAS. This gives 5 ATP for 2 NADH, whereas GPS gives 3ATP for 2NADH. PDH (5ATP) and TCA (20 ATP) are the same

Which steps regulate oxidative phosphorylation

glycolysis steps 3 and 10, PDH, TCA cycle steps 1,3,and 4, oxidative phosphorylation

Inhibitors and activators of oxidative phosphorylation (allosteric)

ATP and NADH (inhibitors)

AMP and ADP (activators)

coordinated regulation of glycolysis, pyruvate oxidation, citric acid cycle and oxidative phosphorylation by ATP

What are the 2 uncouplers that cause a “short circuit”

2,4-DNP - synthetic

UCP1 protein - natural

What is a proton blocker, stopping ATP synthesis?

Oligomycin

How does DNP affect oxygen consumption and ATP synthesis

significantly increases ATP consumption and ATP synthesis over time. DNP translocates H+ using alternative path “short circuit”

What does uncoupling mean?

electron transport and atp synthesis are disconnected

What does UCP1 do?

Uncouples

What happens when UCP1 is overexpressed

ATP production reduced

Redox energy converted into heat production

Necessary for hibernating animals (bears)

non-shivering thermogenesis in human

found in brown adipose tissue, generates heat not ATP

Synthetic uncoupler (DNP)

weight loss, very dangerous medication

inherited mitochondrial diseases in humans cause____. They usually originate in _____ cells and ______ cells.

decreased atp production; neuronal cells and skeletal muscle cells