Citric acid cycle & ETC

what are the two main functions of Coenzyme A?

1) activation of the acyl groups for transfer by nucleophilic attack

2) activation of the alpha-hydrogen of the acyl group for abstraction as a proton

Identify these three points in the metabolic map

biological energy movement starting from sunlight to photosynthesis

What is the central role of the citric acid cycle?

to generate reducing equivalents:

• OXIDIZE acetyl-CoA to generate ATP (energy) and NADH / FADH2 (high energy electron carrieres)

Other than glucose — glycolysis —> pyruvate, what other molecules can become acetyl-CoA for the TCA cycle

Fatty acids and amino acids

in cellular respiration, where is the site of citric acid cycle vs. oxidative phosphorylation

inside mitochondrial matrix for TCA and through membrane for oxidative phosphorylation

name the enzyme classes in order starting from citrate

Is Orange Orange Truly Orange LOL

1. isomerase

2. oxidoreductase

3. oxidoreductase

4. transferase

5. oxidoreductase

6. lyase

7. oxidoreductase

8. lyase

—> first step starts at 8

What are the products of oxidizing one Acetyl CoA?

ATP, 2CO₂, 8e^-

chemical structure of acetyl CoA?

Describe the first step of TCA cycle & spontaneity

big negative delta G

• acetyl Co-A is condensated with oxaloacetate to form citrate

• control point

• catalyzed by lyase citrate synthase, an allosteric enzyme that is inhibited by NADH, ATP, and succinyl-CoA

• citrate synthase removes proton from CH3 group of acetyl-CoA —> negative charge forms bond to carbonyl carbon of oxaloacetate

• reaction driven strongly forward by hydrolysis of CoA

Describe the second step of the TCA cycle & spontaneity

positive delta G

Isomerase (aconitase, iron-sulfur cluster) catalyzes reaction where OH group (as water) is removed from citrate and then added to the neighbouring carbon.

aconitase:

Fe2+ binds to vacant position of the cluster —> activates catalyst

added iron atom coordinates the citrate C-3 carbonxyl and hydroxyl groups and acts as lewis acid, accepting electron pair from hydroxyl group making it better leaving group

How and why are redox-active metals tightly regulated in the cell?

regulated by labile pools, enzymes, chaperones, storage, and subcellular organelles

can cause oxidative stress:

Describe step 3 of TCA cycle & spontaneity

small negative delta G

• control point in the cycle: linked to ET chain by NADH production

• the first (1/4) oxidation steps in the cycle

• carbon of isocitrate w/ the OH group is converted to a carbonyl group with oxidoreductase

• NAD —> NADH + H+

Describe step 4 of TCA cycle & spontaneity

small negative delta G

• control point in the cycle

• 2nd (2/4) oxidation step in the cycle

• oxidoreductase catalyzes oxidation that makes NAD+ —> NADH + H+ and CO2

• produces high energy thioester bond to CoA

Describe step 5 of TCA cycle & spontaneity

• transferase catalyzes phosphate molecule from solution displacing CoA, forming high energy bond to succinate

• this phosphate is passed from GDP —> GTP (in plants/bacteria, ATP is formed instead)

Describe step 6 of TCA cycle & spontaneity

delta G is 0 kJ/mol

• 3rd (3/4) oxidation step in the TCA cycle

• oxidoreductase (succinate dehydrogenase, in eukaryotes —> only enzyme in cycle that is membrane bound) catalyzes reaction where FAD removes two H atoms from succinate to form fumarate (double bond)

Describe step 7 of TCA cycle & spontaneity

small negative Delta G

• Lyase (fumarase aka fumarate hydratase) + water to place hydroxyl group

Describe step 8 of TCA cycle & spontaneity

Delta G positive, but in cells oxaloacetate is continually removed by exergonic citrate synthase (step 1) ** coupling of reaction!

• 4th (4/4) oxidation step

• last reaction of TCA cycle

• oxidoreductase (L-malate dehydrogenase) catalyzes oxidation of malate to oxaloacetate

total number of ATP produced by the catabolism of one molecule of glucose by glycolysis, the citric acid cycle, and reoxidation of NADH and QH2

32

What happens during substrate level phosphorylation, when does it occur?

enzyme transfers a phosphate group from substrate (PEP) molecule to ADP —> ATP

the PEP is derived from the catabolism of glucose

substrate level phosphorylation occurs during glycolysis and in TCA cycle

TCA cycle provides intermediates for biosynthesis, give three examples

• alpha-ketoglutarate transaminated to make glutamate —> make purine nucleotides

• Succinyl-CoA can be used to make porphyrins

• fumarate and oxaloacetate can be used to make several amino acids and pyrimidine nucleotides

The electron transport chain is a series of _______ _______ reactions that transfer electrons from ______ and ______ to ______

The electron transport chain is a series of coupled redox reactions that transfer electrons from NADH and FADH₂ to oxygen

Describe the change in free energy by glycolysis and the TCA cycle

Describe the structure and function of mitochondrion

What is the chemiosmotic hypothesis?

ATP is synthesized in mitochondria and chloroplasts by using a proton gradient across a membrane

1. proton pumping (using electrons) across inner membrane (from matrix to intermembrane space) to create proton gradient (high H+ outside, low H+ concentration inside)

2. pH gradient and membrane potential provides proton-motive force that catalyzes ATP synthesis

Overview of carbohydrate metabolism and the movement of electrons. Contrast substrate level phosphorylation with oxidative phosphorylation

substrate level phosphorylation: forms ATP by transferring phosphate from an intermediate catabolism substrate to ADP

oxidative phosphorylation: forms ATP using energy derived from the redox reactions in ETC

oxidant vs. reductant

oxidant = oxidizing agent = accepts e-

reductant = reducing agent = donates e-

Where in the cell do ETC and oxidative phosphorylation occur?

inner mitochondrial membrane

What does “cellular respiration” include, define it

TCA cycle and oxidative phosphorylation

respiration = ATP-generating process where an inorganic compound (eg. O2) is the ultimate electron acceptor

Chemical equation of the reaction that generates the energy for creating a proton gradient in ETC

high energy electrons transferred to oxygen —> H2O and energy

the electron transfer potential of an electron is measured as ______. Describe how this value is calculated and understood.

reduction potential E₀^’ or redox potential

• negative E = strong reducing agent, readily donates

• positive E = strong oxidizing agent, readily accepts

• 

where n = number of electrons transferred and F is faraday constant

What is the condition for standard oxidation reduction potential?

pH 7 25^oC

What apparatus can be used to measure redox potential? how does it work?

Describe the organization of parts in the ETC (protein complexes, enzymes, electron movement)

• four protein complexes in the inner mitochondrial membrane

• lipid soluble coenzyme (UQ,CoQ) and water soluble protein (cyt c) shuttle between the four protein complexes

• high energy electrons (in the form of NADH and FADH2 from TCA) generally fall in energy from complexes I and II to IV

• O2 reduction —> H2O in IV

Describe the change in free energy (relative to O2) across the four proteins in ETC

What metal is a component of all four ETC protein complexes (and in cytochrome C)

Iron

Describe the electron affinity E_o^’ of ETC components (the four proteins) moving down the chain

electron affinity increases as you go down the chain of complexes

Describe the exact electron + proton passage over the four complexes

1. electrons reach Q (ubiquinone/Coenzyme Q) through complexes I and II; when going through I, 4H+ is pumped out

2. QH2 serves as a mobile carrier of electrons and protons, passing e to complex III; 4H+ are pumped out

3. complex III passes to cytochrome c

4. complex IV transports e from cytochrome c to O2 —> H2O; 2H+ pumped out

Name complex 1 of ETC, what does it do?

NADH: ubiquinone oxidoreductase

catalyzes 2 simultaneous coupled reactions

1. H^- (hydride) transfer from NADH + H (from matrix) to CoQ (exergonic)

2. transfer 4 H⁺ from matrix to intermembrane (endergonic)

—> a proton pump driven by the energy of electron transfer

ubiquinone vs. ubiquinol

different oxidation states of coenzyme Q, the mobile electron carrier

ubiquinol (QH2) is the reduced version (after taking electro from complex I) of ubiquinone (Q)

Describe the conformational changes in complex I for transporting protons from the matrix to the cytosol

energy of electron transfer allows proton to cross complex I near the interface between its hydrophilic and hydrophobic domains

—> long helical rod (magenta) reorients subunits to release 3H+ into intermembrane space

Name complex II and describes its structure and what it does

succinate dehydrogenase

2 prosthetic groups and 4 diff proteins

electrons pass from FAD to an Fe-S center and then to ubiquinone

NO PROTON PUMPING

1. removes 2 H from succinate to give fumarate + 2H+

2. reduces (gives electrons) Coenzyme Q

Which complex (I or II) is the entry point for cofactor FADH₂ ?

complex II

Describe the arrangement of redox centers in complex II

Name complex III and describe its function and structure

Ubiquinone: cytochrom c oxidoreductase

• couples the transfer of electrons from ubiquinol to cytochrome c

• transfers 4 H+ total into intermembrane

Describe cytochrome c, its structure, and how it transports electrons

• soluble protein in intermembrane space

• single heme at center of structure, linked to the rest of the protein via 2 sulfur atoms

• 3rd sulfur from a methionine coordinates the iron

• transports electron from complex III to the copper center of complex III

the four ETC complexes may function as supercomplexes, what does this mean?

Supercomplexes are larger, stable associations of two or more protein complexes that are involved in the mitochondrial electron transport chain. These complexes, such as Complexes I, III, IV, and V, are the building blocks of the electron transport chain, which is responsible for generating ATP through oxidative phosphorylation. Supercomplexes are thought to play a role in optimizing the electron transport chain's efficiency and reducing the production of reactive oxygen species

what is the energy stored in the proton gradient called? Describe the two different components of this energy

proton motive force (pmf)

1. chemical potential energy: difference in concentration of a chemical species in two regions separated by the membrane

2. electrical potential energy: results from separation of charge when protein moves across the membrane