TCA Cycle

Aerobic organisms use oxygen to:

extract energy from nutrient molecules.

Which generates more energy, fermentation or respiration?

Respiration generates significantly more energy than fermentation and was a critical turning point in the evolution of life.

Why use oxygen for the TCA Cycle?

It is an excellent electron receptor for two major reasons: Availability-abundant & Power-highly electronegative

Is the TCA Cycle a true Cycle? Yes or No, and Why?

Yes. The TCA cycle is a true metabolic cycle because it is a closed loop that regenerates its starting material, oxaloacetate, in each turn.

Oxygen was released by: cyanobacteria ~2.3 billion years ago but did not accumulate in the atmosphere for several hundred million years.

How are modern organisms classified?

Modern organisms are classified based on how they cope with ROS or use oxygen in energy generation.

Obligate anaerobes

Grow only in the absence of oxygen and use fermentation for energy production.

Aerotolerant anaerobes

Depend on fermentation, have detoxifying enzymes and antioxidant molecules to handle ROS.

Facultative anaerobes

Have not only biochemical mechanisms to detoxify ROS, but can use oxygen as an electron receptor when available.

Obligate aerobes

Highly dependent on oxygen for energy production, ROS protection from elaborate detoxifying mechanisms composed of enzymes and many antioxidant molecules.

Obligate anaerobes are typically found:

deep in the soil’s highly reductive environment.

Aerotolerant anaerobes are typically found:

in the upper layer of the soil.

Aerobic metabolism consists of the following processes:

Citric acid cycle, Electron transport pathway & Oxidative phosphorylation.

Aerobic metabolism occurs within:

the mitochondria. This is a form of compartmentalization.

Most of the free energy is captured by:

The Electron Transport Cycle.

Electrons are transferred from:

a redox pair with a more negative reduction potential to those with

more positive reduction potential.

Which two enzymes for nicotinic acid?

nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADPH).

Both structures of NAD & NADPH contain:

adenosine and the N-ribosyl derivative of nicotinamide linked together though a pyrophosphate group.

NADPH provides:

reducing power for anabolic reactions.

NAD helps in:

the shuffling of protons, is an essential cofactor, DNA repair, epigenetics, and intercellular calcium signaling (innervation).

Riboflavin: a component of two coenzymes:

flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).

FMN and FAD function as:

a tightly bound prosthetic groups of flavoproteins and can act as a donor or acceptor of two hydrogen atoms

FMN can transfer how many hydrogen atoms at a time?

One. It plays a key role in the link between the reactions in the mitochondrial matrix and the ETC.

What is the difference between a Coenzyme and a Prosthetic Group?

PGs are more tightly bound because of their covalent bonds.

The Citric Acid Cycle is:

a series of reactions that release chemical energy stored in the two-carbon acetyl group of acetyl-CoA

Acetyl-CoA is synthesized:

from pyruvate, the product of fatty acid catabolism, and from certain reactions in amino acid metabolism.

Fatty Acids can come into the Cycle through:

TCA, but not gluconeogenesis because they don’t make sugars.

The TCA Cycle is composed of:

8 reactions occurring in two stages.

Stage 1 of TCA Cycle:

two-carbon acetyl group of acetyl-CoA react with oxaloacetate and 2 CO2 released (Step 1 - 5)

Stage 2 of TCA Cycle:

oxaloacetate is regenerated (Step 6 - 8)

In Reaction 1:

2 Carbons come in = Citrate

“2 Carbons come in = Citrate “ means: Oxaloacetate reacts with acetyl CoA and H2O to yield citrate and CoA.

The steps of Reaction 1 are:

an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, which is then hydrolyzed to citrate and CoA.

Reaction has no ATP because:

Synthases don’t use ATP.

Reaction 1’s Favorability? Regulation?

Highly Favorable. Helps push the Cycle forward in Steps 6, 7, & 8. Highly Regulated.

In Reaction 2:

Citrate to IsoCitrate via Aconitase.

“Citrate to IsoCitrate via Aconitase” means:

The enzyme aconitase (more formally, aconitate hydratase) catalyzes the reversible

transformation of citrate to isocitrate, through the intermediary formation of the tricarboxylic acid cis-aconitate by dehydration.

The carbon-carbon double bond of cis-aconitate:

is rehydrated to form the more reactive secondary alcohol, isocitrate.

Purpose of Reaction 2:

To make Citrate more energetically favorable for the next step = IsoCitrate.

Is Reaction 2 favorable?

No.

In Reaction 3:

Oxidation to Alpha-K

“Oxidation to Alpha-K” means: Isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate to form α-ketoglutarate.

The steps oxidative decarboxylation of are:

(1) Isocitrate is oxidized to oxalosuccinate (2) Decarboxylation of oxalosuccinate to enol intermediate (3) Enol intermediate rearranges to a-ketogluterate

Reaction 3’s Favorability? Regulation?

Favorable. Highly Regulated. Pays for Reaction 2.

In Reaction 4:

“Oxidation to Succinyl-CoA”

“Oxidation to Succinyl-CoA” means:

Another oxidative decarboxylation, in which a-ketoglutarate is converted to succinyl-CoA and O2 by the action of the a-ketoglutarate dehydrogenase complex.

In Reaction 4, NAD+ serves as a:

electron acceptor.

The a-ketoglutarate dehydrogenase complex:

a-ketoglutarate dehydrogenase (oxidation), dihydrolipoyltranssuccinylase (moving), and dihydrolipoyl dehydrogenase (oxidation again).

Reaction 4’s Favorability? Regulation?

Highly Regulated. Highly Favorable.

In Reaction 5:

“Cleavage to Succinate”

“Cleavage to Succinate” means:

Cleavage of succinyl-CoA is coupled to substrate-level phosphorylation.

Cleavage of the high-energy thioester bond to form succinate is:

reversible and catalyzed by succinyl-CoA synthetase (succinate thiokinase).

In mammals, cleavage of succinyl-CoA is coupled to:

substrate-level phosphorylation of ADP or GDP. Two enzyme forms expressed in many tissues: one for ATP and one for GTP.

Reaction 5 Favorability?

It’s close to equilibrium. Thus, it is reversible.

ADP and GDP are:

Energetically Equivalent. The bonds are what matter.

The Completion of the TCA Cycle (Oxidation of Succinate to Oxaloacetate) consists of:

of three reactions. These reactions include: (1) Oxidation of a single bond to a double bond, (2) Hydration across the double bond, and (3) Oxidation of the resulting alcohol to a ketone.

This trio of reactions in the Completion of the TCA Cycle will be seen again in:

(1) Fatty acid breakdown and synthesis (2) Amino acid breakdown and synthesis.

In Reaction 6:

“Oxidation to Fumarate"

“Oxidation to Fumarate" means:

An FAD-dependent oxidation of a single bond in succinate to a double bond in fumarate.

Reaction 6’s Mechanism involves: hydride removal by FAD and a deprotonation.

Why does Reaction 6 use FAD and not NAD+?

The reaction is not sufficiently exergonic to reduce NAD+. FAD is easier.

Succinate dehydrogenase is:

actually part of the electron transport pathway in the inner mitochondrial membrane.

The electrons transferred from succinate to FAD (to form FADH2):

are passed directly to ubiquinone (UQ) in the electron transport pathway.

FAD is covalently bound to:

succinate dehydrogenase which links the C-8a carbon of FAD and the N-3 of a His residue of the enzyme.

FMN and FAD are found as:

tightly bound prosthetic groups in flavoenzymes.

Flavin coenzymes can exist:

in any of three oxidation states, and this allows flavin coenzymes to

participate in one-electron or two-electron transfer reactions.

Flavoproteins catalyze:

many reactions in biological systems and work with many electron donors and acceptors.

Succinate Dehydrogenase contains:

three types of Fe-S centers – a 4Fe-4S center, a 3Fe-4S center, and a 2Fe-2S center.

Iron-Sulfur Clusters cycle:

between net oxidation states that differ by 1, so clusters participate in one electron redox reactions (Fe2+ and Fe3+)

Succinate Dehydrogenase is bound to:

inner membrane of the mitochondrion.

How do electrons from FADH2 enter the electron transport chain? Why?

Through Fe-S clusters. To contribute to ATP Synthesis.

Electron Wire

Multiple redox centers. Distance of redox centers in proteins is optimized to facilitate electron transfer.

Citrate is a poor substrate for oxidation (T/F)

True.

Because Citrate is Poor substrate for oxidation, Aconitase:

isomerizes citrate to yield isocitrate which has a

secondary -OH, which can be oxidized.

What is the Stereochemistry of Reaction 2?

Aconitase removes the pro-R H of the pro-R arm of citrate.

Aconitase doesn’t use an iron-sulfur cluster (T/F). False.

Aconitase has a [4Fe-4s] cluster:

that is bound to the protein by the side chains of three cysteine residues.

What activates Aconitase?

Binding of Fe2+ to the vacant position of the cluster.

The added Iron to Aconitase:

coordinates the C-3 carboxyl and hydroxyl groups of citrate and acts as a Lewis acid, accepting an electron pair from the hydroxyl group and making it a better leaving group.

In Reaction 7:

Trans-Hydration to L-Malate

“Trans-Hydration to L-Malate” means:

Fumarase Catalyzes the trans-Hydration of Fumarate to Form L-Malate

How does Trans-Hydration work?

Hydration occurs across the newly formed double bond. Hydration involves trans-addition of the water across the double bond.

Are we certain of the mechanism of Fumerase?

No.

What are the Regulated Steps of TCA Cycle?

1, 3, & 4.

In Reaction 8:

“Oxidation to Oxaloacetate”

“Oxidation to Oxaloacetate” means:

a NAD+-dependent oxidation. The carbon that gets oxidized is the one that received the -OH in the previous reaction.

Favorability of Reaction 8?

Energetically Expensive. Paid for by Step 1.

Is the concentration of oxaloacetate in the mitochondrial matrix quite low? or quite high?

Low.

The malate dehydrogenase reaction is pulled forward by:

the favorable citrate synthase reaction.

How do the concentration gradients also push forward Reaction 8?

The product is kept low concentration, keeping it going forward.

Oxidation of glucose to CO2 is a _____-electron oxidation.

24

Electrons from glucose oxidation feed into:

the electron transport pathway, driving synthesis of ATP.

The Oxidation of pyruvate yields:

30 ATP per molecule of glucose.

How does Acetyl-CoA enter the TCA Cycle?

Pyruvate is decarboxylated to Acetyl-CoA. Acetyl-CoA then enters the TCA cycle. For each glucose two Acetyl-CoA enter the TCA cycle.

In eukaryotes, pyruvate is transported in the mitochondrion because:

all the downstream reactions occurs there

Fatty acids enter the TCA Cycle:

at the level of Acetyl-CoA.