Cellular Respiration refers to themolecular processes involved inO2 consumption and CO2

formation by cells.

Oxidation of activatedacetyl groups. Energyis conserved in the formof NADH and FADH2.

Oxidation oforganic fuelmolecules toacetyl CoA.

Oxidation of Pyruvate to Acetyl CoA and CO2

Pyruvate, derived from glucose by glycolysis, is oxidized to yieldacetyl CoA and CO2 by a complex of three enzymes known as thepyruvate dehydrogenase complex.

Enzymes: pyruvate dehydrogenase dihydrolipoyl transacetylase dihydrolipoyl dehydrogenase

Cofactors: thiamine pyrophosphate lipoamide

FAD NAD+

CoA

Located in the mitochondrial matrix. Thus pyruvate, madein cytoplasm, must first be transported across the inner membrane.

dehydrogenase

dehydrogenase1 23

4

This is also catalyzed by pruvate dehydrogenase.

There are four steps in the conversion of pyruvate to acetyl CoA:

Step 1: Pyruvate is decarboxylated after it combines with thiaminepyrophosphate (TPP). This reaction is catalyzed by pyruvatedehydrogenase (E1).

pyruvate + TPP hydroxyethyl-TPP + CO2

An important feature of this step is that the carbon atom betweenthe nitrogen and sulfur atoms in the thiazole ring is significantlyacidic, so that it can dissociate its proton to form a carbanion.

(A cofactor of E1)

It is the carbanionic form which then adds to the carbonyl groupof pyruvate.

The positive charge on the ring nitrogen functions as an electronsink to stabilize the negative charge that will be transferred to the ring and is necessary for the subsequent decarboxylation.

Protonation results in the formation of hydroxyethyl TPP.

Step 2: The hydroxyethyl group attached to TPP is oxidized to forman acetyl group and is concomitantly transferred to the lipoamide on E2 resulting in the product acetyllipoamide.

The oxidant in this reaction is the disulfide group of lipoamide which is reduced to its sulfhydryl form.

The oxidant

This is the prosthetic group on dihydrolipoyl transacetylase (E2).

This reaction is catalyzed by pyruvate dehydrogenase (E1).

Step 3: The acetyl group is transferred from acetyllipoamide to CoAthereby forming acetyl CoA. This reaction is catalyzed by the enzymedihydrolipoyl transacetylase (E2). The energy rich thioester bond is preserved.

Step 4: Lipoamide is oxidized via the enzyme dihydrolipoyl dehydrogenase (E3). During this reaction 2 electrons are transferredto FAD, a prosthetic group on the enzyme, and then to NAD+.

This is the reduced form of the lipoyl group.

FAD is the prosthetic group on E3.

The pyruvate dehydrogenase complex is immense. It has a massof 7 – 8.5 million daltons making it larger then a ribosome.

Its core consists of the transacetylase polypeptide chains with the pyruvate dehydrogenase and the dihydrolipoyl dehydrogenase bound to the outside of the core.

The important principle here is that the structural integration of these three enzymes makes possible the coordinated catalysis of the reaction.

Intermediates are transferred to each active site via the lipoamide prosthetic group of the transacetylase.

Thus the lipoyl moiety of E2 can interact with the TPP unit of E1 andwith the FAD unit of E3.

•A dietary deficiency in thiamine can lead to the disease Beriberi.

•It is a neurologic and cardiovasular disorder.

•Seen in malnourished individuals.

•Unable to oxidize pyruvate normally, thus organs such as brainwhich are heavily dependent upon aerobic oxidation of glucosefor energy production are principally affected.

•Often see elevated pyruvate in the blood of such individuals.

Regulation of the Pyruvate Dehydrogenase Complex

1) Allosteric Regulation:

a) ATP, NADH, and acetyl CoA inhibit. This inhibition is greatlyenhanced by the presence of long-chain fatty acids.

b) AMP, CoA, and NAD+ activate.

2) Covalent Modification: The complex is inhibited by the reversiblephosphorylation of a Ser on E1.

i) A Mg2+ -ATP-dependent protein kinase phosphorylates the complex (inactivating E1). The kinase is allosterically activated by ATP and inhibited by pyruvate.

ii) A Mg2+- and Ca2+-dependent phosphoprotein phosphatase removes the phosphate group (reactivating E1). Ca2+ stimulatesthe phosphatase, thereby activating the dehydrogenase.

Regulation of the Pyruvate Dehydrogenase Complex

Increasing the ratio of: NADH/NAD+

Acetyl CoA/CoA ATP/ADP

promotes phosphorylation and thus inactivation of the complex.

Thus the PDH complex is turned off when the energy charge is high and biosynthetic intermediates are abundant.

Reactions of the Citric Acid Cycle

General Overview: Glycolysis is a linear pathway in contrast to the citric acid cycle. Per turn of the cycle:

2 carbon atoms enter as an acetyl unit and 2 carbon atoms leaveas CO2.

4 Redox reactions occur which result in the transfer of 3 hydrideions (i.e., 6 electrons) to NAD+ and one pair of hydrogen atoms(i.e., 2 electrons) to FAD. (Will yield 11 ATPs via oxidative phos-phorylation).

One high energy bond (GTP) is formed and the starting oxaloacetate is regenerated.

The cycle consists of 8 coupledreactions which will now be discussed.

Reaction 1: Acetyl CoA (a 2 carbon compound) condenses with oxaloacetate (a 4 carbon compound) to form citrate. Catalyzed bycitrate synthase.

Reaction 2: The enzyme aconitase catalyzes the reversible

isomerization of citrate to isocitrate via formation of the

intermediate cis-aconitate. The isomerization is accomplished

via a dehydration followed by a hydration step.

The resulting isocitrate is able to undergo oxidative decarboxylation.

Reaction 3: Isocitrate is oxidatively decarboxylated to α-ketoglutarate in a reaction catalyzed by isocitratedehydrogenase.

•The first of 4 redox reactions.

•Important in determining the overall rate of the cycle.

**

Unstableintermediate

Reaction 4: α-ketoglutarate is oxidatively decarboxylated to succinyl CoA by the α-ketoglutarate dehydrogenase complex.NAD+ function as the electron acceptor.

•Very similar to the PDH complex, requiring the same cofactors.

•Second redox reaction, generates NADH.

•Unlike PDH, it’s not regulated by phosphorylation.

Reaction 5: Succinyl CoA, like acetyl CoA, has a strongly negativefree energy of hydrolysis of its thioester bond. Thus, energy released via cleavage of the thioester bond of succinyl CoAis coupled to the phosphorylation of GDP. This is the only exampleof a “substrate level phosphorylation” in the cycle.

The resulting GTP can be used: i) as a phosphoryl group donor in protein synthesis; ii) in signal transduction; iii) as a phosphoryl donor to make ATP:

GTP + ADP GDP + ATPnucleoside diphosphokinase

Reaction 6: Succinate is oxidized to fumarate via succinatedehydrogenase.

FAD is the hydrogen acceptor such that two hydrogen atomsare removed from the substrate.

Succinate dehydrogenase is an iron-sulfur protein that is located within the inner mitochondrial membrane and is directly linked to the electron transport chain.

This is the thirdredox reaction.

Reaction 7: The reversible hydration of fumarate to L-malate is

catalyzed by fumarase. This enzyme catalyzes the stereospecific

hydration of the trans double bond of fumarate, but does not act

on maleate, the cis isomer of fumarate.

X

X

Reaction 8: The last reaction of the cycle consists of the oxidation ofmalate to oxaloacetate via NAD-linked L-malate dehydrogenase.

ΔG˚’ = 29.7 kJ/mol

•Fourth redox reaction. Generates NADH.

•Although equilibrium lies far to the left under standard conditions, in the cell the highly exergonic citrate synthase reaction rapidlyremoves oxaloacetate, thereby driving this reaction to the right.

Stoichiometry of the Citric Acid Cycle

The net reaction of the cycle is:

Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O

2 CO2 + 3 NADH + FADH2 + GTP + 2 H+ CoA

Per turn of cycle:

2 carbon atoms emerge as CO2;Not the same carbons that enteredas acetyl CoA.

4 pairs of hydrogen atoms leave the cycle via 4 redox reactions.

Generate 3 NADH and 1 FAD.

IDH

α-KGDH

Succ. CoA Synth.

SDH

MDH

Although the cycle directly generates 1 ATP, the flow of electrons fromNADH and FADH2 into the respiratory chain and ultimately to O2 will generate many molecules of ATP.

Note:

•O2 does not directly participate inthe cycle, but it is needed to regenerateNAD+ and FAD in mitochondria.

•Cycle only operates under aerobicconditions.

•In contrast, glycolysis has both anaerobic and anaerobic mode sinceNAD+ can be regenerated duringthe reduction of pyruvate to lactate.

Complete oxidation of glucose occurs with ~40% efficiency.