Redox Reactions in Metabolism:

Standard reduction potentials, coenzymes in metabolism, and pyruvate dehydrogenase

Bioc 460 Spring 2008 - Lecture 27 (Miesfeld) NADH Acetyl-CoA Redox reactions in living cells provide metabolic energy using NAD+/NADH Vitamins are organic compounds in nature that were discovered through dietary deficiency diseases such as beriberi The PDH reaction uses a ball and chain mechanism to generate acetyl-CoA

Key Concepts

in Redox Metabolism

• Reduction potentials are a measurement of electron affinity . Compounds with a very high affinity for electrons are oxidants, e.g., O 2 , and have a positive reduction potential (Eº’>0). Very strong reductants are compounds that readily give up electrons, e.g., NADH, and have a negative reduction potential (Eº’<0). Electrons flow from reductants to oxidants (electrons flow toward compounds with higher Eº’ values).

• Coenzymes are organic compounds that provide reactive chemical groups to enzymes , many coenzymes were discovered as vitamins through the study of dietary deficiency diseases. Most coenzymes, such as nicotinamide adenine dinucleotide (NADH) and thiamin pyrophosphate (TPP), are noncovalently associated with enzymes.

• The pyruvate dehydrogenase (PDH) complex is a mitochondrial metabolic machine that converts pyruvate to acetyl-CoA in a favorable reaction (  Gº’ = -33.4 kJ/mol). The PDH reaction is in the mitochondrial matrix and captures decarboxylation energy in the form of NADH.

Redox reactions transfer electrons

Redox reactions (oxidation-reduction) in the citrate cycle are a form of energy conversion involving the transfer of electron pairs substrates to the carrier molecules NAD + and FAD . from organic The energy available from redox reactions is due to differences in the electron affinity of two compounds and is an inherent property of each molecule based on molecular structure.

Coupled redox reactions consist of two half reactions:

1) an

oxidation reaction

(loss of electrons) 2) a

reduction reaction

(gain of electrons).

Conjugate redox pairs

Compounds that accept electrons are called

oxidants

and are reduced in the reaction, whereas compounds that donate electrons are called

reductants

and are oxidized by loss of electrons.

Each half reaction consists of a

conjugate redox pair

represented by a molecule with and without an electron (

e-

).

Fe 2+ /Fe 3+ is a conjugate redox pair in which the reductant that loses an

e-

ferrous ion during oxidation to generate a (Fe 2+ ) is the ferric ion (Fe 3+ ) the oxidant:

Fe

2+

<--> Fe

3+

+

e-

Similarly, the reductant cuprous ion oxidant cupric ion (Cu 2+ ) plus an

e-

(Cu + ) can be oxidized to form the in the reaction:

Cu

+

<--> Cu

2+

+

e-

Conjugate redox pairs

Two half reactions are combined to form a redox reaction. For example, the transfer of an

e-

from from Fe 2+ (the reductant) to Cu 2+ (the oxidant) to form Fe 3+ and Cu + .

Fe

2+

Cu

2+

<--> Fe

3+

+

e-

+ <--> Cu

e-

+

Fe

2+

+ Cu

2+

<--> Fe

3+

+ Cu

+ The Fe was oxidized and the Cu was reduced in a redox reaction in which the

e-

was the shared intermediate.

This Fe-Cu redox reaction takes place within the cytochrome c oxidase complex in the electron transport system of the inner mitochondrial membrane.

Aerobic respiration is the transfer of electrons from glucose to O

2

to form CO

2

and H

2

O

The more electrons a carbon atom has available to donate, the more reduced (less oxidized) it is. Hydrogen is less electronegative than carbon, and therefore electrons in C H bonds are considered "owned" by the carbon. Oxygen is more electronegative than carbon and the electrons in C-O and C=O bonds are all "owned" by the oxygen atom.

Redox reactions in the citrate cycle involve the transfer of

e-

pairs to generate NADH and FADH

2 The reduction of NAD + to NADH involves the transfer of a

hydride ion (:H )

, which contains 2

e-

and 1 H + , and the release of a

proton (H + )

into solution NAD + + 2

e-

+ 2 H + <--> NADH + H + In contrast, FAD is reduced by

sequential addition

(1 e- and 1 H + )

of one at a time to give the fully reduced FADH 2

hydrogen

product FAD + 1

e-

+ 1 H + <--> FADH + 1

e-

+ H + <--> FADH 2 Enzymes that catalyze biochemical redox reactions are strictly called

oxidoreductases

, however, since

most

oxidation reactions involve the loss of one or more hydrogen atoms, they are often called

dehydrogenases

.

Reduction potential (E) is a measure of the electron affinity of a given redox pair

Biochemical standard reduction potentials (Eº’) are determined under standard conditions using an electrochemical cell that measures the relative

e-

affinity of a test redox pair compared to the hydrogen half reaction .

Two half cells are connected by a galvanometer which measures the flow of electrons between two electrochemical cells. An agar bridge between the two half cells allows ions to flow and balance the charge to keep the electron circuit intact.

Fe 3+ has a higher

e-

affinity than H +

Standard reduction potentials are expressed as half reactions and written in the direction of a reduction reaction . Redox pairs with a positive Eº’ have a higher affinity for electrons that redox pairs with a negative Eº’.

Electrons move from the redox pair with the lower Eº’ (more negative) to the redox pair with the higher Eº’ (more positive) .

The hydrogen half reaction is set as the standard with a Eº’ = 0 Volts.

The amount of energy available from a coupled redox reaction is defined as



Eº’

By convention, the 

E

º' of a coupled redox reaction is determined by subtracting the

E

º' of the oxidant (

e-

acceptor) from the

E

º' of the reductant (

e-

donor) using the following equation: 

E

º' = (

E

º'

e-

acceptor ) - (

E

º'

e-

donor ) The 

E

º' for a coupled redox reaction is

proportional to the change in free energy

 Gº' as described by the equation (n is the number of

e-

):  Gº' = -

nF



E

º' If 

E

º' > 0, then the reaction is favorable since  Gº' will be negative. A coupled redox reaction is favorable when the reduction potential of the

e-

acceptor is

more positive

than that of the

e-

donor.

Calculating the



Gº’ for a citrate cycle oxidation reaction using the



Eº’ of the half reactions

The oxidative decarboxylation of isocitrate by the enzyme isocitrate dehydrogenase in the third reaction of the citrate cycle: Isocitrate + NAD + <-->  -ketoglutarate + CO 2 + NADH + H + Using the Eº’ values from the table with the half reactions as reductions: NAD +  -ketoglutarate + CO 2 + H + + 2

e-

+ 2

e-

+ 2 H + ---> NADH (Eº’ = -0.32 V) ---> isocitrate (Eº’ = -0.38 V) And now calculate  Eº’ considering that NAD+ is the e- acceptor and isocitrate is the

e-

donor (electrons move from low Eº’ to higher Eº’): 

E

º' = (

E

º'

e-

acceptor ) - (

E

º'

e-

donor ) 

E

º'

= (-0.32 V) - (-0.38 V) =

+0.06 V

Another way to get the same answer

If it makes more sense to you to write the two half reactions in the

direction of the overall net reaction

, then simply reverse the Eº’ value for the isocitrate oxidation and add the two Eº’ values together: Writing each half reaction in the direction of the net reaction: NAD + + H + + 2

e-

isocitrate --->  -ketoglutarate + CO 2 ---> NADH (Eº’ = -0.32 V) + 2

e-

+ 2 H +

(Eº’ = +0.38 V)

Isocitrate + NAD + <-->  -ketoglutarate + CO 2 + NADH + H + 

E

º'

= (-0.32 V) + (+0.38 V) =

+0.06 V

This is the method used in the Berg textbook (pg. 508), although in that case, they calculate the  Gº’ values first, and then add the  Gº’ values together.

Now use this  Eº’ value to calculate the  Gº’ for the reaction  Gº' = -

nF



E

º'  Gº' = -2 (96.48 kJ/molV) +0.06 V  Gº' = -11.6 kJ/mol A value for 

G

º’ < 0 confirms that this coupled redox reaction is favorable, i.e., it is favorable to oxidize isocitrate and reduce NAD + .

In order to calculate the

actual reduction potentials

for conjugate redox pairs, you need to use the

Nernst equation

and know the actual concentration of the oxidant (

e-

acceptor) and the reductant ( inside the cell (the mitochondrial matrix in this case):

e-

donor)

E

=

E

º' + RT ln [

e-

acceptor]

nF

[

e-

donor]

Pyruvate Dehydrogenase

Pyruvate that is destined for the citrate cycle, or fatty acid synthesis, is converted to acetyl CoA by the enzyme

pyruvate dehydrogenase (PDH)

. Acetyl-CoA has only two metabolic fates in the cell, and therefore, its production by PDH must be tightly regulated. • acetyl-CoA can be metabolized by the citrate cycle to convert redox energy to ATP by oxidative phosphorylation • acetyl-CoA can be used as a form of stored energy by conversion to fatty acids that are transported to adipocytes (fat cells) as triglycerides.

The pyruvate dehydrogenase complex catalyzes the

oxidative decarboxylation of pyruvate

to form CO 2 and acetyl-CoA in a reaction that requires three enzymes (E 1 , E 2 , and E 3 ), and five coenzymes (NAD + , FAD, CoA, TPP, and lipoic acid), that work together to catalyze five linked redox reactions. Pyruvate + CoA + NAD + ---> acetyl-CoA + CO 2 + NADH  Gº’ = -33.4 kJ/mol

Coenzymes provide additional chemical groups to enzymes that facilitate catalysis

Why are human vitamin deficiencies rare in developed countries?

Nicotinamide adenine dinucleotide (NAD

+

)

NAD is derived from the water-soluble vitamin

niacin

which is also called vitamin B 3 . NAD + , and its phosphorylated form NADP + , are involved in over 200 redox reactions in the cell which are characterized by the transfer of 2

e-

hydride ions (:H ). as

Catabolic redox reactions

primarily use the conjugate redox pair NAD + /NADH and a

nabolic redox reactions

use NADP + /NADPH. Note that the "+" charge does not refer to the overall charge of the NAD molecule, but rather only to the charge on the ring N in the oxidized state.

Nicotinamide adenine dinucleotide (NAD

+

)

Severe niacin deficiency causes the disease

pellagra

which was first described in Europe in the early 1700s amongst peasants who relied on cultivated corn as their primary source of nutrition. Pellagra is rare in Mexico because corn used for tortillas is traditionally soaked in lime solution (calcium oxide) prior to cooking and this releases niacin from its bound form upon heating.

Since corn obviously contains niacin, why would eating corn "cause" pellagra?

Flavin adenine dinucleotide (FAD)

FAD is derived from the water-soluble vitamin

riboflavin

which is also called vitamin B 2 . Riboflavin is the precursor to FAD and to the related molecule flavin mononucleotide (FMN). Riboflavin, is found in dairy products and is destroyed by light, which is one reason why milk is stored in light-tight containers.

FAD is reduced to FADH 2 by the transfer of two electrons in the form of hydrogen atoms . Unlike NAD, FAD can accept one electron at a time and form a partially reduced intermediate called a semiquinone (FADH).

Coenzyme A (CoA)

CoA is derived from the water-soluble vitamin

pantothenic acid

which is also called vitamin B 5 . CoA is absolutely essential for life as it is required for energy conversion by the citrate cycle, it is also a cofactor in fatty acid, acetylcholine, heme and cholesterol biosynthetic pathways. The primary role of CoA is to function as a carrier molecule for

acetate units

in the form of

acetyl-CoA

.

acetate group

Coenzyme A (CoA)

Acetyl-CoA consists of a central pantothenic acid functional  -mercaptoethylamine group unit that is linked to a derived from

cysteine

, and to adenosine 3,5-diphosphate . The acetate unit is covalently attached to CoA through an activated thioester bond which has a high standard free energy of hydrolysis making it an ideal acyl carrier compound. Attachment of acetate units to the reduced form of CoA requires reactions with high  Gº' values , for example, PDH (  Gº' = -33.4 kJ/mol) and  -ketoglutarate dehydrogenase (  Gº' = -33.5 kJ/mol).

Thiamin pyrophosphate (TPP)

FAD is derived from the water-soluble vitamin

thiamin

(or thiamine) which is also called vitamin B 1 . A carbon atom on the thiazole ring of TPP is the functional component of the coenzyme and is involved in aldehyde transfer. Thiamin is absorbed in the gut and transported to tissues where it is phosphorylated by the enzyme thiamin kinase in the presence of ATP to form thiamin pyrophosphate (TPP) and AMP.

Thiamin pyrophosphate (TPP)

Thiamin deficiency was first described in Chinese literature over four thousand years ago and is the cause of

beriberi

, a disease characterized by anorexia, cardiovascular problems and neurological symptoms. Beriberi has been found in populations that rely on white polished rice as a primary source of nutrition because milling rice removes the bran which contains thiamin. Fish and silkworms contain the enzyme

thiaminase

, which degrades thiamin. Cooking these foods destroys the thiaminase and alleviates the symptoms of beriberi caused by ingesting too much raw fish and silkworm.



-Lipoic acid (lipoamide in proteins)

 -Lipoic acid is a coenzyme synthesized in plants and animals as a 6,8-dithiooctanoic acid. The role of  lipoic acid in metabolic reactions is to provide a reactive disulfide that can participate in redox reactions within the enzyme active site.  -Lipoic acid is not considered a vitamin because it is synthesized at measurable levels in humans, however, because of its potential to function as an antioxidant in the reduced form,  -lipoic acid is promoted as a nutritional supplement.

High levels of



-lipoic acid are found in broccoli, spinach, and tomato.



-Lipoic acid (lipoamide in proteins)

Lipoamide, the naturally occurring form of  lipoic acid, is a

covalent linkage

of  lipoic acid to a lysine  amino group on proteins . The long hydrocarbon chain bridging  -lipoic acid and lysine provides a flexible extension to the reactive thiol group.

The E2 subunit of the pyruvate dehydrogenase complex contains the lipoamide at the end of a polypeptide tether which functions as a "

ball and chain

" that moves the lipoamide back and forth across a 50 Å span in the interior of the complex.

The pyruvate dehydrogenase (PDH) complex is a highly efficient metabolic machine

The conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase complex is an

oxidative decarboxylation

reaction that represents another amazing example of protein structure and function. The eukaryotic pyruvate dehydrogenase complex contains multiple subunits of three different catalytic enzymes that work together as a metabolic machine.

Note that TPP, lipoamide, and FAD are all regenerated.

The pyruvate dehydrogenase (PDH) complex is a highly efficient metabolic machine

Three of the coenzymes are covalently linked to enzyme subunits, with TPP attached to the

E1 pyruvate dehydrogenase

subunit, lipoamide is the functional component of the

E2 dihydrolipoyl transacetylase

subunit, and FAD is covalently bound to the

E3 dihydrolipoyl dehydrogenase

subunit. The two other coenzymes, CoA and NAD + , are transiently associated with the E2 and E3 complexes, respectively.

The pyruvate dehydrogenase (PDH) complex is a highly efficient metabolic machine

The pyruvate dehydrogenase reaction can be broken down into

five distinct catalytic steps

: 1. Decarboxylation 2. Transfer of the acetyl group to lipoamide 3. Formation of acetyl-CoA 4. Redox reaction to form FADH 2 5. Redox reaction to form NADH

5 4 1 2 3

The pyruvate dehydrogenase (PDH) complex is a highly efficient metabolic machine

The E1, E2 and E3 subunits of the mammalian PDH complex are packed together in a huge ~400 Å diameter sphere with a combined molecular weight of ~7800 kDa. The stoichiometry of the E1:E2:3 subunits (22:60:6) is consistent with there being

60 active sites

in the pyruvate dehydrogenase complex.

The pyruvate dehydrogenase (PDH) complex is a highly efficient metabolic machine

The lipoamide moiety of the E2 subunit is attached near the end of a ~200 amino acid long segment of the protein that functions as both a structural linker connecting the E2 and E1 subunits, and as a type of lipoamide "

ball and chain

." The linker region in the E1-binding domain serves as a "

pivot

" for the ball and chain.

Arsenite is a naturally occurring inhibitor of lipoamide

Inadvertent ingestion arsenite can lead to an untimely death by irreversibly blocking the catalytic activity of lipoamide-containing enzymes such as the PDH and  ketoglutarate dehydrogenase complexes.

Chronic arsenic poisoning can come from environmental sources such as arsenic-contaminated drinking water and result in the appearance of ulcerous skin lesions and an increased risk of a variety of cancers.

Arsenite is a naturally occurring inhibitor of lipoamide

Since the 1990s it has been documented that millions of people in India have been chronically exposed to toxic levels of arsenic in contaminated drinking water shallow hand-pumped wells.

obtained from During the 1970s and 1980s, UNICEF and other relief organizations helped drill thousands of wells in small Indian villages as an

humanitarian effort

to circumvent public water supplies that had become biologically contaminated. About ten years later when large numbers of villagers in the Ganges delta region developed skin lesions and cancers, it was realized that these wells contained water with toxic levels of arsenic. Massive efforts were undertaken to

close down contaminated wells

and to develop purification systems to reduce the arsenic to safe levels in other water supplies.