Transcript Bioenergetics and Metabolism

Redox Reactions in Metabolism:
Standard reduction potentials, coenzymes in
metabolism, and pyruvate dehydrogenase
Bioc 460 Spring 2008 - Lecture 27 (Miesfeld)
NADH
Acetyl-CoA
Vitamins are organic compounds
in nature that were discovered
through dietary deficiency
diseases such as beriberi
Redox reactions in living
cells provide metabolic
energy using NAD+/NADH
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., O2, 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 from organic
substrates to the carrier molecules NAD+ and FAD.
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-).
Fe2+/Fe3+ is a conjugate redox pair in which the ferrous ion (Fe2+) is the
reductant that loses an e- during oxidation to generate a ferric ion (Fe3+)
the oxidant:
Fe2+ <--> Fe3+ + eSimilarly, the reductant cuprous ion (Cu+) can be oxidized to form the
oxidant cupric ion (Cu2+) plus an e- in the reaction:
Cu+ <--> Cu2+ + e-
Conjugate redox pairs
Two half reactions are combined to form a redox reaction. For example,
the transfer of an e- from from Fe2+ (the reductant) to Cu2+ (the oxidant) to
form Fe3+ and Cu+.
Fe2+ <--> Fe3+ + eCu2+ + e- <--> Cu+
Fe2+ + Cu2+ <--> Fe3+ + 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 O2 to form CO2 and H2O
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 CH 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 FADH2
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 of one hydrogen
(1 e- and 1 H+) at a time to give the fully reduced FADH2 product
FAD + 1 e- + 1 H+ <--> FADH + 1 e- + H+ <--> FADH2
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.
Fe3+ 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 Eº' of a coupled redox reaction is determined by
subtracting the Eº' of the oxidant (e- acceptor) from the Eº' of the
reductant (e- donor) using the following equation:
Eº' = (Eº'e- acceptor) - (Eº'e- donor)
The Eº' 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Eº'
If Eº' > 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 + CO2 + NADH + H+
Using the Eº’ values from the table with the half reactions as reductions:
NAD+ + H+ + 2 e- ---> NADH
(Eº’ = -0.32 V)
-ketoglutarate + CO2 + 2 e- + 2 H+ ---> 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’):
Eº' = (Eº'e- acceptor) - (Eº'e- donor)
Eº' = (-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- ---> NADH
(Eº’ = -0.32 V)
isocitrate ---> -ketoglutarate + CO2 + 2 e- + 2 H+ (Eº’ = +0.38 V)
Isocitrate + NAD+ <--> -ketoglutarate + CO2 + NADH + H+
Eº' = (-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Eº'
Gº' = -2 (96.48 kJ/molV) +0.06 V
Gº' = -11.6 kJ/mol
A value for Gº’ < 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 (e- donor)
inside the cell (the mitochondrial matrix in this case):
E = Eº' + 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 CO2 and acetyl-CoA in a
reaction that requires three enzymes (E1, E2,
and E3), and five coenzymes (NAD+, FAD,
CoA, TPP, and lipoic acid), that work together
to catalyze five linked redox reactions.
Pyruvate + CoA + NAD+ ---> acetyl-CoA + CO2 + 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 B3. 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- as
hydride ions (:H-).
Catabolic redox reactions
primarily use the conjugate
redox pair NAD+/NADH and
anabolic 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 B2.
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 FADH2 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 B5.
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 unit that is linked to a
functional -mercaptoethylamine group 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 B1. 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 FADH2
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 obtained from
shallow hand-pumped wells.
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.