Bioenergetics and Metabolism

1
Redox Reactions in MetabolismStandard 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
The PDH reaction uses a ball and chain mechanism
to generate acetyl-CoA
Redox reactions in living cells provide metabolic
energy using NAD/NADH
2
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 (Eºgt0).
  Very strong reductants are compounds that readily
  give up electrons, e.g., NADH, and have a
  negative reduction potential (Eºlt0). 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.

3
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).??
4
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 lt--gt Fe3 e-
Similarly, the reductant cuprous ion (Cu) can be
oxidized to form the oxidant cupric ion (Cu2)
plus an e- in the reaction

Cu lt--gt Cu2 e-
5
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 lt--gt Fe3 e- Cu2 e- lt--gt Cu Fe2
Cu2 lt--gt 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.
6
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 C-H bonds are
considered "owned" by the carbon. Oxygen is
more electronegative than carbon and the
electrons in C-O and CO bonds are all "owned" by
the oxygen atom.
7
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 lt--gt 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 lt--gt FADH 1 e- H
lt--gt 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.
8
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
9
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.
10
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' gt 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.
11
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 lt--gt ?-ketoglutarate
  CO2 NADH H
• Using the Eº values from the table with the half
  reactions as reductions
• NAD H 2 e- ---gt NADH (Eº
  -0.32 V) ??-ketoglutarate CO2 2 e- 2 H
  ---gt 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

12
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- ---gt NADH (Eº
  -0.32 V) ?isocitrate ---gt ?-ketoglutarate
  CO2 2 e- 2 H (Eº 0.38 V)
• Isocitrate NAD lt--gt ?-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.
13
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 lt 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

14
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.
15
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 ---gt acetyl-CoA CO2
NADH ?Gº -33.4 kJ/mol
16
Coenzymes provide additional chemical groups to
enzymes that facilitate catalysis
Why are human vitamin deficiencies rare in
developed countries?
17
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.
18
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?
19
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).
20
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
21
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. Attachmen
t 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).
22
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.
23
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.
24
?-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.
25
?-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.
26
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.
27
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.
28
The pyruvate dehydrogenase (PDH) complex is a
highly efficient metabolic machine

• The pyruvate dehydrogenase reaction can be broken
  down into five distinct catalytic steps
• Decarboxylation
• Transfer of the acetyl group to lipoamide
• Formation of acetyl-CoA
• Redox reaction to form FADH2
• Redox reaction to form NADH

5
4
1
2
3
29
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 E1E23
subunits (22606) is consistent with there being
60 active sites in the pyruvate dehydrogenase
complex.
30
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.
31
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.
32
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.