Biological oxidation I
• Macroergic compound
• Redox in metabolism
• Respiratory chain
• Inhibitors of oxidative phosphorylation
• Metabolism consists of catabolism and
• Catabolism: degradative pathways
– Usually energy-yielding!
• Anabolism: biosynthetic pathways
The ATP Cycle
• ATP is the energy currency of cells
• In phototrophs, light energy is transformed
into the light energy of ATP
• In heterotrophs, catabolism produces ATP,
which drives activities of cells
• ATP cycle carries energy from
photosynthesis or catabolism to the energyrequiring
processes of cells
“High energy” bonds
Phosphoanhydride bonds (formed by splitting out H 2 O
between 2 phosphoric acids or between carboxylic and
phosphoric acids) have a large negative DG of hydrolysis.
Phosphoanhydride linkages are said to be "high energy"
bonds. Bond energy is not high, just DG of hydrolysis.
"High energy" bonds are represented by the "~" symbol.
~P represents a phosphate group with a large negative DG
• Phosphocreatine (creatine phosphate), another
compound with a "high energy" phosphate linkage, is
used in nerve and muscle for storage of ~P bonds.
• Phosphocreatine is produced when ATP levels are high.
• When ATP is depleted during exercise in muscle,
phosphate is transferred from phosphocreatine to ADP,
to replenish ATP.
• Phosphoenolpyruvate (PEP), involved in ATP
synthesis in Glycolysis, has a very high DG of P i
• Removal of P i from ester linkage in PEP is spontaneous
because the enol spontaneously converts to a ketone.
• The ester linkage in PEP is an exception.
Other examples of phosphate esters with low but
negative DG of hydrolysis:
• the linkage between phosphate and a hydroxyl
group in glucose-6-phosphate or glycerol-3-
• ATP has special roles in energy coupling and P i transfer.
• DG of phosphate hydrolysis from ATP is intermediate
among examples below.
• ATP can thus act as a P i donor, and ATP can be synthesized
by P i transfer, e.g., from PEP.
ATP (to ADP)
of phosphate hydrolysis (kJ/mol)
• A thioester forms between a carboxylic acid and a thiol
(SH), e.g., the thiol of coenzyme A (abbreviated CoA-SH).
• Thioesters are ~ linkages. In contrast to phosphate esters,
thioesters have a large negative DG of hydrolysis.
• The thiol of coenzyme A can react with a carboxyl
group of acetic acid (yielding acetyl-CoA) or a fatty
acid (yielding fatty acyl-CoA).
• The spontaneity of thioester cleavage is essential to the
role of coenzyme A as an acyl group carrier.
• Like ATP, CoA has a high group transfer potential.
Coenzyme A includes
in amide linkage to the
carboxyl group of the B
The hydroxyl of
pantothenate is in ester
linkage to a phosphate
The functional group is
the thiol (SH) of
“High energy” (macroergic) compounds
exemplifying the following roles:
• Energy transfer or storage
ATP, PP i , polyphosphate, creatinephosphate
• Group transfer
ATP, Coenzyme A
• Transient signal
Oxidation and reduction
• Oxidation of an iron atom involves loss of an electron (to
an acceptor): Fe 2+ (reduced) Fe 3+ (oxidized) + e -
• Since electrons in a C-O bond are associated more with
O, increased oxidation of a C atom means increased
number of C-O bonds.
• Oxidation of C is spontaneous.
Increasing oxidation number of C
Redox in Metabolism
• NAD + collects electrons released in
• Catabolism is oxidative - substrates lose
reducing equivalents, usually H + ions
• Anabolism is reductive – NAD(P)H
provides the reducing power (electrons) for
NAD + , Nicotinamide
is an electron acceptor
in catabolic pathways.
The nicotinamide ring,
derived from the
vitamin niacin, accepts
2 e - and 1 H + (a
hydride) in going to the
reduced state, NADH.
NADP + /NADPH is
similar except for P i .
NADPH is e donor in
NAD + /NADH
The electron transfer reaction may be
summarized as :
NAD + + 2e + H + NADH.
It may also be written as:
NAD + + 2e + 2H + NADH + H +
FAD (Flavin Adenine Dinucleotide), derived from the
vitamin riboflavin, functions as an e acceptor. The
dimethylisoalloxazine ring undergoes reduction/oxidation.
FAD accepts 2 e - + 2 H + in going to its reduced state:
FAD + 2 e - + 2 H + FADH 2
• NAD + is a coenzyme, that reversibly
binds to enzymes.
• FAD is a prosthetic group, that remains
tightly bound at the active site of an
Oxidation of the coenzyme Q
• Electron Transport: Electrons carried by
reduced coenzymes are passed through a
chain of proteins and coenzymes to drive
the generation of a proton gradient across
the inner mitochondrial membrane
• Oxidative Phosphorylation: The proton
gradient runs downhill to drive the
synthesis of ATP
• It all happens in or at the inner
• Four protein complexes in the inner
• A lipid soluble coenzyme (UQ, CoQ) and a water
soluble protein (cyt c) shuttle between protein
• Electrons generally fall in energy through the
chain - from complexes I and II to complex IV
Sequence of electron carriers in the respiratory chain
Complex II, does not
Complexes of Respiratory chain
Complex Name No. of
46 FMN, 9 Fe-S centers
5 FAD, cyt b 560
, 3 Fe-S
Complex III CoQ-cyt c
Complex IV Cytochrome
11 cyt b H
, cyt b L
, cyt c 1
13 cyt a, cyt a 3
, Cu A
, Cu B
• Electron transfer from
NADH to CoQ
• Path: NADH FMN
Fe-S UQ FeS
• Four H + transported
out per 2 e-
Role of FMN: Since it can accept/donate either 1 or 2 e - ,
FMN has an important role in mediating electron transfer
between carriers that transfer 2 e - (e.g., NADH) and
carriers that can only accept 1 e - (e.g., Fe 3+ ).
• aka succinate dehydrogenase (from TCA cycle!)
• aka flavoprotein 2 (FP 2 ) - FAD covalently bound
• four subunits, including 2 Fe-S proteins
• Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S, 2Fe-2S
• Path: succinate FADH 2 2Fe 2+ UQH 2
• Net reaction: succinate + UQ fumarate + UQH 2
CoQ-Cytochrome c Reductase
• CoQ passes electrons to cyt c (and
pumps H + ) in a unique redox cycle
known as the Q cycle
• The principal transmembrane protein
in complex III is the b cytochrome
• Cytochromes, like Fe in Fe-S clusters,
are one- electron transfer agents
• UQH 2 is a lipid-soluble electron
• cyt c is a water-soluble electron
Heme is a prosthetic group of cytochromes. Heme contains an iron
atom embedded in a porphyrin ring system. The Fe is bonded to 4 N
atoms of the porphyrin ring. Hemes in the three classes of cytochrome
(a, b, c) differ slightly in substituents on the porphyrin ring system. A
common feature is two propionate side-chains.
Cytochrome c Oxidase
• Electrons from cyt c are used in a four-electron
reduction of O 2 to produce 2H 2 O
• Oxygen is thus the terminal acceptor of
electrons in the electron transport pathway -
• Cytochrome c oxidase utilizes 2 hemes (a and
a 3 ) and 2 copper sites
• Complex IV also transports H +
Coupling e - Transport and
This coupling was a mystery for many years
• Many biochemists squandered careers searching
for the elusive "high energy intermediate"
• Peter Mitchell proposed a novel idea - a proton
gradient across the inner membrane could be
used to drive ATP synthesis
• Mitchell was ridiculed, but the chemiosmotic
hypothesis eventually won him a Nobel prize
• Proposed chemiosmotic hypothesis
– revolutionary idea at the time
1961 | 1978
proton motive force
Moving unit (rotor) is c ring and
Remainder is stationary (stator)
c ring subunit
„a‟ subunit binds
to outside of ring
has 1 a subunit
2 b subunits, and
F 0 contains the proton channel
ring of 10-14 c subunits
F 1 subunit has 5 types of
( 3 , b 3 , , , ), displays
and b are members of
The Chemiosmotic Theory of oxidative phosphorylation,
for which Peter Mitchell received the Nobel prize:
Coupling of ATP synthesis to respiration is indirect,
via a H + electrochemical gradient.
Chemiosmotic theory - respiration:
Spontaneous e transfer through complexes I, III, & IV is
coupled to non-spontaneous H + ejection from the matrix.
H + ejection creates a membrane potential (DY, negative
in matrix) and a pH gradient (DpH, alkaline in matrix).
Chemiosmotic theory - F 1
Non-spontaneous ATP synthesis is coupled to spontaneous
H + transport into the matrix. The pH and electrical gradients
created by respiration are the driving force for H + uptake.
H + return to the matrix via F o
"uses up" pH and electrical
ATP must be transported out of the mitochondria
• ATP out, ADP in - through a "translocase"
• ATP movement out is favored because the
cytosol is "+" relative to the "-" matrix
• But ATP out and ADP in is net movement of a
negative charge out - equivalent to a H + going in
• So every ATP transported out costs one H +
• One ATP synthesis costs about 3 H +
• Thus, making and exporting 1 ATP = 4H +
What is the P/O Ratio?
i.e., How many ATP made per electron pair through
• e - transport chain yields 10 H + pumped out per
electron pair from NADH to oxygen
• 4 H + flow back into matrix per ATP to cytosol
• 10/4 = 2.5 for electrons entering as NADH
• For electrons entering as succinate (FADH 2 ), about
6 H + pumped per electron pair to oxygen
• 6/4 = 1.5 for electrons entering as succinate
Shuttle Systems for e -
Most NADH used in electron transport is cytosolic
and NADH doesn't cross the inner mitochondrial
• What to do?
• "Shuttle systems" effect electron movement
without actually carrying NADH
• Glycerophosphate shuttle stores electrons in
glycerol-3-P, which transfers electrons to FAD
• Malate-aspartate shuttle uses malate to carry
electrons across the membrane
Respiratory chain =
+ electron transport
Inhibitors of Oxidative
• Rotenone inhibits Complex I - and
helps natives of the Amazon rain forest
• Cyanide, azide and CO inhibit
Complex IV, binding tightly to the
ferric form (Fe 3+ ) of a 3
• Oligomycin are ATP synthase
Uncoupling e- transport and
• Uncouplers disrupt the tight
coupling between electron
transport and oxidative
phosphorylation by dissipating
the proton gradient
• Uncouplers are hydrophobic
molecules with a dissociable
• They shuttle back and forth
across the membrane, carrying
protons to dissipate the
Uncouplers and Inhibitors
There are six distinct types of poison which may
affect mitochondrial function:
1. Respiratory chain inhibitors (e.g. cyanide,
antimycin, rotenone and TTFA) block
respiration in the presence of either ADP or
2. Phosphorylation inhibitors (e.g. oligomycin)
abolish the burst of oxygen consumption after
adding ADP, but have no effect on uncouplerstimulated
3. Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP)
abolish the obligatory linkage between the respiratory
chain and the phosphorylation system which is observed
with intact mitochondria.
4. Transport inhibitors (e.g. atractyloside, bongkrekic
acid, NEM) either prevent the export of ATP, or the
import of raw materials across the the mitochondrial
5. Ionophores (e.g. valinomycin, nigericin) make the inner
membrane permeable to compounds which are ordinarily
unable to cross.
6. Krebs cycle inhibitors (e.g. arsenite, aminooxyacetate)
which block one or more of the TCA cycle enzymes, or
an ancillary reation.
Inhibitors of respiratory chain
Name Function Site of action
retenone e transport inhibitor Complex I
amytal e transport inhibitor Complex I
antimycin A e transport inhibitor Complex III
cyanide e transport inhibitor Complex IV
carbon monoxide e transport inhibitor Complex IV
azide e transport inhibitor Complex IV
2,4-initrophenol uncoupling agent transmembrane H+ carrier
pentachlorophenol uncoupling agent transmembrane H+ carrier
oligomycin inhibits ATP-ase OSCP protein