biological oxidation i - top recommended websites...oxidation and reduction oxidation of an iron...
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Biological oxidation IRespiratory chain
• Metabolism
• Macroergic compound
• Redox in metabolism
• Respiratory chain
• Inhibitors of oxidative phosphorylation
Outline
Metabolism
• Metabolism consists of catabolism and
anabolism
• Catabolism: degradative pathways
– Usually energy-yielding!
• Anabolism: biosynthetic pathways
– energy-requiring!
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 energy-requiring processes of cells
“High energy” bonds
Phosphoanhydride bonds (formed by splitting out H2O
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
of hydrolysis.
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 Pi
hydrolysis.
• Removal of Pi 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-
phosphate.
• ATP has special roles in energy coupling and Pi transfer.
• DG of phosphate hydrolysis from ATP is intermediate
among examples below.
• ATP can thus act as a Pi donor, and ATP can be synthesized
by Pi transfer, e.g., from PEP.
Compound
DGo of phosphate hydrolysis (kJ/mol)
Phosphoenolpyruvate (PEP)
Phosphocreatine
Pyrophosphate
ATP (to ADP)
Glucose-6-phosphate
Glycerol-3-phosphate
• 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.
Some other
“high energy”
bonds:
• 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
b-mercaptoethylamine,
in amide linkage to the
carboxyl group of the B
vitamin pantothenate.
The hydroxyl of
pantothenate is in ester
linkage to a phosphate
of ADP-3'-phosphate.
The functional group is
the thiol (SH) of
b-mercaptoethylamine.
“High energy” (macroergic) compounds
exemplifying the following roles:
Energy transfer or storage
ATP, PPi, polyphosphate, creatinephosphate
Group transfer
ATP, Coenzyme A
Transient signal
cAMP
Oxidation and reduction
Oxidation of an iron atom involves loss of an electron (to
an acceptor): Fe2+ (reduced) Fe3+ (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
• Catabolism is oxidative - substrates lose
reducing equivalents, usually H+ ions
• Anabolism is reductive – NAD(P)H
provides the reducing power (electrons) for
anabolic processes
NAD+, Nicotinamide
Adenine Dinucleotide,
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 Pi.
NADPH is e donor in
synthetic pathways.
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+ FADH2
NAD+ is a coenzyme, that reversibly
binds to enzymes.
FAD is a prosthetic group, that remains
tightly bound at the active site of an
enzyme.
Oxidation of the coenzyme Q
Respiratory ChainAn Overview
• Electron Transport: Electrons carried byreduced 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 thesynthesis of ATP
• It all happens in or at the inner mitochondrial membrane
Electron Transport
• Four protein complexes in the inner
mitochondrial membrane
• A lipid soluble coenzyme (UQ, CoQ) and a water
soluble protein (cyt c) shuttle between protein
complexes
• Electrons generally fall in energy through the
chain - from complexes I and II to complex IV
27
Sequence of electron carriers in the respiratory chain
Complex I
proton pump
Complex II, does not
pump protons
Complex III
proton pump
Complex IV
proton pump
Coenzyme Q
electron shuttle
Cytochrome c
electron shuttle
Complex Name No. of
Proteins
Prosthetic Groups
Complex I NADH
Dehydrogenase
46 FMN, 9 Fe-S centers
Complex II Succinate-CoQ
Reductase
5 FAD, cyt b560, 3 Fe-S
centers
Complex III CoQ-cyt c
Reductase
11 cyt bH, cyt bL, cyt c1,
Fe-SRieske
Complex IV Cytochrome
Oxidase
13 cyt a, cyt a3, CuA, CuB
Complexes of Respiratory chain
Complex INADH-CoQ Reductase
• Electron transfer from
NADH to CoQ
• Path: NADH FMN
Fe-S UQ FeS
UQ
• 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., Fe3+ ).
Complex IISuccinate-CoQ Reductase
• aka succinate dehydrogenase (from TCA cycle!)
• aka flavoprotein 2 (FP2) - FAD covalently bound
• four subunits, including 2 Fe-S proteins
• Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S, 2Fe-2S
• Path: succinate FADH2 2Fe2+ UQH2
• Net reaction: succinate + UQ fumarate + UQH2
Complex IIICoQ-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
• UQH2 is a lipid-soluble electron
carrier
• cyt c is a water-soluble electron
carrier
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.
Complex IVCytochrome c Oxidase
• Electrons from cyt c are used in a four-electron
reduction of O2 to produce 2H2O
• Oxygen is thus the terminal acceptor of
electrons in the electron transport pathway -
the end!
• Cytochrome c oxidase utilizes 2 hemes (a and
a3) and 2 copper sites
• Complex IV also transports H+
Coupling e- Transport and
Oxidative Phosphorylation
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
2005-2006
Peter Mitchell• Proposed chemiosmotic hypothesis
– revolutionary idea at the time
1961 | 1978
1920-1992
proton motive force
ATP Synthase
ATP synthase
subunit
c ring subunit
subunit b subunit
F1 subunit has 5 types of
polypeptide chains
(3, b3, , , ), displays
ATPase activity
and b are members of
P-loop family
F0 contains the proton channel
ring of 10-14 c subunits„a‟ subunit binds
to outside of ring
Exterior column
has 1 a subunit
2 b subunits, and
the subunit
Moving unit (rotor) is c ring and
Remainder is stationary (stator)
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 - F1Fo ATP synthase:
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 Fo "uses up" pH and electrical
gradients.
ATP-ADP Translocase
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
the chain?
• 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 (FADH2), 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
membrane
• 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 =
oxidative phosphoryltion
+ electron transport
Inhibitors of Oxidative
Phosphorylation
• Rotenone inhibits Complex I - and
helps natives of the Amazon rain forest
catch fish!
• Cyanide, azide and CO inhibit
Complex IV, binding tightly to the
ferric form (Fe3+) of a3
• Oligomycin are ATP synthase
inhibitors
UncouplersUncoupling e- transport and
oxidative phosphorylation
• Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient
• Uncouplers are hydrophobic molecules with a dissociable proton
• They shuttle back and forth across the membrane, carrying protons to dissipate the
gradient
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
uncouplers.
2. Phosphorylation inhibitors (e.g. oligomycin)
abolish the burst of oxygen consumption after
adding ADP, but have no effect on uncoupler-
stimulated respiration.
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
inner membrane.
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.
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
Inhibitors of respiratory chain