Download Oxidative Phosphorylation

Oxidative Phosphorylation
Voet & Voet: Chapter 22
Lecture
17
Biochemistry 2000
Slide 1
Glucose “combustion”
Seguin & Lavoisier (1789) ...
in general, respiration is nothing but the slow combustion of carbon and
hydrogen, which is entirely similar to that which occurs in a lamp or lighted
candle ...
Combustion of Glucose
C6H12O6
+
6 O2
→
6 CO2
+
G'º = -2823 kJ/mol
6 H2O
Electron Transfer in Glucose Combustion
C6H12O6
+
6 H2O
→
6 CO2
+
24 H+
6 O2
+
24 H+
+
24 e-
→
12 H2O
+
24 e- (glucose oxidation)
(oxygen reduction)
In living system, the electrons of glucose are not transferred directly to oxygen.
Rather they are transferred in a multistep pathway that harnesses the libertated
energy to form ATP
Lecture
17
Biochemistry 2000
Slide 2
Catabolism of Proteins, Fats
and Carbohydrates
The citric acid cycle (also called Krebs or
tricarboxylic cycle) is the “hub” of the
metabolic system.
It accounts for the majority of
carbohydrate, fatty acid and amino acid
oxidation.
- 3 carbon sugars are oxidized to and
released as CO2
Glucose Combustion
Glycolysis provides 4 e- (2 NADH)
Citric Acid cycle produces 20 e- (8 NADH + 2 FADH2)
Respiration
24 e- enter electron transfer chain
Lecture
17
Biochemistry 2000
Slide 3
Anatomy of Mitochondrion
Mitochondrion is the “Power Plant” of eucaryotic
cells
Citric Acid Cycle, Fatty Acid Oxidation, Oxidative phosphorylation
(e - transfer and majority of ATP synthesis) occur within
mitochondrion
Two specialized membranes
1 – smooth outer membrane permeable to small molecules & ions
2 – convoluted inner membrane (large surface area) impermeable
to most small molecules & ions
Proteins of electron transport and oxidative
phosphorylation are embedded in the inner membrane
Mitochondrial Matrix
Densely packed, gel-like (~50 % H2O) composition containing high
concentrations of enzymes and soluble metabolic intermediates
Lecture
17
Biochemistry 2000
Slide 4
Electron Carriers
Universal 2 Electron Carriers
NAD+
NADP+
FAD+
FMN+
+
+
+
+
2 H+
2 H+
2 H+
2 H+
+
+
+
+
2 e2 e2 e2 e-
→
→
→
→
NADH + H+
NADPH + H+
FADH2
FMNH2
1 Electron Carriers
Cytochromes (Fe3+) + e- → Cytochromes (Fe2+)
FeS proteins (Fe3+, typically) + e- → FeS proteins (Fe2+, typically)
Mediators of Electron Transport
FMN+ + H+ + e- → FMNH* ; FMNH* + H+ + e- → FMNH2
Q + H+ + e- → QH* ; QH* + H+ + e- → QH2
Mediators allow transfer of electrons between 2 e- and 1e- carriers
Lecture
17
Biochemistry 2000
Slide 5
Universal
Electron
Carriers
NAD+/NADH (and FAD+/FADH2) are universal electron carriers
Funnel e- from numerous metabolic pathways into the respiratory chain
Typically dehydrogenase enzymes transfer electrons to NAD+ or FAD+
Lecture
17
Biochemistry 2000
Slide 6
FMN (flavin mononucleotide)
+
FMN+ can be an integral part of an enzyme (prosthetic) or a
soluble compound
As a prosthetic, FMN+ can accept and transfer either 1 or 2 eSoluble FMN+ only accepts 2 eAs a prosthetic, FMN+ mediates e- transfer between electron
carriers that accept two electrons (eg. NAD+ or FAD+) and
those that only accept one electron (eg. Fe3+)
Note: FMN+ has the same structure as FAD+ without the AMP
Lecture
17
Biochemistry 2000
Slide 7
Ubiquinone
Ubiquinone (Coenzyme Q, CoQ or Q) is
very hydrophobic due to its long
alkyl tail
Dissolves within the hydrophobic core of
the membrane
Isoprenoid tail (n=10) is longer than
membrane is wide and must adopt a
folded structure
Quinone ring of ubiquinone can accept
1 e- (semiquinone radical) or 2 e(ubiquinol)
Mediates electron transfers between 1 and 2
e- carriers
Mobile carrier (unlike FMN) that can diffuse
through membrane
Lecture
17
Biochemistry 2000
Slide 8
Cytochromes – 1 e- carrier
Cytochromes are a family of small proteins
Contain a heme a prosthetic group
Fe of heme is coordinated by four N atoms of
the porphyrin ring
Fe3+ is reduced to Fe2+ when it accepts an
electron
Three classes of cytochromes (a, b, c) have
slightly different heme prosthetics
Results in different standard reduction
potentials (E'°)
Lecture
17
Biochemistry 2000
Slide 9
Fe•S Proteins
Many e- transfer proteins contain one or more Iron sulfur centers (Fe•S)
Fe•S centers are prosthetic groups
Contain 1-4 Fe atoms complexed with elemental S atoms and coordinated by cysteine
residues
In one case, the Fe atom is coordinated by 2 His residues (Reiske Fe•S protein)
Regardless of number of Fe atoms, an Fe•S center can only accept 1 eClose proximity of Fe atoms prevents more than one e- from being accepted
4Fe•4S
Lecture
17
3Fe•4S
2Fe•2S
Biochemistry 2000
Fe
Reiske
(2Fe•2S)
Slide 10
Respiratory (e transfer) Chain
-
Respiratory (e- transfer) chain is composed of 4 complexes, CoQ and
cytochrome c
There are several different routes through respiratory chain depending upon the
electron donor
Lecture
17
Biochemistry 2000
Slide 11
Routes through Respiratory Chain
Electron transfer from NADH
utilizes complex I, III, IV, CoQ and
cytochrome c
Electron transfer from succinate
(citric acid cycle intermediate)
utilizes complex II, III, IV, CoQ
and cytochrome c
Other electron transfers utilize
complex III, IV, CoQ and
cytochrome c
Lecture
17
Biochemistry 2000
Slide 12
Respiratory Chain (NADH)
(1) NADH transfers 2 e- to complex I (FMN → Fe S → Fe S)
(2) Complex I transfers 2 e- to Ubiquinone
(3) Ubiquinone transfers 2 e- to complex III (CytbL → CytbH → Fe S → Cytc1)
(4) Complex III transfers 1 e- to Cytc (x2)
(5) Cytc transfers 1 e- to complex IV (CuACyta → CuBCyta3) (x2)
(6) Complex IV transfers 2 e- to ½ O2
10 H+ transported across membrane for 2 e- (2 H+ from matrix not shown)
Lecture
17
Biochemistry 2000
Slide 13
Complex I
Overall Reaction
NADH + 5H+N + Q → NAD+ + QH2 + 4H+P
Complex I uses energy of electron transfer
to transport 4 H+ from the mitochondrial
matrix to the intermembrane space
Complex I is a proton pump
Very large protein complex containing at
least 46 proteins in mammals
Figure: Electron Microscopy reconstruction of
Complex I
Lecture
17
Biochemistry 2000
Slide 14
Q cycle & Complex III
Q-cycle transfers e- from
ubiquinone to complex III in
two steps
In each step, 2 e- are donated by QH2
and 1 e- is returned (Q or Q•-)
Overall reaction is not balanced
Complex III requires a pool of QH2 in
order to function
Complex III is another proton
pump
Lecture
17
Biochemistry 2000
Slide 15
Complex IV
Yet another proton pump
Overall Reaction
4 cyt c (red) + 8 H+N + O2 → 4 cyt c (ox) + 4H+P + 2H2O
Cytochrome c passes 4 e- (one at a time) to
Cu-S center (CuA)
CuB and cytochrome a3 form a binuclear
center (Fe-Cu) that passes e- to O2
Lecture
17
Biochemistry 2000
Slide 16
Summary of Respiration
Energy of electron transfer is
conserved in a proton
gradient
For each 2 e- transferred from
NADH → O2 , 10 H+ are
transported across inner
membrane
 G'° = -220 kJ/mol
Proton gradient is referred to as Proton
Motive Force
Actively respiring mitochondria have
 ~ 0.15 – 0.20 V and  pH = 0.75
 G ~ 20 kJ/mol (or 200 kJ for 10 mol of H+)
Lecture
17
Biochemistry 2000
Slide 17
Chemiosmotic Model
(Conversion of PMF to ATP)
Proton Motive Force is utilized
to synthesize ATP (30- 50
kJ/mol)
FoF1 ATP Synthase couples
transport of H+ into matrix
with ATP synthesis
Fo is a transmembrane pore
F1 is a soluble ATPase
Lecture
17
Biochemistry 2000
Slide 18
Mitochondrial F0F1 ATP Synthase
F1 generally includes 5 subunits (    
- 3  subunits are regulatory
- 3  subunits are catalytic
- 3 ADP + Mg2+ binding sites are at the 
interface (mostly )
F0 generally includes 3 subunits (ab2c10)
- The 10 c subunits form pore
Parts of F1 rotates relative to F0 during the
catalytic cycle
- conformational change facilitates H+ transport and
ATP synthesis
Lecture
17
Biochemistry 2000
Slide 19
Binding Change Mechanism
Binding Change Mechanism of ATP synthesis
(for simplicity: only  subunits are shown above)
F1 contains irregularly shaped “shaft” ( subunit) that rotates relative to the 
subunits
- rotation of shaft is driven by flow of H+ through F0
There are three active site conformations (loose, tight and open)
- as the shaft rotates, the  subunits change conformation
- at any time, each active site adopts a different conformation
(a) loose
(b) tight
(c) open
Lecture
17
– loosely binds ADP + Pi
– tightly binds substrate and forms ATP
– favors release of ATP
Biochemistry 2000
Slide 20
Proton Transfer
“c” subunit (F0) rotates with “shaft” while
“a” subunit is stationary
“a” subunit forms 2 H+ wires (half channels)
that combine to transfer H+ across
membrane
Cartoon of
“a” subunit H+
wires
H+ is added on intermembrane side and H+
is released on matrix side
Rotation of c subunits is required to relay
protons between H+ wires
Lecture
17
Biochemistry 2000
Slide 21