Department of Chemistry and Biochemistry Biochemistry 3300 University of Lethbridge
III. Metabolism Oxidative Phosphorylation
Biochemistry 3300 Slide 1 Biochemical Anatomy of Mitochondria
Transmembrane channels allow small molecules (< 5 kD) and ions to pass through the outer membrane.
Convolutions of the inner membrane provides large surface area. → depending on the tissue they are more or less profuse
Specific transporters carry pyruvate, fatty acids and amino acids into the matrix for access to the citric acid cycle.
Biochemistry 3300 Slide 2 Universal Electron Acceptors Collect Electrons Dehydrogenases (catabolism) transfer electrons to universal electron carriers which funnel electrons into the respiratory chain M mitochondria C cytosol
Biochemistry 3300 Slide 3 Electron Carriers I. Nicotinamide Adenine Dinucleotide
Optical Test
NAD+ / NADP+ accept a hydride (H-) and a proton is released
Biochemistry 3300 Slide 4 Electron Carriers II. Flavins
FMN (Flavin Mononucleotide) is a prosthetic group of some flavoproteins.
Similar in structure to FAD (Flavin Adenine Dinucleotide), but lacking the adenine nucleotide.
When free in solution, FMN (like FAD) accepts - + 2 e + 2 H to form FMNH2.
Biochemistry 3300 Slide 5 Electron Carriers II. Flavins
When bound as a prosthetic, FMN (and FAD) can accept 1 e- to form the 'half-reduced' semiquinone radical.
The semiquinone can then accept a 2nd e- to yield FMNH2.
FMN (and FAD) mediating e- transfer between carriers that transfer 2e- (e.g., NADH) & those that can accept only 1e- (e.g., Fe+++).
Biochemistry 3300 Slide 6 Electron Carriers III. Ubiquinone
Coenzyme Q (CoQ, Q, ubiquinone) is very hydrophobic. Located in the hydrocarbon core of membranes.
CoQ contains a long isoprenoid tail, with multiple units (typically n = 10) having a carbon skeleton comparable to that of isoprene.
The isoprene tail of Q10 is longer than the width of a lipid bilayer (likely folded into compact shape)
Biochemistry 3300 Slide 7 Electron Carriers – Ubiquinone
The quinone ring of coenzyme Q can be reduced to the quinol in a 2e- reaction:
+ Q + 2 e + 2 H QH2.
When bound to special sites in respiratory complexes, CoQ can accept 1 e− to form a semiquinone radical (Q·−).
Thus CoQ, like FMN (& FAD), can mediate between 1 e− & 2 e− donors/acceptors.
Coenzyme Q functions as a mobile e- carrier within the mitochondrial inner membrane.
Biochemistry 3300 Slide 8 Electron Carriers IV. Cytochromes
Cytochromes contain a Heme prosthetic group.
Heme contains an iron atom in a porphyrin ring system. The Fe is bonded to 4 N atoms of the porphyrin ring and is the redox center.
Hemes in the 3 classes of cytochrome (a, b, c) have different porphyrin ring substituents (eg. proprionate)
Only heme c is covalently linked to the protein cytochrome c via thioether bonds to cysteine residues.
Biochemistry 3300 Slide 9 Electron Carriers - Cytochromes
Cytochrome c Heme iron undergoes 1 e- transition between ferric and ferrous states: Fe+++ + e- ↔ Fe++ Met80 Heme Fe interacts with: - 4 N of polyporphyrin ring and - 2 axial ligands above & below heme
X His18 N N
Fe
N N PDBid 5CYT Y
Axial heme Fe ligands: His18 and Met80 Axial ligands alter reduction potential of heme Fe
Biochemistry 3300 Slide 10 Electron Carriers IV. Cytochromes
Heme prosthetic group absorbs light at characteristic wavelengths
Absorbance spectra can follow the redox state of the heme (same as for all other electron carriers)
Many cytochromes are subunits of large integral membrane complexes containing multiple electron carriers - located within mitochondrial inner membrane
Cytochrome c is a small, water-soluble protein with a single heme group
Biochemistry 3300 Slide 11 Electron Carriers V. Iron-sulfur Centers
Iron-sulfur centers (Fe-S) are prosthetic groups containing 1-4 iron atoms complexed to elemental & cysteine S atoms.
Electron transfer proteins may contain multiple Fe-S centers.
4-Fe centers have a tetrahedral structure, with Fe & S atoms alternating as vertices of a cube.
Biochemistry 3300 Slide 12 Electron Carriers V. Iron-sulfur Centers
Iron-sulfur centers transfer only one electron!! (even when they have more than one Fe)
Eg., a 4-Fe center might cycle between redox states described as: 3Fe+++, 1Fe++ (oxidized) + 1 e- 2Fe+++, 2Fe++ (reduced)
Iron-sulfur proteins where one Fe atom is coordinated by two His residues are named Rieske iron-sulfur proteins.
Biochemistry 3300 Slide 13 Electron Carriers
Electron carriers that are organic compounds have lower standard reduction potentials than heme iron electron carriers
Note: Fe:S electron carriers tend to have intermediate standard reduction potentials
Biochemistry 3300 Slide 14 Respiratory Chain
Most respiratory chain proteins are embedded in the inner mitochondrial membrane (or in the cytoplasmic membrane of aerobic bacteria).
Biochemistry 3300 Slide 15 Respiratory Chain Complexes
Protein components of the electron-transfer chain are primarily organized as large, transmembrane (or membrane associated) protein complexes
Biochemistry 3300 Slide 16 Respiratory Chain
4H+
Electron transfer from NADH to O2 involves multi-subunit inner membrane complexes I, III & IV, plus CoQ & cyt c.
Within each complex, electrons pass sequentially through a series of electron carriers.
CoQ is located in the lipid core of the membrane. There are also binding sites for CoQ within protein complexes.
Cytochrome c resides in the intermembrane space. It alternately binds to complex III or IV during e- transfer.
Biochemistry 3300 Slide 17 Respiratory Chain
4H+
The standard reduction potentials of constituent e- carriers are consistent with the e- transfers observed.
Biochemistry 3300 Slide 18 Inhibitors of Electron Transport
Respiratory chain inhibitors include:
Rotenone (a rat poison) & Amytal Complex I Antimycin A Complex III CN- & CO Complex IV
- Any of these sites will block e transfer from NADH to O2.
Experimental setup?
How do we measure 'Electron Transfer Chain' activity
Biochemistry 3300 Slide 19 Effect of Inhibitors on Electron Transport
Oxygen electrode: O2 selective membrane permits measurement of [O2]
O2 produced in sample chamber is reduced by anode generating a measurable current
Biochemistry 3300 Slide 20 Electron Transport Inhibitors
Experiment (sample chamber of O2 electrode): Buffered mitochondria solution with excess ADP + Pi are equilibrated
Reagents added and [O2] is monitored over time
Example experiment: 1 - Hydroxybutyrate is substrate that allows TCA cycle to function; NADH is source of electrons - O2 levels will decrease as e are transferred to
complex IV where O2 is reduced 2 - Rotenone or amytal inhibit Complex I stopping the electron transfer reactions
O2 levels remain constant as electrons do not reach
complex IV where O2 is reduced 3 - Succinate provides electrons via Complex II - O2 levels will decrease as e are transferred from
complex II to complex IV where O2 is reduced 4 - Antimycin inhibits complex III
O2 levels remain constant as electrons do not reach
complex IV where O2 is reduced 5 - TMPD/Ascorbate provide electrons to cyctochrome C - O2 levels will decrease as e are transferred from
cytochrome C to complex IV where O2 is reduced 6 - CN- (or CO) inhibit complex IV
O2 levels remain constant as O2 is not reduced
Biochemistry 3300 Slide 21 Complex I
Bovine complex I at 17 Å resolution. Complex I catalyzes oxidation of NADH, with reduction of coenzyme Q:
+ → + NADH + H + Q NAD + QH2 And the transfer of 4 H+ across the membrane:
Grigorieff, N. (1998). J. Mol. Biol., 277, 1033-1046 Overall: + → + + NADH + 5H N + Q NAD + QH2 + 4H P Complex I is a proton pump that uses the energy of electron transfer for the vectorial movement of protons across the membrane.
Complex I : L-shaped and contains six iron sulfur centers and a FMN-containing protein. No high-resolution crystal structure of mammalian complex which includes > 46 proteins.
Biochemistry 3300 Slide 22 Complex I
NADH interacts with a solvent exposed domain of the mitochondrial matrix.
Coenzyme Q binds within the membrane domain.
Fe-S centers are in the NADH-binding domain & in a connecting domain closer to the membrane segment. The initial electron transfers are:
+ + NADH + H + FMN ↔ NAD + FMNH2
+ FMNH2 + (Fe-S)ox ↔ FMNH· + (Fe-S)red + H
Biochemistry 3300 Slide 23 Complex I
After Fe-S is reoxidized by transfer of the electron to the next iron-sulfur center in the pathway:
+ FMNH· + (Fe-S)ox FMN + (Fe-S)red + H
Electrons pass through a series of iron- sulfur centers in complex I, eventually to coenzyme Q.
− + Coenzyme Q accepts 2 e and picks up 2 H to yield the fully reduced QH2.
Biochemistry 3300 Slide 24 Complex II
Succinate Dehydrogenase of the TCA Cycle is also called complex II or Succinate-CoQ Reductase.
FAD is the initial electron receptor.
FAD is reduced to FADH2 during oxidation of succinate to fumarate.
FADH2 is then reoxidized by transfer of electrons through a series of iron-sulfur centers to Coenzyme Q, yielding QH2.
Biochemistry 3300 Slide 25 Complex II
X-ray crystallographic analysis of E. coli complex II indicates a linear arrangement of electron carriers within complex II, consistent with the predicted sequence of electron transfers:
→ → → → FAD FeS1 FeS 2 FeS 3 CoQ
In this crystal structure oxaloacetate (OAA) is bound in place of succinate.
PDBid 1NEK
Biochemistry 3300 Slide 26 Path of Electrons to Ubiquinone
Other substrates for mitochondrial dehydrogenases pass their e- into the respiratory chain at the level of ubiquinon, but not through complex II.
Example:
Fatty acyl-CoA electrons via Acyl-CoA dehydrogenase (β oxidation) via ETF (electron transferring flavoprotein) via ETF:ubiquinone oxidoreductase to Reduced CoQ
Biochemistry 3300 Slide 27 β Oxidation
Mitochondria contain four acyl-CoA DH with different fatty acyl-CoA specificities: Glu376 short (C4 to C6)
medium (C6 to C10) long (between medium & very long)
very long (C12 to C18)
PDBid 3MDE
The FADH2 is reoxidized by the mitochondrial electron transport chain.
Biochemistry 3300 Slide 28 Complex III
Complex III (cytochrome bc1 complex)
Accepts electrons from
coenzyme QH2 that are generated by electron transfer in complexes I & II (and by other dehydrogenases)
Couples the transfer of electrons to cytochrome c with the vectorial transport of protons from the matrix to the inermembrane space.
Cytochrome c1, a prosthetic group within complex III, reduces cytochrome c, which is the electron donor to complex IV.
Biochemistry 3300 Slide 29 Complex III – The Q cycle
The “Q cycle” depends on: (1) mobility of CoQ in the lipid bilayer (2) CoQ binding sites that stabilize the semiquinone radical, Q·− .
Biochemistry 3300 Slide 30 Complex III – The Q cycle
− + It takes 2 cycles to reduced Q to QH2; 2e are transferred and 2H are extracted from the matrix compartment.
In 2 cycles, 2 QH2 enter the pathway & one is regenerated.
Biochemistry 3300 Slide 31 Complex III
Cytochrome c1
Rieske protein Rieske iron-sulfur center (Fe-S) has a flexible link to the rest of the complex.
e
n Heme bL
a − r - it changes position during e transfer. b Heme b m H
e
M
Rieske Fe-S extracts an e− from CoQ,
and moves closer to heme c1, to which it transfers the e−.
PDBidBiochemistry 1BE3 3300 Slide 32 Complex IV
Cytochrome oxidase (complex IV) carries out the irreversible reaction:
+ - O2 + 4 H + 4 e → 2 H2O
The four electrons are transferred into the complex one at a time from cytochrome c.
Large enzyme (13 SU; 204 kD) Bacteria contain a form that is much simpler (3-4 SU).
Comparison of the two forms suggests that three are critical to the function.
Biochemistry 3300 Slide 33 Complex IV
Mitochondrial subunit II contains two Cu ions coordinated to two Cys residues.
Subunit I contains two heme groups (a & a3) and CuB
Heme a3 and CuB form binuclear center → accepts electrons from 'heme a'
and transfers them to O2
The overall reaction: + + 4 cyt c (red) + 8 H N + O2 → 4 cyt c (ox) + 4H P + 2H2O Note: reaction has been doubled to balance equation w/o fractions.
Biochemistry 3300 Slide 34 Metal Center Ligands in Complex IV
CuA Accepts electrons from cytochrome C and passes electrons to 'heme a'
CuA ligands include His, Met, Cys and backbone amines
'Heme a' (right) Axial ligands are His N atoms. Heme a is held in place between 2 transmembrane α-helices by its axial His ligands. 'Heme a' transfer electrons to the binuclear center ('heme a3' and CuB)
Biochemistry 3300 Slide 35 Metal Center Ligands in Complex IV
Heme a3, is adjacent to CuB and has only one axial ligand (His)
CuB ligands are His side chains
O2 binds at the open axial ligand position of heme a3, adjacent to CuB.
Electrons are passed to the binuclear center (from 'heme a') where O2 is reduced.
The open axial ligand position of heme a3 makes it susceptible to binding of CN− , CO, or the radical signal molecule ·NO. All three compounds inhibit cytochrome oxidase (complex IV) activity.
Biochemistry 3300 Slide 36 Summary
Complexes I and II (and other dehyrogenases) pass electron to Q
QH2 serves as mobile carrier of electrons that are passed to Complex III
Complex III passes electrons to the mobile carrier cytochrome c.
Complex IV transfers electrons from cytochrome c to O2
Electron flow through Complexes I, III and IV is coupled to H+ transport across the membrane Biochemistry 3300 Slide 37 Energy from the respiratory chain is Conserved in a Proton Gradient
Transfer of two electrons from NADH through the respiratory chain:
NADH → NAD+ + H+ + 2e- 0.320 + - ½ O2 + 2H + 2e → + H2O 0.817
∆ 0 + + E’ = 1.14 V NADH + H + ½ O2 → NAD + H2O
Biochemistry 3300 Slide 38 Energy from the respiratory chain is Conserved in a Proton Gradient
The standard biochemical free-energy change is:
∆G’0 = - n F ∆E’0
= -2(96.5 kJ/V · mol)(1.14V)
= -220 kJ/mol
In the cell where the actual [NADH]/[NAD+] ratio is kept above 1 the real free-energy change is substantially more negative.
→ much of the energy is used to pump protons out of the matrix
Biochemistry 3300 Slide 39 Energy from the respiratory chain is Conserved in a Proton Gradient
For each pair of electrons transferred to O2 protons are pumped, 4 H+ by Complex I 4 H+ by Complex III, and 2 H+ by Complex IV
Total 10 H+ per e- pair → formation of a proton gradient
Biochemistry 3300 Slide 40 Energy from the respiratory chain is Conserved in a Proton Gradient
Energy stored in such a gradient can be termed proton-motive force.
It has two components: (1) Chemical potential energy (∆pH) → due to concentration difference (2) Electrical potential energy (∆ψ) → due to charge separation
In actively respiring mitochondria ∆ψ = 0.15 – 0.20 V ∆pH = 0.75
=(5.70 kJ/mol)∆pH + (96.5 kJ/V·mol)∆ψ Given that the free-energy change for pumping protons outward is ~20 kJ/mol (H+) it would require ~200 kJ/mol to pump 10 H+
Biochemistry 3300 Slide 41 Exception: thermogenesis
The mitochondria of plants, fungi, and EasternEastern skunk skunk cabbage cabbage unicellular eukaryotes have electron transfer systems that are essentially the same as those in in animals.
They also contain alternative enzymes: - → e are directly transferred to O2 → energy is released as heat without H+ pumping
Biochemistry 3300 Slide 42 Chemiosmotic Model
When electrons flow spontaneously down the electrochemical gradient, energy is made available to do work. → ATP synthesis
There is enough free energy stored in the proton gradient to drive the synthesis of ATP (50 kJ/Mol)
What is the chemical mechanism that couples the two processes?
Biochemistry 3300 Slide 43 Chemiosmotic Model
Proton-motive force drives the synthesis of ATP as protons flow into the matrix through a proton pore associated with an ATP synthase.
+ + ADP + Pi + n H P → ATP + H2O + n H N
Biochemistry 3300 Slide 44 Testing the Chemiosmotic Model
Energy of substrate oxidation generates a proton gradient, that drives the ATP synthesis → inhibitors of the electron transport chain influence ATP synthesis
Follow O2 consumption
(O2 Electrode) and ATP synthesis
Biochemistry 3300 Slide 45 Testing the Chemiosmotic Model
How do we explain this result?
DNP is a proton ionophore:
Destroys the proton gradient by transports protons across the membrane
“Uncouples” proton gradient and ATP synthesis
Biochemistry 3300 Slide 46 Testing the Chemiosmotic Model
Artificially electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrate as electron donor.
Example: Mitochondrial suspensions in buffered solutions (slowly adopt the pH of the buffer)
Lowering the pH of the solution (in the absence of electron donors) and in the presence of valinomycin allows ATP formation using endogenous mitochondrial ADP + Pi
Note: Valinomycin is a K+ ionophore that eliminates the electric term of the electrochemical potential which would oppose the proton gradient over time
Biochemistry 3300 Slide 47 Mechansim of ATP Synthesis
F1Fo ATP Synthase of mitochondria, chloroplasts, bacteria: + – F1Fo couples ATP synthesis (at F1) to gradient driven H transport (ie. opposite direction of electron transfer proton pumping)
Kinetic studies reveal the reaction is reversible:
Enz-ATP (Enz-ADP+Pi)
-1 -1 Keq = k1/k-1 = 10 s /24 s =0.42
If there is no ∆pH or ∆ψ to
drive the forward reaction, Keq favors the reverse reaction, ATP hydrolysis (ATPase).
Biochemistry 3300 Slide 48 ATP Synthase Has Two Functional Domains
Electron Microscopy: F1 appears as "lollipops" on the inner mitochondrial membrane, facing the matrix.
Urea wash (panel C): Gentle wash with denaturants removes
F1 from mitochondrial inner membrane
Biochemistry 3300 Slide 49 ATP Synthase Has Two Functional Domains
SMP F1 of intact mitochondria faces the interior or mitochondrial matrix
Roles of functional domains were established in studies of submitochondrial particles (SMP).
Mitochondria treated with ultrasound:
Inner membrane fragments and then reseals as vesicles with F1 on the outside!. These SMP are said to be inside out (inverted vesicles).
Biochemistry 3300 Slide 50 ATP Synthase Has Two Functional Domains
Inverted membrane vesicles from the inner mitochondrial membrane still contain the intact respiratory chain. → catalyze electron transfer
F1, the catalytic subunit, if separated from SMP catalyzes ATP hydrolysis SMP → Spontaneous reaction
If F1 is removed from SMP electron transfer from NADH to O2 continues but no H+ gradient is produced.
Membrane still contains Fo which acts as a proton pore. + Adding back F1 restores normal low permeability to H .
Biochemistry 3300 Slide 51 Inhibitors
+ Inhibitors of F1Fo, that block H transport coupled to ATP synthesis or hydrolysis, include:
– oligomycin, an antibiotic
– DCCD (dicyclohexylcarbodiimide), a reagent that reacts with carboxyl groups in hydrophobic environments, forming a covalent adduct.
Oligomycin and DCCD inhibit the ATP Synthase by interacting with Fo.
Both inhibitors block the Fo pore and prevent protons from crossing the membrane when depleted of F1.
Biochemistry 3300 Slide 52 The Structure of Mitochondrial F1
The complete subunit composition of the ATP Synthase was first established in E. coli, which has an operon that encodes genes for all subunits.
F1 in E. coli consists of 5 polypeptides with
stoichiometry α3, β3, γ, δ, e (named in order of decreasing mol. weights).
α & β subunits (513 & 460 aa in E. coli) are homologous.
Three nucleotide-binding catalytic sites, located at αβ interfaces but predominantly involving residues of the β subunits. Each α subunits contains an additional tightly bound ATP not involved in catalysis. Adenine nucleotides bind to α & β subunits with Mg++.
Biochemistry 3300 Slide 53 The Structure of Mitochondrial F0
Fo is a complex of integral membrane proteins. – The stoichiometry of subunits
in E. coli Fo is a, b2, c10.
E. coli
Mammalian F1Fo is slightly more complex than the bacterial enzyme. Since names were originally assigned based only on apparent MW, some subunits were given different names in different organisms. – Bovine δ subunit is homologous to E. coli ε subunit. – Bovine "OSCP" is homologous to E. coli δ subunit. – Bovine ε subunit is unique.
Biochemistry 3300 Slide 54 Mitochondrial ATP Synthase Complex
Bovine mitochondrial F1 Yeast mitochondrial Fo
PDBid 1BMF PDBid 1QO1 Biochemistry 3300 Slide 55 The Binding Change Mechanism
Binding change mechanism proposed by Paul Boyer (Nobel Prize).
Accounts for the existence of 3 catalytic sites in F1.
For simplicity, only the catalytic β subunits are shown
It is proposed that an irregularly shaped shaft (green) linked to Fo rotates relative to the ring of 3 β subunits. + The rotation is driven by flow of H through Fo.
Biochemistry 3300 Slide 56 The Binding Change Mechanism
The conformation of each β subunit changes sequentially (and simultaneously) as it interacts with the rotating shaft.
Loose Tight
Eg., the upper subunit (yellow) sequentially changes from:
a loose conformation in which the active site can loosely bind ADP + Pi a tight conformation in which substrates are tightly bound and ATP is formed an open conformation that favors ATP release.
At any one time, each β subunit is at a different stage of the catalytic cycle
Biochemistry 3300 Slide 57 Supporting Evidence
Bovine F1 (DCCD- treated)
90°
PDBid 1E79
Crystal structure of F1 was solved by J. E. Walker (Shared Nobel Prize).
The γ subunit includes a bent helical loop that constitutes a "shaft" within the ring of a & b subunits.
Shown is bovine F1 treated with DCCD to yield crystals in which more of the stalk is ordered, allowing structure determination. Colors: α, β, γ, δ, ε.
Biochemistry 3300 Slide 58 Supporting Evidence
Bovine F1 (DCCD- treated)
90°
PDBid 1E79 Note the wide base of the rotary shaft, including part of γ as well as δ and ε subunits.
Recall that the bovine δ subunit, which is at the base of the shaft, is equivalent to ε of bacterial F1.
Biochemistry 3300 Slide 59 Supporting Evidence
90°
PDBid 1COW
In crystals of F1 not treated with DCCD, less of the shaft structure is solved, but ligand binding may be observed under more natural conditions.
The 3 β subunits are found to differ in conformation & bound ligand.
Biochemistry 3300 Slide 60 Supporting Evidence
Bound to one β subunit is a non-hydrolyzable ATP analog (assumed to be the tight conformation). Bound to another β subunit is ADP (loose). The third β subunit has an empty active site (open).
This is consistent with the binding change model, which predicts that each β subunit, being differently affected by the irregularly shaped rotating shaft, will be in a different one of 3 stages of the catalytic cycle.
Biochemistry 3300 Slide 61 ATP
ATP Regulatory ATP (white) bound to α subunits
ATP
Empty
ADP
ATP PDBid 1COW
Biochemistry 3300 Slide 62 Supporting Evidence - Rotation of the γ Shaft
Rotation of the γ shaft relative to the ring of α & β subunits was demonstrated by Noji, H. et al., Nature 386, 299-302 (1997).
β subunits of F1 were tethered to a glass surface.
A fluorescent-labeled actin filament was attached to the protruding end of the γ subunit. Video recordings showed the actin filament rotating like a propeller. The rotation was ATP-dependent.
Biochemistry 3300 Slide 63 Supporting Evidence Rotation of the γ Shaft
The rotation is ATP-dependent. → stepping
20 nM ATP 200 nM ATP (slow) (fast)
Biochemistry 3300 Slide 64 Supporting Evidence Rotation of the γ Shaft
The rotation also load dependent
The larger the actin filament ….
…the slower the rotation
Biochemistry 3300 Slide 65 Rotation of the γ Shaft
Studies using varied techniques have shown ATP-induced rotation to occur in discrete 120° steps, with intervening pauses.
Some observations indicate that each 120° step consists of 90° & 30° substeps, with a brief intervening pause. Proposals have been made correlating these substeps with particular
stages of the reaction cycle, such as ATP binding and Pi release.
Biochemistry 3300 Slide 66 Subunit Arrangement in the F1FO Complex
Mitochondrial ATP Synthase E. coli ATP Synthase
Each of the 2 Fo b subunits is predicted to include 1 trans-membrane α- helix & a very polar, charged α-helical domain that extends out from the membrane.
Biochemistry 3300 Slide 67 Coupling between ATP Synthesis and Proton Flow
The a subunit of Fo (271 amino acid residues in E. coli) is predicted from hydropathy plots, to include several transmembrane α-helices.
It has been proposed that the a-subunit forms 2 half- channels or proton wires (each a series of protonatable groups or embedded waters), that allow passage of protons between the two membrane surfaces & the bilayer interior.
Biochemistry 3300 Slide 68 Proton Transfer
The c subunit of Fo has a structure with 2 transmembrane α-helices & a short connecting loop. The small c subunit (79 aa in E. coli) is also called proteolipid, because of its hydrophobicity. One α-helix includes an Asp or Glu residue whose carboxyl reacts with DCCD (Asp61 in E. coli). Mutation studies have shown that this DCCD-reactive carboxyl, in the middle of the bilayer, is essential for H+ transport through Fo.
An essential arginine residue on one of the trans-membrane a-subunit α- helices has been identified as the group that accepts a proton from Asp61 and passes it to the exit channel.
Biochemistry 3300 Slide 69 Proton Transfer
As the ring of 10 c subunits rotates, the c- subunit carboxyls relay protons between the 2 a-subunit half-channels. This allows H+ gradient-driven H+ flux across the membrane to drive the rotation.
Biochemistry 3300 Slide 70 Biochemistry 3300 Slide 71 Proton Motive Force - Part II
Proton motive force also drives transport processes.
The inner mitochondrial membrane is generally impermeable to charged
species → but ADP and Pi are needed in the matrix and ATP is used outside!
The adenine nucleotide translocase.
integral inner membrane complex
ADP3- (intermembrane space) is exchanged for ATP4- (antiporter)
Translocase moves 3 negative charges in and 4 out → favoured by the electrochemical gradient → neutralizes a portion of the electrical gradient
Biochemistry 3300 Slide 72 Proton-Motive Force - Part II
A second transport system essential for oxidative phosphorylation: → phosphate translocase
Facilitates the symport of - + H2PO4 and H into the matrix.
Transporting one H+ across the membrane helps drive process but consumes some of the proton gradient
Biochemistry 3300 Slide 73 NADH entry into the Mitochondrium
NADH generated by dehydrogenases in the cytosol (Glycolysis) has to be transported into the mitochondria matrix.→ Malate-aspartate shuttle
NADH is not truly transported Into the mitochondria
Instead it is consumed in the intermembrane space and regenerated in the mitochondria
Most active in liver, kidney & heart.
Biochemistry 3300 Slide 74 NADH entry into the Mitochondria
Skeletal muscle and brain tissue use the glycerol 3-phosphate shuttle.
Mitochondria of plants have an externally oriented NADH dehydrogenase.
→ transfers e- directly to ubiquinone.
Unlike malate-asparate shuttle:
Glycerol-3-phosphate shuttle only pumps 6 protons / NADH as it bypasses complex I
Biochemistry 3300 Slide 75 Inhibition of F1Fo ATP Hydrolysis
When a cell is deprived of oxygen the transfer of electrons to O2 ceases.
What might happen?
→ e- dependent proton pumping ends → proton motive force soon collapses → ATP synthase could start to hydrolyze ATP
Hydrolysis of ATP in the absence of O2 would rapidly lead to cell death!!
This is prevented by a small (84 aa) protein (IF1)
Biochemistry 3300 Slide 76 Inhibition of F1Fo ATP Hydrolysis
IF1 binds simultaneously to two ATP synthase molecules.
IF1 is only inhibitory in its dimeric form → dimerization occurs at (slightly) lower pH
How does this regulate inhibition?
Under oxygen starvation, pyruvate and lactate are accumulate (both are acids) → lowers pH in the cytosol and the mitochondrial matrix
Biochemistry 3300 Slide 77