III. Metabolism Oxidative...

Biochemistry 3300 Slide 1

III. Metabolism

Oxidative Phosphorylation

Department of Chemistry and BiochemistryUniversity of Lethbridge

Biochemistry 3300


Biochemical Anatomy of Mitochondria

Transmembrane channels allow smallmolecules (< 5 kD) and ions to passthrough the outer membrane.

Convolutions of the inner membraneprovides large surface area.→ depending on the tissue they are

more or less profuse

Specific transporters carry pyruvate,fatty acids and amino acids intothe matrix for access to thecitric acid cycle.


Universal Electron Acceptors Collect Electrons

Dehydrogenases (catabolism) transfer electrons touniversal electron carriers which funnel electrons into the respiratory chain M mitochondria

C cytosol


Electron Carriers I. Nicotinamide Adenine Dinucleotide

Optical Test

NAD+ / NADP+ accept a hydride (H-) and a proton is released


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.


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+++).


Electron CarriersIII. 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)


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.


Electron CarriersIV. 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.


Electron Carriers - Cytochromes

PDBid 5CYT

His18

Met80

Cytochrome c Heme iron undergoes 1 e- transition between ferric and ferrous states:

Fe+++ + e- ↔ Fe++

Heme Fe interacts with: - 4 N of polyporphyrin ring and - 2 axial ligands above & below heme

Axial heme Fe ligands: His18 and Met80

X N N Fe N N Y

Axial ligands alter reduction potential of heme Fe


Electron CarriersIV. Cytochromes

Heme prosthetic group absorbs light atcharacteristic wavelengths

Absorbance spectra can follow the redox state of the heme(same as for all other electron carriers)

Many cytochromes are subunits of largeintegral membrane complexes containingmultiple electron carriers

- located within mitochondrial inner membrane

Cytochrome c is a small, water-solubleprotein with a single heme group


Electron CarriersV. 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.


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 residuesare named Rieske iron-sulfur proteins.


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


Respiratory Chain

Most respiratory chain proteins are embedded in the inner mitochondrial membrane (or in the cytoplasmic membrane of aerobic bacteria).


Respiratory Chain Complexes

Protein components of the electron-transfer chain are primarily organized as large, transmembrane (or membrane associated) protein complexes


Respiratory Chain

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.

4H+


Respiratory Chain

The standard reduction potentials of constituent e- carriers are consistent with the e- transfers observed.

4H+


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


Effect of Inhibitors on Electron Transport

Oxygen electrode: O2 selective membrane permits measurement of [O

2]

O2 produced in sample

chamber is reduced by anode generating a measurable current


Electron Transport Inhibitors

Experiment (sample chamber of O2 electrode):

Buffered mitochondria solution with excess ADP + Pi are equilibratedReagents added and [O

2] is monitored over time

Example experiment:1 - Hydroxybutyrate is substrate that allows TCA cycle to function; NADH is source of electrons O

2 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 O

2 levels remain constant as electrons do not reach


3 - Succinate provides electrons via Complex II O

2 levels will decrease as e- are transferred from

complex II to complex IV where O2 is reduced

4 - Antimycin inhibits complex III O

2 levels remain constant as electrons do not reach


5 - TMPD/Ascorbate provide electrons to cyctochrome C O

2 levels will decrease as e- are transferred from

cytochrome C to complex IV where O2 is reduced

6 - CN- (or CO) inhibit complex IV O

2 levels remain constant as O

2 is not reduced


Complex I

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.

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:

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.

Bovine complex I at 17 Å resolution.

Grigorieff, N. (1998). J. Mol. Biol., 277, 1033-1046


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+


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.


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.


Complex II

PDBid 1NEK

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.


Path of Electrons to Ubiquinone

Other substrates for mitochondrialdehydrogenases pass their e-

into the respiratory chain at thelevel of ubiquinon, but not throughcomplex II.

Example:

Fatty acyl-CoA electronsvia

Acyl-CoA dehydrogenase (β oxidation)via

ETF (electron transferring flavoprotein)via

ETF:ubiquinone oxidoreductaseto

Reduced CoQ


β Oxidation

Mitochondria contain four acyl-CoA DH with different fatty acyl-CoA specificities:

short (C4 to C6)medium (C6 to C10)

long (between medium & very long)very long (C12 to C18)

Glu376

PDBid 3MDE

The FADH2 is reoxidized by the mitochondrialelectron transport chain.


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 electronsto cytochrome c with the vectorialtransport of protons from thematrix to the inermembrane space.

Cytochrome c1, a prosthetic group within complex III, reduces cytochrome c, which is the electron donor to complex IV.


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·−.


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.


Complex III

PDBid 1BE3

Rieske protein

Me

mb

ran

e

Cytochrome c1

Heme bL

Heme bH

Rieske iron-sulfur center (Fe-S) has a flexible link to the rest of the complex.

- it changes position during e− transfer.

Rieske Fe-S extracts an e− from CoQ, and moves closer to heme c1, to which it transfers the e−.


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 muchsimpler (3-4 SU).

Comparison of the two forms suggests that three are critical to the function.


Complex IV

Mitochondrial subunit II contains two Cu ions coordinated to two Cys residues.

Subunit I contains two heme groups (a & a3) and Cu

B

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 tobalance equation w/o fractions.


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 a

3' and Cu

B)

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 O

2 is

reduced.

Metal Center Ligands in Complex IV

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.


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 acrossthe membrane


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

NADH + H+ + ½ O2 → NAD+ + H2O∆E’0 = 1.14 V



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 realfree-energy change is substantially more negative.

→ much of the energy is used to pump protons out of the matrix



For each pair of electrons transferred to O2 protons are pumped,4 H+ by Complex I4 H+ by Complex III, and2 H+ by Complex IV

Total 10 H+ per e- pair → formation of 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

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+

=(5.70 kJ/mol)∆pH + (96.5 kJ/V·mol)∆ψ



Exception: thermogenesis

Eastern skunk cabbageThe mitochondria of plants, fungi, and unicellular eukaryotes have electron transfer systems that are essentiallythe same as those in in animals.

They also contain alternativeenzymes:→ e- are directly transferred to O2

→ energy is released as heat without H+ pumping

Eastern skunk cabbage


Chemiosmotic Model

When electrons flow spontaneously down the electrochemical gradient, energy is made available to do work. → ATP synthesis

There is enough freeenergy stored in theproton gradient to drivethe synthesis of ATP(50 kJ/Mol)

What is the chemicalmechanism that couplesthe two processes?


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


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



How do we explain this result?

DNP is a proton ionophore:

Destroys the proton gradientby transports protons acrossthe membrane

“Uncouples” proton gradientand ATP synthesis



Artificially electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrateas electron donor.

Note: Valinomycin is a K+ ionophore that eliminates theelectric term of the electrochemical potential which wouldoppose the proton gradient over time

Example:Mitochondrial suspensions in buffered solutions(slowly adopt the pH of the buffer)

Lowering the pH of the solution (in the absenceof electron donors) and in the presence of valinomycin allows ATP formation usingendogenous mitochondrial ADP + P

i


Mechansim of ATP SynthesisF1Fo 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)

If there is no ∆pH or ∆ψ to drive the forward reaction, K

eq

favors the reverse reaction, ATP hydrolysis (ATPase).

Kinetic studies reveal thereaction is reversible:Enz-ATP (Enz-ADP+Pi)

Keq

= k1/k-1 = 10 s-1/24 s-1

=0.42


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 removesF

1 from mitochondrial inner membrane



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).

SMPF1 of intact mitochondria faces the interior or mitochondrial matrix



SMP

If F1 is removed from SMP electron transfer from NADH to O2

continues but no H+ gradient is produced.

F1, the catalytic subunit, if separated from SMP catalyzes ATP hydrolysis → Spontaneous reaction

Inverted membrane vesicles from the inner mitochondrial membrane still contain the intact respiratory chain. → catalyze electron transfer

Membrane still contains Fo which acts as a proton pore. Adding back F1 restores normal low permeability to H+.


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.


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++.


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.


Mitochondrial ATP Synthase Complex

Bovine mitochondrial F1

PDBid 1BMF

Yeast mitochondrial Fo

PDBid 1QO1


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.


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


Supporting Evidence

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.

Bovine F1

(DCCD- treated)


Supporting Evidence

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.

Bovine F1

(DCCD- treated)

90°

PDBid 1E79


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.


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.


ATP

ATPATP

ADP

ATPPDBid 1COW

Empty

RegulatoryATP (white)bound toα subunits


Supporting Evidence - Rotation of the γ Shaft

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.

Rotation of the γ shaftrelative to the ring of α & β subunits was demonstrated byNoji, H. et al., Nature 386, 299-302 (1997).

β subunits of F1 were tethered to a glass surface.

http://www.res.titech.ac.jp/~seibutu/main_.html

http://www.k2.ims.ac.jp/Publications.html





Supporting EvidenceRotation of the γ Shaft

The rotation is ATP-dependent. → stepping

20 nM ATP (slow)

200 nM ATP (fast)


Supporting EvidenceRotation of the γ Shaft

The rotation also load dependent

The larger the actin filament ….

…the slower the rotation


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.


Subunit Arrangement in the F1FO Complex

E. coli ATP SynthaseMitochondrial 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.


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.


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.


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.



Proton Motive Force - Part II

Proton motive force also drives transport processes.

The inner mitochondrial membrane is generally impermeable to chargedspecies → 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) isexchanged for ATP4- (antiporter)

Translocase moves 3 negative charges in and 4 out → favoured by the electrochemical gradient→ neutralizes a portion of the electrical gradient


Proton-Motive Force - Part II

A second transport system essential for oxidative phosphorylation:→ phosphate translocase

Facilitates the symport ofH2PO4

- and H+ into the matrix.

Transporting one H+ across themembrane helps drive process butconsumes some of the proton gradient


NADH entry into the Mitochondrium

NADH generated by dehydrogenases in the cytosol (Glycolysis) has tobe transported into the mitochondria matrix.→ Malate-aspartate shuttle

Most active in liver, kidney & heart.

NADH is not truly transportedInto the mitochondria

Instead it is consumed in theintermembrane space andregenerated in the mitochondria


NADH entry into the Mitochondria

Skeletal muscle and brain tissue use the glycerol 3-phosphate shuttle.

Mitochondria of plantshave an externally orientedNADH dehydrogenase.

→ transfers e- directly to ubiquinone.

Unlike malate-asparate shuttle:

Glycerol-3-phosphate shuttleonly pumps 6 protons / NADHas it bypasses complex I


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)


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

Date post:	30-Oct-2020
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

III. Metabolism Oxidative...

Documents