BIO 212 Topic 14: The Mitochondrial Inner Membrane—Electron Transport Chain and Oxidative Phosphorylation

The mitochondrion

• responsible for ATP synthesis/cellular respiration

• Krebs cycle in the mitochondrial matrix

• Oxidative phosphorylation in the membrane

• Two membranes—outer and inner, both derived from the ancestral bacteria

Outer mitochondrial membrane

• composition: 1:1 protein ot lipid ratio by weight (is about typical for a membrane)

• highly permeable to due to the presence of beta barrel porins, which have openings wide enough to pass anything less than 5,000 Da (including protons, ATP, etc.)

• if the outer membrane is permeable to H+, how does the cytosol not become more acidic/IMS become more basic?

  • relative volume of mitochondria and cytoplasm

  • similar pH of IMS and cytoplasm

Inner mitochondrial membrane

• folds in to form invaginations that increase surface area (cristae)

• composition—3:1 protein to lipid ratio by weight

• HIGHLY impermeable; charged and polar things must be transported via transporter proteins

• total surface area in the human body is about 14,000 square meters!

Mitochondrial matrix

• includes about 2/3 of all mitochondrial proteins, and hundreds of different enzymes

• location of citric acid cycle

Mitochondrial proteins

• mitochondrial DNA only codes for 13 polypeptides, each of which is found in the ETC or in ATP synthase

• other proteins must be imported

Mitochondrial DNA

• coiled, circular, very small

• 16,529 bp (compared to 3.2 billion in human genome)

• 13 structural genes, 22 tRNA genes, 2 rRNA genes

• has more copies of its DNA than human cells (10-12 copies per mitochondria = higher levels of expression)

Oxidative phosphorylation

• oxidative = electrons are oxidized; energy lost by the electrons as they move through the ETC is used to fuel H+ transport (create a membrane potential), which is used to power ATP via ATP synthase (do work)

• phosphorylation = ADP is phosphorylated to make ATP

ATP structure

• adenine

• ribose (adenine + ribose = adenosine)

• 3 phosphate groups (alpha = adenylic acid/AMP, beta = ADP, gamma = ATP)

Glycolysis

• 1 glucose —> 2 pyruvate

• Generates 2 ATP, 2 NADH

• Occurs in cytosol

Pyruvate processing (oxidative decarboxylation of pyruvate)

• 2 pyruvate —> 2 acetyl coA + 2 CO2

• Generates 2 NADH

• Occurs in mitochondrial matrix

Citric acid (Krebs) cycle

• 2 acetyl coA —> 2 acetyl CoA (cycle)

• Generates 2 ATP, 6 NADH, 2 FADH2, 2 CO2

• 8 steps

• Occurs in mitochondrial matrix

• can be fueled by carbohydrate breakdown (i.e. pyruvate), but also the degradation of fatty acids and amino acids

ETC

• Two energy electrons are taken from NADH and FADH2 (one pair per molecule) and moved sequentially along the electron transport chain via redox reactions until they reduce O2 to H2O

• energy released is used to power active transport of H+ across the inner membrane, creating a gradient whose dissipation is used to power ATP synthesis (among some other things)

• moves in order of increasing redox potential of ETC components

• are found in all living organisms

Redox potential

• measure of potential energy

• measure of how badly a molecule wants an electron

• ex: NADH is a pretty good electron donor, but O2 is not because it wants all its electrons

Reduction potential

• the tendency of a chemical species to be reduced (gain electrons)

Where are ETCs found?

• bacteria, protists, (archaea?)—plasma membrane

• Fungi, animals (mitochondria)

• plants (mitochondria, chloropalsts)—chloroplasts make ATP for their own use

Complex I (NADH dehydrogenase complex)

• uses energy from a pair of electrons supplied by NADH to pump four protons into the inter-membrane space

• passes electrons to coenzyme Q

• called NADH dehydrogenase because the removal of electrons is also often accompanied by protons/hydrogens

• 7 mtDNA subunits, 38 nDnA subunits—huge! and has a MM of over 900,000 Da

• Two prosthetic groups—FMN and Fe-S

Prosthetic groups

• small, tightly-bound non-amino acid units required for the biological function of some proteins

• are involved in electron passage; rather than binding directly to the protein, electrons bind to these prosthetic groups

• serve as a place for the electron to step as they move through the electron transport chain

• can be small metal ions (cofactors) or small organic molecules (coenzymes)

Fe-S

• prosthetic group (inorganic metal ions—cofactors)

• iron sulfur complexes

• are complexed to cysteines—held tightly to the protein, but are not part of it

• oxidized form is Fe3+

• reduced form is Fe2+

FMN (flavin mononucleotide)

• small organic nucleotide prosthetic group (coenzyme)

• phosphate, linear pentose sugar, and the nitrogenous base flavin (derived from riboflavin/vitamin B2, which is also used in the synthesis of the coenzyme FADH2)

• oxidized form = quinone state, 2 double bonds

• intermediate free radical (semiquinone state) = 1 double bond, 1 free radical

• reduced form = hydroquinone state, 1 double bond

Complex III (cytochrome b-c1 complex)

• used energy from the electrons supplied by coenzyme Q to pump four more protons into the intermembrane space

• electron handoff from coenzyme Q to complex III is complicated, so sometimes (about 2% of the time), electrons escape, introducing free radicals (oops!)

• called cytochrome (cell color) because of its red-brown color, which results from its heme-containing proteins (heme porphyrin ring is highly conjugated)

• 1 mtDNA subunit, 10 nDNA

• Has Fe-S and heme prosthetic groups

Heme groups

• cofactors which covalently bind to proteins via cysteines

• composed of a porphyrin ring with an iron ion in the center

• like Fe-S, oxidized form has Fe3+ and reduced form has Fe2+ (transfer of one electron)

Complex IV (cytochrome oxidase complex)

• uses energy from the electrons (supplied by cytochrome C) to pump two protons into the intermembrane space

• passes the “used” electrons to ½ O2 and 2 H+ to form H2O—oxygen is the terminal electron acceptor

• step in the ETC characterized by the largest drop in potential energy

• 3 mtDNA subunits, 10 nDNA

• has copper ion (2+, 1+) and heme prosthetic groups

Ubiquinone (coenzyme Q)

• electron carrier

• lipid/polyisoprenoid

• has a head group with four double bonds—double bonds can be reduced!

• oxidized form — quinone state, 4 double bonds

• intermediate free radical — ubisemiquinone state, 3 double bonds and a free radical

• reduced form — ubiquinol state, 3 double bonds

Cytochrome C

• peripheral membrane protein (104 amino acids)

• held to the membrane by electrostatic attractions to the polar heads—is highly basic and has lots of positive charges to hold tightly to the negative phospholipid head groups

• electron carrier

• heme prosthetic group

Discovery of order of electron flow (ETC pathway)

• hemes and quinones have different absorption spectra in oxidized vs. reduced forms because of conjugation and shifting of double bond location during oxidation and reduction reactions; based on the absorption profile, we can tell whether the prosthetic group is oxidized or reduced

• used chemical inhibitors of electron flow to study—road blocks put at different points to see what is reduced and what is oxidized (everything before the “road block” remains reduced; everything after can continue moving the ETC)

Rotenone

• inhibitor of complex I

• widely used as an insecticide in agriculture

• poses significant risk to long-term agricultural workers

Antimycin A

• inhibitor of complex III

Complex IV inhibitors

• cyanide

• CO (although affinity is not very high compared to affinity for hemoglobin, so you will probably suffocate first)

• Azides (N3-); blocks final transfer of electrons to O2, so the entire ETC halts and the body rapidly depletes its ATP

FADH electrons

• lower energy than NADH

• enter the ETC at a correspondingly lower point (complex II)

• pumps 6 protons per electron pair (four from complex III, two from IV)

Complex II (succinate dehydrogenase)

• is also an enzyme in the Krebs cycle

• 0 mtDNA subunits, 4 nDNA

• brings electrons from FADH2 into the system; is not a pump

• prosthetic groups are Fe-S and FAD

• transfers two electrons directly to ubiquinone, so these electrons never pass through complex I because they are not high enough energy

Concentration difference (ΔpH) over IMM

• matrix pH is about 7.6-8.2

• IMS is about 7.2

• the IMS is so huge that it stays about at cytosolic pH, but the matrix is depleted of protons, so it becomes more basic

Charge difference (ΔΨ) over the IMM

• mitochondrial matrix is negative relative to the IMS

Chemiosmotic hypothesis, 1961

• proposed by Peter Mitchell

• H+ gradient drives ATP synthesis directly (opposed prevailing conviction that some phosphorylated intermediate hands its own phosphate off to ADP to make ATP)

• H+ gradient formed from electron transfers creates a proton-motive force that powers ATP synthesis

ATP synthase

• first observed in the early 1960s via TEM

• 15,000 ATP synthases in the typical liver mitochondrion (of which there are about 1500)

ATP synthase structure

• Fo base

• F1 head

• roughly proposed by Paul Boyer, 1979 (no evidence)

• visualized by John Walker via x-ray crystallography in 1994 (before cryo-EM)

Fo base

• is the letter o, not zero—stands for oligomycin, which is the inhibitor that blocks H+ transport through the complex

• ab₂c_10-14

• transmembrane region of the ATP synthase

a subunit (Fo)

• consists of two hydrophilic half-channels, one which feeds protons to the c ring (entrance channel) and one which allows protons to exit the c ring and pass into the mitochondrial matrix

• allows passage of H+ across the inner membrane

b subunit(s) (Fo)

• connect the functional units together and makes sure the F1 portion does not fly off the Fo base

• contacts the F1 gamma subunit

c subunits (Fo)

• make up a ring that acts as a rotating proton channel connecting the two half-channels

• very mechanical structure—one of the few “wheels” that have evolved in biology

• aspartic residue (Asp61) in the subunit binds to a proton, causing it to undergo conformations that cause the ring to rotate—captures potential energy from the protons as kinetic energy

• motions spin the gamma subunit

F1 head

• is the coupling factor—couples energy released from the proton gradient with energy required for ATP synthesis

• α3β3γδε

• projects into the matrix

• three enzymatic sites (αβ) pairs

• held to the Fo unit by b, δ, and ε subunits

γ (gamma) subunit

• connects Fo to F1

• asymmetrical/irregular shaped, so as it turns with the c ring, it presents a different face to the alpha beta pairs to which it is bound, inducing a cycle of conformational changes to accommodate the spinning

Alpha beta pairs

• enzymatic sites; undergo conformational changes (each pair has three distinct conformations)

• binding change mechanism: open —> loose —> tight —> back to loose

• fairly slow as far as enzymes go because of its complexity; catalyzes synthesis of about 100 ATP per second

• ATP formation is spontaneous—many enzyme reactions involve at least one side chain in the enzyme covalently bonding to the substrate in the transition state; when the product is formed, the covalent bond is cleaved and the product is released

  • Since there is no evidence of ATPase doing this, it implies that the reaction occurs as long as ADP and Pi are close enough

Open conformation (alpha beta pair)

• active site is open; ADP and Pi enter the site and ATP leaves

Loose conformation (alpha beta pair)

• ADP and Pi are bound to the ATP synthase and are aligned within the active site

Tight conformation (alpha beta pair)

• ATP is bound

Rotational catalysis

• each catalytic site (alpha beta pair) is in a different conformation

• note that the subunits stay in place relative to the others, while the gamma subunit moves/rotates and induces the conformational changes

• movement of three protons across the membrane makes an ATP

• evidence—how do we visualize the spinning?

Rotational catalysis experiment

• attached a fluorescent actin filament to the gamma subunit

• added exogenous ATP to power the spinning of the gamma subunit (remember that reactions, even enzyme-catalyzed reactions, are reversible—only depends on concentration)

• flooding of ATP reverses reaction, gamma subunit spins (in the opposite direction); ATP synthase becomes a H+ ATPase

• beta subunits were attached to the glass coverslip via His-tags

• observed that the actin filament spins, although not smoothly (corresponds with conformational change)

Crystal structure of ATP synthesis

• published 1994

• F1-ATPase at 2.8 angstrom resolution (the width of an ATOM is just a couple of angstroms!)

• John Walker

• showed that the F1 structure was consistent with Boyer’s proposed method of energy coupling between H+ gradient and ATP synthesis

ATP synthasome

• ATP synthase molecules are often found in non-covalently associated pairs, which seem to cause a bend in the inner mitochondrial membrane—bends in the cristae are often very rich in ATP synthase complexes

• pair is also noncovalently associated with the ADP/ATP transporter

Stoichiometry of ATP synthesis

• theoretically, 36 or 38 ATP molecules can be formed per glucose—but in reality, about 30 ATP molecules are formed per glucose

• Some of the electrochemical (pH, charge) gradient is depleted to do other types of work

• pH gradient drives the transport of phosphate and pyruvate from the intermembrane space into the matrix (pyruvate/phosphate up gradient and H+ down gradient); depletes the charge and concentration gradient

• the charge/voltage gradient drives ADP-ATP exchange; ATP (four negative charges at physiological pH) leaves the matrix and ADP enters (three negative charges), creating a net movement of one negative charge out which depletes the electrical gradient

ATP synthesis in the body

• about 2 × 10²6 ATP molecules—hundreds of mols—are synthesized per day in a human at average activity

• the overall efficiency of converting energy in glucose to energy in ATP is about 42%, which is very efficient—consider a series of 5 reactions, each with an efficiency of 90%—would give an overall efficiency of about 59%, and the Krebs cycle alone is 8 reactions

• PE is lost to random thermal motion/entropy/increased disorder (the first law of thermodynamics)

Uncouplers

• pump/transport protons, but do not do any work—essentially wastes the proton gradient

• proton ionophores/proton shuttles

2,4 dinitrophenol (DNP)

• pharmacological uncoupler

• lipid soluble (can dissolve in the membrane and easily cross)

• weak acid (can be both protonated and deprotonated very easily

• picks up a proton from one side, diffuses across the membrane, and deprotonates in the matrix—“short circuits” the H+ gradient

• potential energy from the gradient is lost as heat

• was widely prescribed as a weight loss drug until people started dying due to problems making ATP, excess heat from loss of potential energy, and because the drug is non-specific—not only did it increase H+ permeability in the mitochondrial membrane, but also the lysosomes

Uncoupling proteins (UCPs)

• whole family of naturally occurring molecules that “waste” the H+ gradient

UCP1 (thermogenin)

• thermogenin = heat genesis (creation)

• pretty much the same function as DNP, but highly regulated expression

• critical in non-shivering thermogenesis

• expressed in the mitochondrial membrane of brown adipose tissue (mammals); brown fat cells are stuffed full of mitochondria with UCP1 to generate heat and maintain temperature homeostasis—causes cells to be brown because they have lots of mitochondria and therefore lots of cytochromes

• especially important for hibernating animals to maintain homeostasis

Mitochondrial diseases

• first cellular organelle linked to a human disease (Luft’s disease)

• have the largest effects on organs and tissues with high energy demand (brain, muscles, heart, etc.)

• symptoms often include dementia, deafness, blindness, problems moving the eyes, drooping eyelids, muscle weakness, poor balance, seizures, heart failure, tremors, vomiting, chronic heartburn, acid reflux, trouble feeling, poor growth, liver problems, and respiratory problems

Luft’s disease

• first human disease to be linked to a cellular organelle

• Rolf Luft (1962) studied a 27 yo female patient who presented with fatigue/muscle weakness, weight loss even on a daily calorie intake of over 3000 kcals, a high body temperature, and the highest BMR ever recorded

• is characterized by isolated mitochondria with high ATPase activity that is only slightly stimulated by 2,4 DNP

• mitochondria are “loosely coupled” (mostly uncoupled) and therefore proton gradient is generated but mostly wasted