Chapter 5 - Aerobic Respiration and the Mitochondrion
How does mitochondrial fission occur?
apparently induced by contact with thin tubules from the ER, which can encircle the mitochondrion like a noose. The ER tubules initial constriction then it is completed through action of soluble protein that are recruited to the outer surface of mitochondrion from the cytosol
How is NADH that is formed during glycolysis in the cytosol transferred into the mitochondria?
enters via the malate-aspartate or glycerol phosphate shuttles
What is formed in peroxisomes that is dangerous?
hydrogen peroxide (H₂O₂) which is a reactive and toxic compound is formed in peroxisomes. It is broken down by the enzyme catalase
What promotes the movement of H⁺ ions through cytochrome oxidase?
in cytochrome oxidase, electrons are transferred one at a time. The energy released by O₂ reduction is presumably used to drive conformational changes that promote the movement of H⁺ ions through the protein
describe an experimental method to look at the sequence of carriers in the ETC
inhibitors can be added. Addition of rotenone blocked the electron transport at a site that left NADH and FMN in the reduced state preventing the transport of electrons to ubiquinone and therefore left cytochrome b, c and a in the oxidized state. This indicates that NAD and FMN are upstream of the rotenone block. In a similar way other blocks such as an antimycin A block and cyanide block can be used to see what carriers remain reduced or oxidized
Describe the formation of ATP utilizing the Na⁺/K⁺ - ATPase
it was seen only in vitro that the Na⁺/K⁺ - ATPase pump with the ions moving along their concentration gradients was able to drive ATP formation from ADP
peroxisomes
membrane bound vesicles that contain oxidative enzymes. They oxidize very long chain fatty acids and synthesize plasmalogens. They form by splitting from preexisting organelles, import preformed proteins and engage in oxidative metabolism
What does the binding change mechanism theory state in regards to the basis of ATP formation
movement of protons through ATP synthase alters the binding affinity of the active site and each active site goes through distinct conformations that have different affinities for substrates and products
What does the fusion and fission of mitochondria determine?
the balance between the fusion and fission of mitochondrial is a major determinant of mitochondrial number, length and degree of interconnection
describe the two components of the proton gradient/proton motive force
the concentration gradient between the matrix and the intermembrane space creates a pH gradient (∆pH) and the separation of charge across the membrane creates an electric/membrane potential. The energy in both components is proton-motive force (∆p)
chemiosmosis
the coupling of H⁺ translocation to ATP synthesis
Ubiquinone
the electon carrier (UQ, coenzyme Q) is a lipid-solube molecule made of five-carbon isoprenoid units
describe the experiment looking at ATP formation with ATP synthase in submitochondrial particles
the mitochondrion was isolated and sonicated to prepare submitochondrial particles. The submitochondrial particles contained ATP synthase still attached with ATP formation and substrates being oxidized. When treated with urea to dissociate the F₁ particles, the substrates were oxidized by no ATP was formed
how are electron carriers arranged in the electron transport chain?
they are arranged in increasingly positive redox potential
Describe the size and shape of mitochondria
typical mitochondria are bean-shaped organelles but may be round or threadlike. The size and number of mitochondria reflect the energy requirements of the cell.
What are the electron transport complexes? What are the enzymes?
1) complex I (NADH dehydrogenase) 2) complex II (succinate dehydrogenase) - this is not actually a part of the ETC 3) complex III (cytochrome bc₁) 4) Complex IV (cytochrome c oxidase)
Catabolic process vs. anabolic process
1) Catabolic process: breakdown of large molecules to produce energy 2) anabolic process: synthesis of large molecules from smaller molecules
describe the basic electron pathway in the electron transport chain
1) electrons enter the chain from either NADH (via complex I) or FADH2 (via complex II) 2) electrons are passed from either complex I or II to ubiquinone 3) electrons are passed from the now reduced ubiquinone (ubiquinol) to complex III 4) electrons passed from complex III to the peripheral protein, cytochrome c (mobile) 5) electrons transferred from cytochrome c to complex IV 6) complex IV transfers the electrons to the terminal electron acceptor, O2, which then is reduced to water
What are the fates of pyruvate? In what conditions does each act?
1) ethanol fermentation (anaerobic) 2) lactate fermentation (anaerobic) 3) the tricarboxylic acid cycle (aerobic)
what are the types of electron carriers?
1) flavoproteins 2) cytochromes 3) three copper atoms 4) ubiquinone 5) iron-sulfur proteins
Compare lactate and ethanol fermentation
1) lactate fermentation: occurs in humans in the muscles when exercising converting pyruvate to lactate in a reaction catalyzed by lactate dehydrogenase 2) ethanol fermentation: occurs in bacteria and yeast and pyruvate undergoes 2 reactions. 1. pyruvate to acetaldehyde by pyruvate decarboxylase and 2. acetaldehyde to ethanol by alcohol dehydrogenase
How many electrons can be accepted by ubiquinone?
2 electrons
how many electrons can be accepted by the FMN group of a flavoprotein?
2 electrons
describe the experiment performed to observe the rotational catalysis of ATP synthesis
A modified portion of the ATP synthase consisting of α₃β₃γ was prepared. A fluorescent actin filament was placed in the γ subunit and exogenous ATP was added. This addition of ATP and use of fluorescence allowed for the observation of rotation (counterclockwise)
What is the most common energy carrier molecule?
ATP - adenosine triphosphate
describe the bind changing mechanism for ATP synthesis
At the start of the cycle, the site is in the open (O) conformation and the substrates ADP + Pi enter the site. Step 1: movement of protons through the membrane induces a shift to the loose (L) conformation in which the substrates are loosely bound Step 2: the movement of additional protons induces a shift to the tight (T) conformation in which the affinity for the substrates increase causing them to be tightly bound to the catalytic site Step 3: the tightly bound ADP and Pi spontaneously condense to form a tightly bound ATP (no conformational changes) Step 4: the movement of additional protons induces a shift to the open (O) conformation in which the affinity of ATP is greatly decreased allowing the product to be released from the site and allowing ADP + Pi to bind again to repeat the cycle
Describe the layers of the mitochondria
Contains an inner and outer membrane which creates 2 enclosed spaces: the intermembrane space and matrix. The inner mitochondrial membrane is divided into the inner boundary membrane and the cristae
how do electrons enter the electron transport chain?
Enter as NADH via complex I or enter as FADH2 via complex II
summarize oxidative phosphorylation in relation to the electron transport chain
In the first step, substrates isocitrate and succinate are oxidized and the electrons are transferred to coenzymes NAD⁺ or FAD to form NADH or FADH₂. The high energy electrons are transferred through a series of electron carriers of increasing postive redox potential that make up the electron transport chain. As the electrons move through the electron transport chain, H⁺ are pumped out across the inner membrane into the intermembrane space. The energy released is used to translocate protons from the matrix to the intermembrane space, establishing a proton gradient across the inner mitochondrial membrane. The low energy electrons are accepted by the terminal electron acceptor, O₂, which then gets reduced to water. In step 2, the protons move down the electrochemical gradient back into the matrix through an ATP-synthesizing complex which can use the energy to form ATP.
What is the importance of Factor 1 (F₁) in ATP formation?
Isolation of the coupling factor 1 showed that it hydrolyzed ATP (behaved as an ATP synthase) which led to conclusion that an ionic gradient establishes a proton-motive force to phosphorylate ADP
complex I
NADH dehydrogenase: it catalyzes the transfer of electrons from NADH to ubiquinone and transports four H⁺ per pair. The electron transfer occurs in the hydrophilipic arm and is coupled by conformational changes to proton translocation across the membrane
describe the three conformational states of the β subunits active site
O - open state: binds loosely to the protein/molecule such as ADP + Pi L - loose state: binds loosely to ADP + Pi T - tense state: ADP and Pi are brought together very close allowing for ATP production to take place spontaneously
What subunits on ATP synthase catalyze the synthesis of ATP
Occurs due to the rotation of the γ subunit which induces changes in the conformation of the catalytic site of the β subunit. The catalytic site passes through 3 states (O, L and T state/conformations)
When does pyruvate undergoing fermentation? What are the types?
Pyruvate will undergo fermentation in the absence of O2. It can either undergo 1) ethanol fermentation 2) lactate fermentation
how many electrons can be accepted by the heme group of cytochrome C?
The Fe³⁺ of the heme can accept 1e⁻ to be turned into the reduced form Fe²⁺
describe the movement of the protons in the F₀ complex
The c ring of the F₀ complex has 8-15 subunits (this varies between organism). Each proton form the intermembrane space enters a half-channel within the a subunit and then binds to an acidic residue that is negatively charged (coo⁻ of Asp61 in e.coli) that is accessible on one of the c subunits. The proton binding induces a conformational change that causes the ring to move by approximately 30°. The proton is carried in almost a full circle (308°) by rotation of the c ring and is then released into a second half-channel (a subunit) that opens into the matrix
Describe the role of the F₀ portion of the ATP synthase in formation of ATP
Utilizes the proton gradient to drive the catalytic machinery. The c subunits of the F₀ base form a ring. The c ring is bound the the γ subunit for the stalk. The protons move through the membrane and rotate the ring. This rotation drives ATP synthesis due to conformational changes in the β from rotation in the γ subunit.
dehydrogenases
a class of enzymes that remove H2 from organic molecules. They utilize NAD+ to shuttle electrons causing it to be reduced
plasmalogens
a class of phospholipids in which one of the fatty acids is linked to glycerol by an ether linkage rather than an ester linkage
cytochrome oxidase
a large complex (13 subunits) that adds four electrons to O₂ to form 2 molecules of H₂O. it is a proton pump which translocates 4 protons into the intermembrane space while the other 4 protons are used to create 2H₂O molecules from the reduced O₂
glyoxysome
a special peroxisome found in plants that can convert fatty acids to glucose by germinating seedlings
flavoproteins
a type of electron carrier that are made up of polypeptides bound to either FAD or FMN
How did oxygen accumulate on earth?
accumulation of oxygen occurred due to cyanobacteria that could use light as an energy source and release O2 as a biproduct (from splitting H2O)
describe the reason for premature aging as related to mitochondrial or peroxisomal function. Describe the experiment
accumulations of mutations in mtDNA is considered a major cause of aging. Mice homozygous for a mutant gene (Polg) which encodes for the enzyme that replicates mtDNA accumulates more mutation than the normal mice. The "mutator" mice appeared normal for the first 6-9 months of age then rapidly developed signs of premature aging such as hearing loss, greying hair and osteoporosis. Their lifespan was reduced by half.
dinitrophenol (DNP) - How does this affect the metabolic rate?
an uncoupler. It uncouples glucose oxidation and ATP formation by increasing the permeability of the inner membrane to H⁺, thus eliminating the proton gradient. The difference in uncoupling proteins (UCPs) account for differences in metabolic rate
complex IV
cytochrome c oxidase: catalyzes transfer of electrons to O₂ and transports 2H⁺ across the inner membrane
What is located in the mitochondrial matrix? What does this allow for?
contains circular DNA molecules, ribosomes and enzymes which allows for RNA and proteins to be synthesized in the mitochondrial matrix
What part of the mitochondria is the machinery from ATP located?
cristae
Complex III
cytochrome bc₁: catalyzes the transfer of electrons from ubiquinone to cytochrome c and transports four H⁺ per pair
how was cytochrome oxidase proven to be a proton pump
cytochrome c was purified and incorporated into an artificial bilayer of a liposome. The medium was seen to become acidified because as electrons were transferred from cytochrome c to cytochrome oxidase, O2 was being reduced to water and protons were translocated from the compartment within the liposome center into the external medium
iron-sulfur proteins
electron carrier that are iron containing proteins in which iron atoms are linked to inorganic sulfide ions.
three copper atoms
electron carriers that are located within a single protein complex and alternate between Cu²⁺/Cu¹⁺
cytochromes
electron carriers that contain heme groups bearing either Fe or Cu metal ions (Fe³⁺/Fe²⁺)
describe the composition of the inner and outer mitochondrial membrane
outer: 50% lipid by weight and contains a large pore forming protein called porin and is even permeable to some proteins inner: more than 75% protein and contains cardiolipin but not cholesterol. Also is generally impermeable, even to small molecules
What molecules were present in the atmosphere before O2
reduced molecules such as H2, NH3 and H2O
How many cytochrome c molecules are required for the reduction of O₂? What is the reaction formula?
reduction of O₂ requires 4 reduced cytochrome c molecules. 4 cyt c²⁺ (reduced) + 8H⁺ (from the matrix) + O₂ (oxidized) → 4 cyt c³⁺(oxidized) + 2H₂O (reduced) + 4H⁺
Do strong oxidizing or reducing agents have an affinity for electrons? How do redox reactions affect free energy?
strong oxidizing agents have a high affinity for electrons while strong reducing agents have a weak affinity for electrons. Redox reactions are accompanied by a decrease in free energy
complex II
succinate dehydrogenase: Not actually a part of ETC but catalyzes the transfer of electrons from succinate to FAD (reducing it to FADH2) to ubiquinone without the transfer of H⁺. This enzyme is also present in TCA cycle
describe the structure of the ATP synthase
the F₁ particle is the catalytic subunit and contains three catalytic sites for ATP synthesis. The F₀ particle attaches to the F₁ and is embedded in the inner membrane of the mitochondira. The F₀ base contains a channel through which protons are conducted from the intermembrane space to the matrix. This was demonstrated in experiments with submitochondrial particles
What is another role of proton-motive force in addition to ATP synthesis?
the H⁺ gradient drives transport of ADP into and out of the mitochondria. ADP is the most important factor controlling respiration rate
