Test #4 Flashcards
ATP requirement for men
8400 kJ (2000 kCal) – 83 kg of ATP
How much ATP do human posses
250 g at any given moment
Compensating the disparity between amount of ATP available and amount of ATP needed
Compensate by constantly recycling ADP and ATP
How many times is a molecule of ATP recyled each day
300 times
How are ATP molecules recycled
Recycled through Oxidative Phosphorylation
Overall Delta G of Reaction of ETC
Exothermic
What does ETC make
Makes Proton gradient –> Resulting proton gradient and transmemebrane electrical potential creates proton motive force
How is ATP synthesized (overall)
ATP is synthesized when electrons flow back to Mitocondrial matrix through enzyme complex
***ATP is synthesized by oxidation of a fuel + phosphorylation of ADP that are coupled by a proton gradient across the mitocondrial membrane
What couples Oxidation of fuel and Phosphorylation of ADP
Coupled by a proton gradient across mictocondrial membrane
Cell respiration
Generation if Hight Transfer Potential Electrons by TCA cycle and flow through respiratory chain
Membrane system of MItocondria
Mitocondria are bound by a double membrane
1. Outter memebrane
2. Highly folded inner memebrane
Inner memebrane of Mitocondria
Folded into a series of interanl ridges called “cristea”
Compartments of Mitocondria
- Intermemebrane space
- Matrix
Mitocondrial Matrix
Bound by inner memebarne – Matrix is the site of most of the reactions of the TCA cycle and Fatty acid oxidation
Where does ETC take plase
ETC takes place in the inner memebrane
Surface area of inner memebrane
Has increased SA because of cristae –> Increases SA of Inner memebrane creates more sites for Oxidative Phosphorylation
Permability of outter memebrane
The outter memebreane is permeable to most small molecules + ions – BECAUSE it has proins –> has pore forming proeteins
Pore forming proteins in outter memebrane
VDAC – Volt dependet anion Chanel –> most prevelant protein in mitocondrial memebarne
***Plays a role is regulated flux of metabolites across the memrbane (Phophates + Cl- + Organic anions + Adenine)
Permability of inner membrane
Inner memebrane = impermaeble
***There is a large family of transporters that shuttel metabolites across the inner memebrane
Two faces of inner mitocondrial memebrane
- Matrix side – N side (Negitive)
- Cytoplasmic side – P (Positive)
Bacterial ETC
Electron proton pumps + ATP are synthesizing complexes are on on the cytoplasmic side
Where is ETC and ATP synthesis
In the mitocondria
***ETC = in the inner memebrane of mitocondria
Where is TCA cycle
In the mitocondrial matrix
What does TCA produce
NADH + FADH2 –> They both go to ETC
Sections of ETC
Split into 4 sections
- Complexes pump Hydrogen into the intermemebrane space –> THEN ATP synthase brings Hydrogen back
Charge of inside of cells
Negitive –> Need to bring in positive charge
Purpose of ATP synthase (overall)
Maintain Homeostasis
Defect in mitocondria
Have disease –> usually die
How are mitocondrial diseases passed on
Usually from mother to Son
(Only need 1 X in son = more likley to get it)
Second name for ETC
Oxidative Phosphorylation
Why does the inner membrane need complexes
Because it is impermeable to most molecules
ETC (Overall)
ETC = a series of coupled redox reactions that transfer electrons from NADH to FADH2
What does NADH and FADH2 reduce
NADH and FADH2 are used to reduce O2 to H2O
***Highly exergonic reaction – done by electron transfer reaction that takes place in ETC
How do we measure electron transfer potentail
Meausre Electron transfer potential based on redox potentoal
The electron transfer potential conversion of NADH and FADH2 in ETC
In oxidative phosphorylation – the electron trasnfer potential of NADH and FADH2 is converted into posphoryl trasnfer potential of ATP
Redox example
Have X –> X-
Have something that can oxidized or reduced
***THe reduction potential can be measired using electron motive force generated by sa,ple half cell connected to 2 standrad refernce cells
Measuring reduction potential
The reduction potential can be measured using electron motive force generated by sample half cell connected to 2 standard reference cells
- The reduction potential (redox potential) is the measure of a molecule’s tendancy to dinate or acceot electrons
Measure of HPTP
Delta G
Measure of ETP
E’ – reduction potential
Strong reducing agent
Readily donates electons
***VERY negitive E0
String oxidizing agent
Readliy acceopts electrons
***Has a positive E0
Free energy change is related to the change in reduction potential (equation)
Delta G = -nFdEo
n = number of electrons transfer
F = Faraday constant
Charge of strong oxidizers and reducers
Slide 7
What does electron flow from NADH to O2 power
Powers the formation of proton gradient
Delta G for reactions of NADH + O2
1/2 O2 + 2H+ + 2 e- –> H2O E = POS
NAD+ + H+ + 2e- –> NADH E = NEG
COMBINED REACTIONS: 1/2O2 + NADH + H+ –> H2O + NAD+ Delta G = -220.1
Overall: The oxidation of 1 NADH can be coupled with the rephosphorylation of MULTIPLE ADP molecules
Driving force of Oxidative Phosphorylation
The electron transfer potential of NADH/FADH2 relative to O2
What is release of energy in reduction of O2 used for?
Release of energy = used to generate H+ gradient that is THEN used for synthesis of ATP and the trasnprot of metabolites across the membrane
Quanatative energy associated with H+ gradient
Delta G = RTln(C2/C1) + ZFdV
- c1 = concentration of the protons on one side of the mebrane AND c2 is the concetration of protons on the side opf the gradient to which the protons are moving
- pH is 1.4 lower than inside –> ln(c2/c1) = 3.2
- dV is the voltage potential across the mebrane –> Memrane potential is 0.14 V
- Z (charge of the proton) = +1 because outside is positive
- R = teh gas constant
- T = temperature (in kelvin)
Overall – dG = 21.8 kJ
What is dG for each H+ transproted out of the matrix
dG = 21.8 kJ
How to calculate Kelvin
Celcius + 273
What makes up the respiratory chain?
4 Comlexes
Four complexes of the respiratory chain
- NADH Oxidoreductase
- Q-cytochrome c oxidoreductase (Complex 3)
- Cytochrome c oxidase (complex 4)
- Succinate Q-reudctase (Complex 2)
How do electrons flow from NADH to O2
Electron flow through three large protein complexes embedded in the inner mitocondrial membrane
What does electron flow through complexes generate?
Electron flow through complexes = exergonic –> powers the transport of H+ across the inner membrane
Succinate Q-reductase (overall)
Contains Succinate dehydriogenase that generate FADH2 in the TCA
***It does NOT pump H+
- Succinate Q - Red. deleivers electrons from FADH2 to Complex 3
Complexes 1,2,3
Appear to be super molecular complex = faccilitates rapid transfer of substrate + prevents the release of intermediates
Purpose of the complexes?
The complexes pump protons out of the mitocondrial matrix = genrates H+ gradient
***Protons then move back through ATP synthatse to generate ATP
What is ATP used for
ATP is used for active transport (Low –> HIGH)
What carreies electrons from one complex to the next?
Two special electron carriers:
1. Coenzyme Q
2. Protein Cytochrome C
Coenzyme Q
Hydrophob Quionone that diffises rap[idly within inner mitochondrial memebrane
Electrons are carried from NADH-Q to Q-cytochrom OR by reduced form of Q
Flow of elerctons from FADH2
Electrons from FADH2 are transfered first to Q THEN Q-cytochrome
Coenzyme Q (Overall)
Quinonine deriavtive with long tail of 5 Carbon isporene units
***Most mammales = have 10 isoprene units
- Q – binds to protons and electrons
- Can exist in several oxidation states
Purpose of the isporene units in Coenzyme Q
Give hydrophobic nature
Oxidation states of Quinone
Quinone = can exist in several oxidation states
START – Fully Oxidized (Q) –> ADD E- AND H+ – GET Semiquinone (QH) –> ADD H+ – GET Semiquinone radical (Q.-) -OR if ADD H+ and e- to QH –> GET QH2 (reduced form of Q)
Protein Cytochrome C
Cyt C = an electron carrier that employs an Oron incprportated into a heme
Small solluble proteon shuttle
**Shuttles elevctrons from Q-Cytochrom C To Cytochrom C Oxidase (Complex 3 –> 4) AND catylyzes reduction of Oxygen in the last part of ETC
**Uses heme prostetic groups
ETC Overall
NADH –> Complex 1 –> e- goes to COmplex 3 –> e- goes to Complex 4 –> Oxyegn is reduced to water
***All of the complexes require Iron
Requirmnet for Cytochrome C
Requires Iron
Protestic groups of Complex 1
- FMN
- Fe-s
Protestic group of complex 2
- Heme Bh
- Heme Bl
- Heme C1
- Fe-s
Prostetoc group of Comlex 3
- Heme a
- Heme a3
- CuA and CuB (Requires COPPER NOT Iron)
Prosthetioc group of complex 4
- FAD
- Fe-s
Where are e- passed to in the protein complexes
The e- are passed to electron carriers in the protein complexes
Q pool
Oxidized and reduced Q are present in the inner Mitocondrial memebrane
Cytochromes generally
Cytochromes are electron transfering proteins that contain a heme group
Heme in Cytochrome C
The Heme iron cycles between Fe2+ and Fe3+ as it accepts and donates electrons
Common part of the ETC
Iron-sulfur clusters
Iron Sulfur clusters
aka “non-heme iron proteins” – prominent electron carries
Purpose of Fe-s
Electron carriers
Types of Fe-S known
- 1Fe –> 1 Fe that is tetrahedrally coordinated to 4 S groups of 4 Cysteins
- 2Fe-2S –> 2 iron and 2 Sulfides + 4 Cysteins
- 4Fe-4S –> 4 Iron and 4 Sulfars + 4 Cysteins
Iron in Fe-S clusters
Cycles between Fe2+ and Fe3+ – as it accepts and donates electrons
What does NADH Q Oxidoreduc contain?
Has 2Fe2S + 4Fe-4S – Fe in these complexes cycle
How do Fe-S undergo redox
Undergo redox without releasing or binding H+
Frataxin
Small mitondrial protein that is crucial for Fe-S synthesis
Frataxin definceiney
results in Freidrweich’s ataxia – affects the nervous system + Heart + Skelotal systems
Freidrich’s ataxia
Affects nervous + heart + Muscle
- Genetic Autosmal Recessive
- metabolism disorder
- Symton – bottom of feet is heard = feet roll
- heart probelm –> have hypertrophic cardiomyopathy = large cells = large heart – blood = fills and the nervous syetm is activated = heart contracts = the blood goes to the body BUT with the bigger cells = thick blood vessels = less blood held in the heart = have less blood being pumped
- Antiseption disorder – means the next generation will get it younger and young – younger generations get the disease faster
- Have GAA repeats in Frataxin gene = abnormal DNA
Where do High-potential Electrons of NADH enter the respiratory chain?
e- enter ETC at NADH Q Oxidoreductase
Electrons get passed form NADH –> Q = forms QH2 (passed by complex 1)
***Electrons carries between NADH and Q uses Flavin Mononumcleotoide and Fe-S proteins
Q –> QH2 THEN the QH2 leaves the enzyme for the Q pool in teh hydrophic ineterior of the inner memebrane
Flow of e- from NADH –> Q
Uses FMN and Fe-S
Protons in Complex 1
4 H+ are pumped out of matrix into memebrane space
NADH + Q + 5H+ –> NAD+ + QH2 + 4H+
Flow of H+ in ETC
Matrix –> Inner membrane space
Reaction of FMN in complex 1
FMN Oxidized –> FMNH2
**Adding 2e- and 2H+
Electron Flow through Complex 1
NADH –> FMN –> Fe-s clusters –> Q = get QH2
What codes for H+ pump?
Encoded by genes in the mitocondria and the nuclues
Shape of NADH Q OR
L-Shaped – horizontal arm lying in the membrane + verticle arme that projects into the matrix
Reaction at Complex 1
- Binding NADH and transfer of 2 e- to FMN –> Get FMNH2
- e- go form FMNH2 –> Fe-S clusters
Structural elements required for proton pump
- Memebrane embded part – has 4 H+ half chanels consisting iof verticle helicies
- one set of half chanels are exposed to the matrix and the other half are exposed to the inner memebrane space
- Encloded Q chamber
- Hydrophobic funnel conects Q chamber and Water chanel – extends the entire length of the memebrane part
Verticle helices in H+ pumps
Linked on matrix side by a long horitonzal helix that connects the metrix half chanels
THE intermemrane space half chanels are joined by a speries of B-hairpin helix connecting elements
Connection of Intememebrane space half channels
Intermemrane space half chanels are joined by a speries of B-hairpin helix connecting elements
What happens when Q acceps e-
The Q exiits near junction of hydrophillic protion and memebrane embedded protion
How do the strucutral elements cooportae to pump H+ into the matrix?
Q accepts 2 e- = generates Q2- –> negitive charge on Q2- interacts electrostatsically with negtive Amino Acids on memebrane embeded arme – the interaction causes confirmational change in long horizontal helix in B-helix elememy –> Confirmational change alters the structure of the verticle helizes -= have a chnage in pKA of Amino acids = allows H+ from the matrix to bind to the Amino Acids and THEN dissocaite into the watert linked chanel –> THEN can enter the intermmebrane space –> THEN Q2- tajes 2 H+ from matrix –> forms QH2 and QH2 leaves Q pool and another cycle can occur
Affect of QH2 forming
removes H+ = contributes to the formation of Proton Motive Force
Other ways to synthesize NADH
- FA degradation
- e- from cytoplasmically generated NADH
What is the entry point for FADH2
Ubiquinol
Where is Succinate Dehydrogenase
Succinate Dehydrogenase = part of Complex 2
FADH2 in ETC
FADH2 –> Fe-2 – reduces Q –> QH2 – QH2 then enters the Q pool
Difference in Complex 2
Complex 2 is NOT a proton pump – means that less ATP is formed in oxidation of FADH2
e- flow from Ubiquinol to Cty C
Electrons flow from ubiquinol to Cyt C through Q-cytochrom c Oxidoreductase
Q-cytochrom C Oxidoreductase
Cataylyzes flow of electrons from QH2 –> Cytc (reduces two molecules of cytochrome C)
***Complex 3 = also a proton pump
Cytochromes in Complex 3
- cyt b
- Cyt c1
Reaction in Complex 3
QH2 + 2CytCox + 2H+ (matrix) –> Q + 2cytcred + 4H+
Net protons pumped in Complex 3
2 protons – smaller because smaller driving force
Hemes of Complex 3
Heme c1 – Uses Cys + Met
Heme BL – Uses His
Heme bH – uses His
Rieske Fe-S – Uses His + Cys
Cytochrome
Electron transfering protein taht contains a heme group
Heme in Cyt b1, C, and C1
iron-Protophorin – Iron in the cyt alternates between + 2 and +3 state
***2 Cyt subunits = have 3 hemes
2 hemes in B (bL and bH)
1 heme in C1
What does Cyt have besides hemes?
In Addition to hemes –> Cyt has 2 fe-2S in center
Center of Complex 3
Has reike Center – unula in that one fe is coordinated by 2 His
Coordination = stabilizes center in reduced form = increases the reduction potential to accept the elerctons from QH2
Why do identical hemes have diffrent electron affinitys?
Identical hemes have different electron affinities because they are in diffreent envirnments
Mutations in Succinate Dehydrigenase
Result in increase in Succinate = facilitates in the development of cancer
Purpose of the Q cycle
The Q cycle funnels electrons from two electron carrier to a one elercton carrier and pumps protons
Electrons in QH2 Vs. cyt C
QH2 – carries 2 e-
cyt C –> Carries 1 e-
Q cycle overall
QH2 –> Cyt C
Q Cycle = Mechanism for coupling of e- transfer from Q to Cyt C to Transmemebramne proton pump
How many types of Q cycle exist?
2 types
Two halves of the Q cycle
Part 1 – One electron from QH2 reduces Cyt C and reacts with Q to form Q-
QH2 –> CytC + Q THEN Q –> Q-
Part 2 – Another QH2 reduces Cty C and Q-
QH2 –> cyt C + Q-
Protons in Q cycle
In one cycle – 4 protons are pumped out of the matrix and two are
Reaction of Q cycle
2QH2 + Q + 2Cty c oxd + 2 H+ (matrix)–> 2Q + QH2 + 2cty C red + 4H+ (ims)
Issue in Q cycle (
QH2 – pases 2 e- to Cyt c BUT Cyt C can only accept 1 electron
Solution: Mechanism for coupling of e- transfer from Q to Cyt C to Transmemebramne proton pump
Q cycle (depth)
2 QH2 molecules bind to complex at the same time –> each give 2 e- and 2H+ –> THE H+ GET RELEASED TO INTERMEMBRANE SPACE
- The first QH2 to exit the pool binds to the first Q binding spot (Q0) and its 2 e- travel through the complex to diofferent destinations -- 1st e- flows to Rieske center THEN to C1 THEN to molecule of oxidized Cyt C = converts cty C to reduced form
2nd e- passes through 2 heme groups of cty b to an oxidize Q in second binding site (Qi) --. reduces the Q to Q radical -- fully oxidizes Q leave the first site and enters the Q pool
- Second molecule of QH2 binds to Q0 site of Ctc C and reacts in the same way at the first -- 1 e= goes to partially rediced Q in Qi --> the radical takes up 2H+ from the matric to form QH2
END: 4 H+ are released into IMPS and 2 H+ are removed from the matrix (removal still contributes to the gradient)
Result of 1 Q cycle
2 QH2 molecules are oxidized to from 2Q molecules –> THEN one Q Molecule is reduced to QH2
Purpose of Q cycles
Solves the problem of funneling elerctons from a 2 e- carrier to a 1 e- carrier
IN essence – its a recycling system that makes use of both electrons effectively
Cytochrome C Use
Catylzyes the reduction of O2 to water
Cytochrom C Oxidase (overall)
Cty C Oxodase acceots 4 electrons from 4 molecules of cyt C to catylyze the reduction of O2 to 2 molecules of water
E- go from Cty C –> Cty C oxidase
Protons in cyt C oxidase
Eight proteons are removedfrom the matrix
- 4 H+ = the chemical proton – REST go to the IMPS
Chemical protons
Protons used to reudce O2
Cyt C Oxidase reaction
4CytCred + 8H+ (matrix) + O2 –> 4Cytc Oxd + 2water + 4H+
END – pump 4 protons into the IMS + use 4 protons to reudce Oxygen
Heme requirements in Cyt C oxidase
Heme a – His
Heme a3 – His
CuA/CuA– uses Cys + His
CuB – His + Tyr
Structure of Cytochromse C Oxidase
Conatins 13 Subunits
Requirments of Cty C Oxidase
- Two heme A Moeities – Heme A and A3
- Two copper ions – the two centers contain 3 Cu ions
Two copper ions in Cyt C Oxidase
- Copper A – have 2 copper ions – the Cu ions are linked by bridging Cysteine residues
- Copper A = intially accepts elecrtons from reduced Cyt C - Copper B – has three His –> One Histidine residue is linked to tyrosine
***Copper centers alternate between reduced Cu and Oxd Cu2+ as they accept and donate e-
Electron flow in Cyt C Oxidase
Cty C –> CuA/CuA –> Heme a –> Heme a3 –> CuB
***Adding two more electrons and 4 H+ generates two molecules of water
What happens when the Fe in heme a3 and CuB are rediced
Bind to oxygen as a peroxide bridge between them
What disease in Tyr associated with?
PKU
Cytochrome C Oxidase
Last of the H+ assemblies – it catylizes the transfer of electrons from reduced Cyt C to O2
What makes ETC aerobic
Requirement of O2 for rxn in complex 4 – reason that humans need to breath
How many electrons are used to reduce O2
4 e- –> 4 e- are funneled to O2 to completeley reduce it to water
What happens when O2 is reduced
H+ are pumped from matrix to teh IMPS
dG of Complex 4
Reaction is thermodynamically favorable – dG = -231.8
***Want to capture as much of the dG as possible in form of the proton gradient for ATP sytnthesis
Heme A in Complex 4
Different than heme in C and C1
Has:
1. A formyl Group instead of CH3
2. A C17 hydrocrabon chain replaces Vinyl Group
3. heme is not covalentley attatched to protein
Complex 4 Mechinsm
- Two molecules of cyt C transfer e- to reduces CuB and Heme A3
- electrons from 2 molecules are red cyt C flow down ETC within Complex 4 – one e- stops at CuB and one at heme a# –> THEY BOTH bind to O2 molecule
- Reduced CuB and Fe in heme a3 bind O2 = forms perpxide bridge
- As O2 binds to e- peroxide bridge forms
- The addition of 2 more e- and 2 more H+ cleaves the peroxide bridge
- Two more e- of Cyt C bind and release e- that travel to active center
- The addition of an e- as well at H+ to each Oxygen reduces the 2 oxugens to Cub2+-OH and Fe3+-OH
- The addition of 2 H+ leads to release of H2O
- reactions with more H+ ions allow the release of 2 molecules of water and resets enzymes to oxidized form
Affect of Oxidizing Cytochrome C
Also converts Oxygen to water
Heme a and Heme a3
Have distict redox potentoals bevause they ate located in different envirnments within Complex 4
Active center in Complex 4
Heme a3 and CuB –> Forms active center at O2 at with O2 –> goes to water
Protons in Complex 4
4H+ come from the matrix = consumtion of 4H+ contibutes to gradient
dG in complex 4
Each H+ moving = 21.8kJ –> 4 = 87.2 kJ –. LESS than the dG avalable from reducihng water – what is the fate of the missing dG –> Cty C Oxidase uses the energy to pump 4 additional H+ from matrix to intermemebrane space = 8H+ are removed from the matrix
Two things that affect Complex 4 mechanism
- Charge nuertrality – is mainatined in the interior of proteins -> THIS adding e- to site = favors H+ binding
- Confirmational Change occurs (espcially for a3-Cub) –> in one confirmation the H+ can enyter prtein from matrix side and in another confirmation they exit to the intermembrane space
Where is most of the ETC organized into
Most of the ETC is organized into the Respirasome Complex
Respirasome
Protein that coordinates complex – reserach shows that the three componenet are arranged in a large comples
***Complex = resparasome
Human Respirasome
Consists of:
1. 2 Complex 1
2. 2 Complex 4
3. 2 Complex 3
4. 2 Cyt C
**The Complex 1 and 4 surrodound the copies of complex 3
**The two copies of cyt C are on the surface of Complex 3
Purpose structure of respirasome
Structures allow for Complex 2 to associated in a gap between Complex 1 and 4
What does respsirasome show
Shows multienzyme system used to increase efficiency
Where is cyt C located in mitocondria
Sits on the external size of the inner membrane
Solution for Toxic derivatives of O2
The toxic derivatives are scavenged by protective enzymes
Toxic derivatives of O2
Superoxide Ions + Peroxide
Partial reduction fo O2
Generates highly reactive Oxygen derivatives – Creates ROS
ROS
reactive oxygen species – made by the partial reduction of O2
***ROS = implicated in a lot of pathological conditions
Types of ROS
- Superoxide Ions
- peroxide Ions
- Hydroxyl Radical
O2 -+ e- –> O2.- (Superoxide ion) + e- –> O22- (peroxide)
Affect of alcholol
Kills nuerons = decreases intellegence
Emphysema
Bronchitus –> COPD – Chronic Absurtutice Pulminary disorder
- Get small cell cracino/Co-cell carcinoma because of Emphysiomia
- 6 months to live
***Casued by ROS
Duchene Muscular
Lose skeoltal muscle and replace it with fatty tissue
- Sympton = cow manuver to get upo
- Die by 11/12 – die because of lung infections
- genetic – X-linked recessive
Diseases associated with ROS
- Emohysema
- Duchene Muscular Dystrophy
- Alcohol Liver disease
Alcholol Liver dieases
Get cirrosious = get cancer
- Live longer but eventually die
- Luver becomes hard –> need liver transplants
Super Oxide Dismutase + Catalse
Help protect against ROS Damage
SOD – 2O2.- + 2H+ –> O2 + H2O2
Catalase – H2O2 –> O2 + H2O
Two forms of SOD in Eukaryots
1 – Magnese containing Mitocondrial from
2 – Copper and zinc containing Cytoplasmic form
Exersize + SOD
Exercise is associated with increased SOD expression
O2 as terminal acceptor
O2 = ideal terminal acceptor because of its high affinity for electrons = provides a large thermodriving form BUT danger lurks in reduction of O2
Dangers in Reductions of O2
4 e- = leads to safe prodycts BUT partial reduction generates hazardous compounds
***Transfer of a single e- or 2 e- is bad –> both potentiall destructive
Stradegy for safe reduction of O2
Catalysts do not release reactive intermediates – cty C does this by holding O2 tightley between Fe and Cu
Issuse – small amounts of ROS
Implications of ROS
ROS have been implicated in the aging process + a growing list of diseases
Cellular defense stradegies against ROS
- Enzyme SOD –> scavenges superoxide radicals and catylezed them to H2O2 and O2
SOD
Superoxide radicals –> H2O2 + O2
- Enzymes preform dismutation reactions – oxidized form of enzyme is reduces by the superoxide to form O2
- reduced form of the enzyme reacts with a second superoxide ion to form peroxide –> takes up 2H+ along the reaction path to make H2O2
Fate of H2O2 formed by SOD
H2O2 is scavenged by catalase
Catalase
Heme protein that catylyzes the dismutation of H2O2 into H2O and O2
Efficiencey of SOD and Catalase
They are very efficient – near diffusion limit
Gluyayhone peroxidase
Also plays a role in scavenging H2O2
Excersize + SOD (depth)
Increase in aerobic metabolism = increse in ROS –> in response rge cell sythesized more protective enzymes – net effect = protection because increase in SOD = protects cells during periods of rests
New use of ROS
Recent evidence suggests that under certain circumstances the controlled generation of ROS = may be implicated imn signal transduction
Exampple – growth factprs have shown to increase ROS levels as part of their signalling pathway + ROS regulates chanells and transcription fcatoprs
***ROS have been implicated in the control of cell diferentation + immuresponse + Autophase
Other cell defense against ROS
Antoxidants E and C – because lipophillic unit L is usefule in protecting memebranes from lipid peroxidation
Proton Motive Force
The proton gradient generated by the oxidation of NADH and FADH2
***Energy rich unequal distrobution = PMF
Used of PMF?
Proton motive force powers the synthesis of ATP
Parts of the proton Motive Force
- Chemical Gradient – pH gradient
- Charge Gradient – created by postive on H+
***Chemiostatic hypothesis = proposes that both components power ATP Synthesis
How did we confirm that the proton gradients can power ATP Sythesis?
Using Heterologous experimental systems
dG of red o2 vs dg of ADP
NADH –> O2 dG = Exo
ADP –> ATP dG = Endo
***The reactions are coupled to drive the synthesis of ATP
Complex that carries out ATP synthesis
Mitocondrial ATPase OT F1/F0 ATPase –> Named like this because it was discovered through the reverse mechanism
How is oxidation of NADH coupled to the Phosphorylation of ATP
Two proposals about How oxidation is coupled to phosphorylations
- e- transfered leads to formation of high covalent intermediate that serves as a compud with HPTP (likes generation of ATP in glycolysis)
- That the e- transfer aids in the formation of an activated protein confirmatyion –> THEN drives synthesis – NO such intermediate has been found
Peter Mitchell
Suggested the Chemiosomatioc Hypothesis
Chemiosomatioc Hypothesis
Suggests that e- transprot and ATP synthsies are coupled by a proton gradeint across the inner mitocondrial memebrane – the transfer of e- through the resporatory chaon leads to the pumping of protons from the matrix to the IMPS –> THEN the H+ flow back into the matrix to establish equillibrium
Idea = that the flow of protons drives ATP synthesis by ATP synthase
Support of Chemiostatsic hypothesis
NOW supported by a lot of evidence
NOW KNOW – the e- transoprt dies geenrate a H+ gradinet + the TMP is 0.14v
pH gradient generated
The pH outside if 1.4 units lower than inside
Demonstrating the Chemiosomatic hypothesis
It was demonstrated using Artifical system – used Bacteriarhodposin
***Synthetioc vesicles with bacteria Rhodopsin and Mitocondrian ATP were sythesized and purofied –> when vesicles were exposed to light ATP was formed –> Showed that the resporaty chain and ATP synthesis are seprate systems that are linked by PMF
What drives ATP synthesis?
Proton motive force
Proton Motive Force Equation
dP (PMF) = Chemical gradients (dpH) + Charge Gradient (dPsi)
What makes up ATP synthase?
- Proton conducting unit
- Catalytic Unit
How is NADH coupled to ATP synthesis?
- e- transport generates PMF
- ATP synthesis by ATPsynthast is powered by PMF
***Coupled by PMF
How is PMF converted into HPTP?
Structure of ATP synthase (overall)
Large complex enzyme resembling a ball on a stick
- Stick = F0 subunit (in inner mitocondrial memebrane)
- Ball = F1 Subunit (protrudes into the matrix)
F1 Subunit
Subunit in ATP synthase with catalytic activity – F1 isolated has ATPase activity
- Conatins 3 active sizes ;ocated on the three Beta Subunits
***F1 protrudes into the mitocondrial matric
Where are the active sites on F1
The active sites are located on the three Beta subunits
F1 Subunit (depth)
F1 unit = has 5 types of PP chains with identical stoicheometry
- Alpha + beta make up most of F1 –> arranged alternativley in a hexameric ring
- Both Alpha and beta bind nucleotides BUT onl Beta has catalytoc actitovity
- Below alpha and beta –> have central Stalk
Central Stalk of F1
HAS Y AND E proteins
- Y –> long helical coil coil that goes through the center of a3b3 hexamer –> y breakls the symetry of the a3b3 hexmoer
Why is each Beta subunit distinct
Each beta Subunit is distinct by virtue of its different interaction with Y
***Distuguishing the three beta subunits = criticakl for understanding sythesis
F0 Subunit of ATP Synthase (depth)
F0 = the hydrophibic segment that spanns inner mitocondrial memebrane
- F0 = have H+ chanel
Chanel in F0
Has a ring of 8-14 Carbon subunits that are embedded in the membrane –> A single subunit binds to the outside of the ring
Ways that F0 abnd F1 are connected
- By the central Stalk
- By exterior colum –> have one of alpha Subunit and 26 of delta subunit
ATP synthates interaction
ATP SYnthases interact with one another to form a dimer –> The dimers then associate to form larger oligiomers of dimers
- Associtiona stabilizes the individual enzumes to rotational forces required for catalysis
Affect of forming dimers
Association of ATP synthase enzy,mes stabilizes the individual enzymes to rotational forces that are required for catalyasis + facilititases the curvature of the inner mitocondrial memebrane
Formation of Cristea
Allows proton pumps of ETC to localize H+ gradinet in vicinity of substrates = higher efficieney
Where are the substartes for ATP synthesis located?
Located at the tips of cristae
***The formation of cristea allows the proton pumps of the ETC to localize the H+ gradient at the tips wherte the substrates are found
Where is the F0 compartment
Embedded in the inner mitocondrial Memebrane
What does F0 have?
Has a proton chanel
What connects F1 and F0?
The Y subunit
What is the outcome of Protons flowing through ATP Synthase
Protons flowing through ATP synthase leads to the release of Tightly bound ATP
What is the name of the ATP synthase mechinsm
The Binding-Change Mechanism
ATP synthase role
Catalyzes the formation of ATP from ADP + Pi
Reaction: ADP3- + HPO42- + H+ –> ATP4- + H2O
What is needed for ATP and ADP to function as substrates
ATP and ADP must be bound to MG2+ to function as substrates
- ATP and ADP in ATP synthase need to be bound to Mg2+
Intermediate in ATP synthase mechinasm
Pentacovalent intemedeiate
ATP synthase mechanism
ADP + Pi + H+ –> Pentacovalent INtermediate –> ATP + H2O
**Slide 48
Binding Charge Mechanism
Accounts for the Synthesis of ATP in response to proton Flow
Confirmations of the three Beta Catalytic Subunits of F1
- O form
- L form
- T form
O form
Nucleatoirs can bind to or be released form the Beta subunit
L form
Nucleotides are trapped in teh Beta Subunit
T form
ATP is synthesized from ADP and Pi in the absence of a proton gradient BUT can’t be released from the enzyme
***Responsible for produces the ATP
What releases the Newly synthesized ATP
Proton flow releases the newly synthesized ATP
- The T form makes the ATP without the proton gradient BUT it can’t release the ATP unless there is a gradient
What is needed for ATP to be formed vs. needed to be releases
ATP forms without H+ flow (without PMF) BUT it will not be released without PMF – need PMF in order to release ATP
Mechanism of ATP formations
Terminal Oxygen atom on ADP attacks the phosphate of Pi to form ATP + H2O
How does the flow of protons drive the synthesis of ATP?
Isotope Exchange experiments
Showed that enzyme bound to ATP can be found in absence of PMF – when ADP and Pi are added to ATP synthase in the presence of H2O(18) –> the O18 was incorporated into the Pi through ATP synthase
- Experiments showed equal amounts of ATP and ADP are bound in equillibruim at cataluytic site even in the abdence of teh H+ gradient
BUT – it showed that ATP does not leave until teh H+ gradient flows through the enzyme
OVERALL – shows that the role of the H+ is not to form ATP BUT it is to release ATP from the substrate
What is teh role of the H+ grandient?
To release ATP from the sythase – NOT to make ATP
Three cataylytic units on F1 at a given point
The 3 beta subunits in F1 – all orefron one of three functions at any given time
PMF and ATP symthase beta units
The PMF – causes the three active sites to sequnetially change functions as the H+ flows through the memebrane mebded part of the enzyme
How do the three Beta subunits respond to H+ flow
What causes the Beta subunits to change function?
The three beta subunits can preform each of the three steps by chnage in confirmations
Steps preformed by the Beta subuit for ATP synthesis
- ADP + Pi binding
- ATP synthesis
- ATP release
F1 parts
- Moving part – rotor –> consists of C ring and gamma stalk
- Stationary part
ATP synthesis (depth)
Rotation of the Gamma subunit = drives the conversion of the 3 forms
***Gamma rotates =
1. T –> O
2. L –> T = enables the transformation of ADP –> ATP
ATP in O = can leave the enzyme and is replaced by ADP – then can rotate again
Rotation of Beta subunits
T –> O –> L – no two are ever present in the same confirmation
***Mechanism suggests that ATP can be synthesized and released by driving gamma subunit in the right direction
- Each subunit cycles through the three confirmations
What turns the gamma subunit?
Proton flow moves the gamma subunit = powers interconversion of forms
Angle and direction of Beta subunit movement
120 Degrees counter clockwise
***120 Degrees = 1ATP
Which beta form produces ATP
T Form – produced if there is a turn –> then it is released and there can be another turn
Rotational Catalysis is…
The worlds smallest motor
Studying ATP sythase
Used experiments using A3B3G
- Beta subunits were engineered to have an amino terminal Poly histidine tag –> have affinity for Nickle = allows teh A3B3 to immobilize on a glass surface that was coated with Nickle
- Gamma subunit was liked to actin filamirs to see it under floruecnce
–> When added ATP = it caused the Actin filaments to rotate clockwise –> the gamma subunit was rotating = driven by hydrolysis of ATP
Clockwise rotation of Gamma subunit
Consistent with predicted mechanism for Hydrolysis
Efficiency of ATP synthase
Near 100% efficiency –> All of the energy released by ATP hydrolysis is converted into rotational movement
Requirment for Actin filaments
Requires Ca2+
Actin vs. Myosin
Actin –> Thin filament
Myosin –> Thick filament
***Both found in skeletal muscles
What powers ATP synthesis
Proton Flow around C ring
Use of Clones A3B3G subunits
When attached to a glass slide they were able to show the movement of the gamma subunit – visualized ATP hydrolysis
Where do protons flow inATP synthase?
Protons flow through F0 component of ATP synthase
Parts of F0 Subunit
Subunit A and C Ring
Structure of Subunit A in F0
Subunit a has two 1/2 chanels (each reach halfway into the A subunit
1. Opens to the intermemebrane space
2. Opens to the matrix
Subunit A and C Ring associatation
Subunit A is attached to the C ring (on outside of the c ring)
Flow of Protons through ATP synthase
H+ enter the half-chanel in SU A that faces the Intermembrane space – in the chanel they bind to a Glutamate or an Aspartate on one of the subunits of the C ring –> Then they leave the C subunit once the C subunit rorates to face the matrix 1/2 chanel –> Then H+ leave into the matrix through the matrix 1/2 chanel
Movement of C ring in F0
First faces the IMPS 1/2 channel –> Then it rorates to face the matrix Half channel
Amino Acids in F0 Subunit of ATPO synthase
Requires Glutamate or Aspartate –> The H+ bind to one of the residues on the Subunits of the C ring in F0
What rotates the C ring in the F0 Subunit?
The proton gradient powers the rotation of the C ring
Affect of the rotation of the C Ring
The rotation of the C ring powers the movements of the Gamma subunit in F1 –> Alters the confirmations of the Beta subunits
What powers the movement of the Gamma subunit in F1?
The rotation of teh C ring due to PMF –> the roation of teh gamma subunits = alters the confirmation of the Beta subunits
PUT IT ALL TOGETHER (ATP synthase)
Gamma subunit rotates such that ATP can be synthesized without proton motive form –> THEN the C ring which moves ONLY because of PMF will move –> The c ring movement due to PMF will cause gamma to move which causes the beta SU to change in confirmation which allows the release of the newly synthesized ATP
Movement of protons ion F0
Intermebrane space –> Matrix
Direction of C ring movement
Clockwise
Hydrogen that goes into C ring vs. H that leaves
NOT the same H
***Look at slide 58
Evidence for rotational mechanism for ATP synthesis
The direction observation of rotory motion of the gamma Subunit
Stationary part of F0?
A subunit
Structure of A subunit (depth)
Has 2 hydrophillic 1.2 chanels that do NOT span the memebrane –> thus H+ can pass BUT it can’t move comp[letley across A in one chanel
How is A SU positioned in relation to C ring
The A SU is positioned such that each 1/2 chanel directley interacts with one C SU in ring
Stucture of C ring (depth)
Each polypeptide forms a pair of alpha spanning helicies
***Have Glu or Asp redidue found in the middle of one of the helicies
Movment of C ring
IF Glu or Asp is charges (unprotinated) –> C SU does NOT move
IN a H+ rich envirnment the H+ will enetr the A SU half chanel and bind to Glu while the H+ poor envirnmemnt of other 1/2 chanel releases an H+
Overall – the C SU binds to H+ –> C SU rotates one C SU –> Rotations brings protinated C form from IMPS chanel to teh deprotinated matrix chanel where it binds to H+ = h+ goes through the chanel
- The movement for H+ through 1/2 chanel from high to low H+ concentration powers rotatioon of C ring
What powers rotation of C ring?
The moevment of H+ 1/2 chanels from high to low H+ concentration
A subunit while the C ring moves
Remains stationary
Proton rich envirnment Vs. poor
Rich (IMPS) – protons enetyers chanel and binds to Glu
Poor (Matrix) – release the protons
Rotational movement of C ring (overall)
Rotational movement brings the deprotanated C SU form the matrix channel to the proton rich IMPS channel
How does rotation of the C ring lead to ATP synthesis?
C ring = rightley linked to the gamma and e SU –> THUS as the C ring turns the Gamma and E SU are truned – rotation of the gamma SU promotes the synthesis of ATP through binding change mechanism
What prevents the hexemer from rotating in Sympaythy with C?
The exterior column formed by 26 chains and 6 SU – prevents hexamer from rotating in sympathy
+
The dimerization and oligiomerization in cristae also stabilize the enzyme from rotational forces
Number of C SU
usually 8-14 –> Significant because it determines the # of protons that muct be transported tp generate ATP
Why is the number of c SU important?
Determines the number of protons that are needed in order to generate ATP
Example – 360 degrees generates 3 ATP –> THIS for 10 c SU – each ATP generated requires 10/3 H+
360 degree rotation of gamma
leads to the synthesis and release of 3 ATP
Number of C rings in vertabrates
Recent evidence shows that the number of C rings in all vertabrates = 8 –> Makes vertabrate ATP synthesis the most efficient sthase known (only 2.7 H+ required)
Assumption for how many H+ flow for 1 ATP
3 H+ flow for 1 ATP
Another function of resporatory chain
To regenerate NAD+ for glycolysis
NAD+ issue in ETC
The inner mitocondrial membrane is impermeable to NADH and NAD+ – need to have a way to shuttle NAD+ to cytoplasm to be used by glycolysis
Solution of NAD+ shuttling issue
Muscle –> e- from the cytoplasmic NADH can enter the ETC using the Glycerol-3Phosphate shuttle
Solution: The e- from NADH rather than NADH itself are carried across the mitocindrial memebrane
Example – introducing teh electrons from NADH into ETC using Glycerol-3-Phosphate shuttle
Purpose of the Glycerol-3-P shuttle
Allows cytoplasmic NADH to enter the ETC
Gleycerol-3-P shuttle (Overall)
e- are transfered from NADH –> FADH2 –> Q = forms QH2
Amount of ATP from NADH
2.5
Amount of ATP from FADH2
1.5 – because DAD is the e- acceptor
Where is the Glycerol-3-P shuttle?
In the cytoplasm
***Used in Muscle
Where does NAD+ go after the Glycerol-3-P shuttle
Goes to the cytoplasm to be used in glycolysis
Two places with Glycerol-3-Phosphate dehydrogenase
- Cytoplasm – for glycolysis
- In the Mitocondria – for shuttle
Glycerol 3 - Phosphate shuttle (depth)
DHAP –> Glycerol-3-P –> DHAP
**When DHAP –> G-3-P – have NADH –> NAD+
**When G-3-P –> DHAP – have FAD –> FADH2 – on the process get Q –> QH2
Where is the Malate Aspartate shuttle
In the heart and liver
Use of Malate Aspartate shuttle
e- from the cytoplasmic NADH are used to generate mitocondrial NADH – getting NADH to the mitocondria
Cytoplasm NADH + Mitocondria NAD+ –> Cytoplasmic NAD+ + Mitcondrial NADH
(Putting NADH in mitocondria)
Parts of the Malate-Aspartate Shuttle
- Two membrane transporters
- Four enzymes
Malate Aspartate Shuttle (depth)
Malate enters matrix through malate shuttle –> Malate is turned to Ocoloacate (GENERATES NADH – NOW HAVE NADH IN THE MATRIX)– Oxoloacetate is combined with Glutamate –> Oxo + Glutamate forms A-KG + Aspartate –> A-KG and Aspartatae leave thriugh their repective trasnproters –> In the intermemebarne space A-KG and Aspartate are combined –> forms Oxoloacete + Glutamate –> Oxoloacate goes to Malate (in the intermemebrane space) = refroms NAD+ in the intermemebrane space + malate can go through for a new cycle
Permeability of the inner memebrane
The inner membrane needs to be impermeable to most small molecules BUT also need to have exchange between the cytoplasm and the mitocondria
How is exchange done in the inner mitocondrial memebarne
using different memebrane spanning transporter proteins
How do cytoplasmic electrons from NADH enter mitocndria
Using shuttles
What NADH is already in the mitocondria
NADH from the TCA cycle and Fatty acid synthesis = already in the mitocondria (because those processes occur in the mitocondria)
Glycerol-3-P shuttle (textbooks)
Overall: Introduces the e- from NADH into the ETC – not intridcuing NADH itself directley
- Transfer e- from NADH –> DHAP = forms Glycerol-3-P (USes Glycerol-3-P dehydrigenase)
- Glycerol-3-P is rexidized to DHAP on outter surface of innter mitocndria memebrane using isozyme of Glycerol-3-P Dehydrogenase – here an e- pair is transfered from FAD –> FADH2
- The reduces FADH2 – transfers e- to G –> e- can enetr the ETC as QH2
Glycerol-3-P dehydrigenase
- Cytoplasmic – Putts e- into DHAP –> forms Glycerol-3-P – forms NAD+
- Mitocndria –> Turns Glycerol-3-P into DHAP – forms FADH2 + QH2
Why use FAD if it is lower yeild?
Because enable e- from NADH to be trasnproted into the mitocodnria NADH gradient
***price of trasnprt is 1 ATP
Where is the Glycerol-3-P shuttle + why
Found in muscle cells –> enables mucslces to sustain a high rate of Oxidative phosphorylation
Some insects + G-3-P shuttle
Some insects lack Lacatae Dehydrigenase and are completley dependents on the Glycerol-3-P shuttle for regeneration of cytoplasmic NAD+
Use of Malate-Aspartate shuttle
Brings e- from cytoplasmic NADH into mitocondria in the heart + liver
Malate-Aspartate shuttle (textbook)
e- are transfered from NADH into the cytoplasm to oxoloacetate –> Forms malate –> Malate traverses the inner mitocondrial emebrane in exchange for A-KG –> THEN get rexoidation of malate using NAD+. in the matrix (catylyzed by matlate dehydrogenase) – forms NADH –. Oxolooacentat does not readily traverse the memebarne = trannsamainan reaction is needed to form aparttate and A-KG that cab be trasnfered
How does Malaate traverse the Inner mitocondrial memebrane
By going through trasnproter AND by exchaning it for A-KG – Malatae goes in and. theA-KG leaves
Malate Dehydrogenase
Malaate –> Oxoloacetente (goes NAD+ –> NADH)
***Oxidizing malate to form Oxoloacetate
How can Aspartate go across the Mitochondrial memebrane
By exchanging for glutamate
Oxoloacate moving across mitocondrial memebrane
Oxoloacateate does not teadioly travser the inner memebarne = need a transamianan reaction
- Glutamate donates NH3 group to Oxoloacetate = forms aspartate and A-KG = can cross memebarne –> THEN in the cytoplasm Asparatate is deaminated to go back to oxoloacetate
Coupling of enter of ADP into mitocondria
The entery of ADP into mitocondria is coupled by the exit of ATP using ATP-ADP Translocase
ADP-ATP Translocase (overall)
Helps couple the entry of ADP into the mitocodnria and the exit of ATP – enables the exchange of cytoplasmic ADP for mitocndrial ATP
- Brings in cytoplasmic ADP
***Enables ADP and ATP to traverse the inner memebrane
- Couples the flows of ADp and ATP
Reaction – ADP (cyt) + ATP (matrix) –> ADP (matrix) + ATP (cyt)
What percent of the inner mitocondria memebrane in ATP-ADP trasnlocase
15% of the inner membrane – very abdundent
***Abudnece is manifestation of the fact that humans exchange the equivilant of theoir weight in ATP each day
What is needed for ATP to leave
ADP much enter the mitocondria
Difference in ATP-ADOP translocase and ATP synthase
In ATP-ADP trasnlocase neither the ATP or the ADP is bound to Mg2+
Energy of ATP-ADp exchange
The ATP-ADP exchange is energyetically expensive –> 25% of the PMF generated by the ETC is conssumed by the exchange process
Eversion
Two molecules going away from each other
Mechanism of ATP-ADP Trabslocase
- ADP enters Translocase (the AS is facing the cytoplasm)
- The Translocase site with ADP flips the other wat – flipes towards the matrix (1st eversion event)
- ADP is release – active sit is still facing the matrix
- ATP from the matrix can enter
- Second eversion occur –> AS flips to face the cytoplasm
- The ATP is released into the cytoplasm
***When ADP comes in teh ATP can be realsed form the matrix
Diffusion of ATP and ADP across inner memebrane
They do not diffuse freely
Purpose of ATP synthase Vs. ADP-ATP translocase
ATP-synthase –> Makes ATp + releases teh ATP intp yje matrix
ADP-ATP translocase –> trasports the ATP from the matrix to cytoplasm and birngs ADP into the matrix to be used by ATP syntahse
ATP in mitocondria vs. nucleus
Cytoplasm + Nucleus = have more ATP that mitocodnria –> testamenet to the efficiencey of the transprt of ADP-ATP translocase
ADP-ATP translocase structure
20 kDalton translocase containing a single nucleotide bidning site that alternativley faces the matrix + the Cytoplasmic sides of the memebarne
Energy of ADP-ATPtranslocase
ATP has one more neg. charge that ADP –> THUS in activatley respiring mitocondria with positive memebrane potential –> ATP trasnprt out of the matrix + ADP in is FAVORED
***ADP/ATP exchage is energetocally expensivce –> 1/4 of the PMF in ETC is consumed in process
Inhibition of translocase
Leads to inhibition of cell respiration
Commonality if Mitocondrial Transporters
All have. acommom Tripartite Structure
Composition of ATp-ADP translocase
ATP-ADP Translocase is composed of three tandom repeating 100 AMino acid domain – each domain conaints two transmemenbarne regions
What is on the inner mitocondiral memebrane
The inner mitocondrial memebrane has many transporters + carriers –> enable the exchange of ions and charges partciles between the matrix and the cytoplasm
Tripartitae structure of Translocase
The AA seq has 3 tandom reptease of 100 AA sequence
- Each repeat has 2 Transmembrane regions
***Tripartite structure has been confiormed by the determination of the 3D structure of the tranporter
- The transpemebrane helicies form a teppee like structure with nucleotide binding site laying at the center
- Each of the three repeats have. asimilar structure
Phophate carries in Inner membrane
Works with ATP/ADP trasnlocase – mediates the electrocal chatge of H2PO4 + OH-
- Combination. ofaction of trasnlocase and Phosphate carrier – leads to exchange of cytoplasmic ADP and Pi for matrix ATP – uses 1 H+
***Transporters PROVIDE ATP SYTHASE with substartes
Phosphate craiiers + translocase + ATP synthase
Phosphate carriers and Translocase work togther to exchage ADp + Pi for matrix ATP –> PROVIDE ATP synthase with substrates
***The three are associated in a large complex
ATP Synthasome
Large complex with translocase + ATP synthase + Phophate carriers
Dicarboxylate carrier
Enables malate + Succinate + Fumarate to be exprote form matrix in exchange for Pi
Tricarboxylate carrier
Excghanges citrate and H+ for malate
How does pyruvate enter mitocondria
Uses heterodimer composed of 2 small transmembrane proteins
What regulates cell repsirtaion
Cell resipiration if regulated primaryly by the need for ATP
***Because ATP is the end product of Cell respiration –> The ATP needs is the ultmate determinate of the rate of repiratory path and its components
ATP in Combustion of gluse
30 Molecules of ATP are mode –> 26 of those 30 are made in ETC
ATP made. inmetalbilsim of glucose
Metabolism of glucose to two molecules of pyruvate –> yeilds 4 ATP
ATP in fermentation
When glucose undergoes fermentation only two molecules of ATP are generated per glucose molecule
What determines the rate of ETC
The need for ATP
When do e- flow through ETC
e- ONLY flow if there is ADP avalble to be converted to ATP
***e- do NOT flow through the ETC unless ADP is avalble to be converted into ATP
Regulation of OP by ADP is…
Repiratory/Acceptor control
***The regulation of OP by ADP is called “Reiratory/Acceotor control”
What is repiratory control an example of
Respiratory control is an example of control using Energy Charge
***Coordiatoon of the compenents of Cell respiration makes regulation possibel
Respiratory control
Means that the rate of OP is controlled by. theamount of ADP avalable
- More O2 consumed = more ADP = more converted to ATP
What is e- transport coupled to
e- transport. ioscoupled to phosphorylation –> means that e- won’t flow thourgh EYC unless ADP is also being phophorylated to ATP
Increased ADP concetration
Increased ADP = increase the rate of OP to meet ATP needs
Rate of oxygen consumption in the mitocondria + ADP
The rate. ofoxygen consumption in the mitocondria = increases when ADP is added –> THEN Oxygen l;evel retruns to intial value when ADP is converted to ATP
ADP levels + TCA
The level. ofADP also affects the rate of TCA – decrese ADP = NADH/FADH2 are not used in ETC –> Makes the TCA slow down because there is less NAD+/FAD+ to feed the cycle
***Low ADP = lower TCA rate
Vs.
Increase ADP –>? Increase OP – NADH and FADH2 are reoxided = increases TCA
Regulation of ATP synthase
Inhibited by Inhibitory factor 1 (IF1)
Inhibitory Factor 1
Conserved protein that inhibites ATP synthase
***Inhibits the Hydrolitic activity of ATP synthatse
- IF1 = prevents ATP hydrolysis when Oxygen is not avalable to accept the e- in ETC
- Inhibits the wastful hydrolysis of ATP
Overexpression of IF1
Overexoressed in some cancers –> facilitates the induction of aerobic glycolysis
Function of IF1
Inhibits the hydrolitic activoty of F0F1 in ATP synthatse
- Tissues deprived of O2 = have no O2 to accept in ETC = can’t geenrtate a PMF –> leads the ATP to be hydrolyszed by synthatse – ROLE of IF1 = to preveny the watsful hydrolysis of ATP by inhibiting the hydrolysis activity of sythase
Result of regulated uncoupling
Uncoupling of ETC and ATP synthesis –> Leads to heat
NOn-shivering Theromogensis
Occurs when the ETC is uncoupled from ATP synetshis –> Generates heat
Uncoupliing protein 1 (UCP1)
facilitates the uncoupling of ETC and ATP synthesis in a regulated fashion
**It is an Integral prtein in the inner mitocondrial memebnrane
**Aka “Thermogenin”
- It is. adimer that resembles ATP/ADP translocase
- It trasnports H+ from the intermmebrane soace to the matrix using Fatty acids
- Genertaes heat by short circuting the mitocondiral proton natter – the energy of the H+ gradient is normally captured as ATP = NOW releases as heat as the H+ flows through UCP1 into the mitocondrial matrix
Where does non-shivering thermogenisis occur
In Brown Adipose Tissue (BAT) – adult humans have non-shivering thermogensis in BAT
Where does uncoupling occur
In BAT
BAT
Rich in mitochondria + has uncoupling occuring there
- Rich in mitocondira because it needs ATP –> The inner mitocondrial memebrane has large amounts. ofUP1
- Amount of BAT decreases as we age
***BAT = specialized tissue for the process. ofnon shivering thermogenisis
Affect of Obesity on BAT
Obesity leads to decrease in BAT
UCP2 and UCP3
Also play a role in energy homeostatis + may be important in regulation of body weight
Kid fatty tissue vs. Adult fatty tissue
Kids have different fatty tissue than adults –> adult fat smells bad
When do you see more BAT
After someone is put in cold environment
Use of uncoupling reactions
Uncoupling reactions are a means to maintain body temperature – espcially in hibernating animals + in some new born animals + in many adult animals (escoailly those adpated to the cold)
Snuck Cabbage
Usues an analagou uncoupking mechanism to heat its floral spikes in early spring = increase sthe amount of evaportaion of the Odorferous molecule yhat attracts insects to fertilize its flowes
Uncoupling in animlas
In animlas uncoupling = in BAT
White Adipose Tissue
Plays. norole in thermogensis BUT serves as energy sourcve + an endocrine gland
UCP1 Activation
The H+ pathway is activated when the core body temperature is begining to decrease
- Alpha adrehenic hormones stimulate the release. fofree Fatty acids from Triglycerides storyed in cytoplasmic lipis
- The long chain FA bind to the cystolic face of UCP1 + Coo- binds to H+ –> Binding causing tsructural change in UCP1 – so the H+ and the COO- now face teh H+ poor envirnment of the matrix = H+ are released –> release of H+ resets the UCP1 to its intial state
Where do humans have BAT
Mostley in theor neck and vchest – adult females have more
***BAT is activated by the cold
how is BAT activated?
Activated by the cold
Non-shiveroing thermogenisis in Pigs
Ancestors of pigs are believed to have lost UCP-1 20 MYO when they inhabited tropical and subtropical envirnments where they couold survive withoiut the non-shivering thermogensis –> As the range of pigs expanded the kack of UCP1 became a liability
- Pigs = unsual mammales in that they have large litters and are hoofed anima=las that build nests for birth
- Charchaterics= appear to be an adaptation to the absence. ofUCP1 –> pigs need to rely on oyher methods as a means of themroegensis (nsting + large litter + shivering)
UCP2 and 3 (textbook)
UCP2 – 50% identical to UCP1
- Found in many tissues
UCP3 – 57% ideitical to UCPO 1 and 25% isdetical to UCP2 –> Found in skeletal Muscle and BAT
Family of uncoupling proteins
All play a role in energy homeostatsis
Genes for UPC2/3
Genes for UCP2/3 –> map to regions of the human and mouse chromosome that have been linked to obesity –> Supports the notion that they function. asa means of regulating body weight
How do posons effct OP
Can inihibit by:
1. Inihibition of the ETC by preventing the generation of PMF
2. Inihibition of ATP synthase
3. Uncoupling of ETC and ATP synthsis
4. Inhibition of ATP export
2,4 Dinitrophenol
Inhibits Oxidative Phophorylation – does so by uncoupling ETC from ATP syntehsis
- Works by carrying protons across the inner mitocodniral membrane down theor concentration gradient and bypassing ATP synthase
Sites of ETC inhibitors
NADH-Q OcdRed –> QH2 – Blocked by Rotenone + Amytal
Q-cyt C OxdRed –> Cty C – Blocked by Antimycon A
Cyt C Oxidase –> O2 – blocked by CN- + N3- + CO
Where do you put CO detector
ON the floor – if it is at high level you would due before it beeps
Retanone + Amytal
Inhibits ETC – block e- transfer
**Amytal = setative
**Retanone = fish + insect poison
- NADH Q OXDRed inhibition = prevents the utilization of NADH as a substrate
- In the presence of Retonones + Amytals –> e- flow form Oxdation of Succinate is impared because e- enter at QH2
Retonones + parkinsons
Retinoes may play a role in the development of Parkinsons
Antimycin A
Interferes with e- flow from Cytochrom Bh
CN- + N3- Vs. CO
CN- + N3- –> react with Ferric acid form of heme a3
CO –> inhibits ferrous forms
**All inhibit ETC = inhibit ATP synthase
**e- flow in Cyt C can be blocked
Inhibition of ATP synthase
Oligomycon (antifungal AB) + DCC – prevent influx of H+ through ATP synthase by bindinh to COO- group pf C ring
- B;ock on of only one C SU by DCC is suffeicent to inhibit the roation of entire C ring = inhibits ATP synthase
***If activley respiring miticindria are expsoded to inhibitor of ATP synthatse –> ETC stops – shows that the ETC. andATP synthase are coupled
Uncoupling of ETC and ATP synthesis
Uncoupling usinhg DNP + other acidic Aromatic coumpunds
- Uncouplers = carry H+ across the membrane down the gradinet
- In the presence of uncpuplers e- trasmpry is mormal but ATP is not formed by ATP synthaste because the PMF is continually diseemsinated
***Loss of resporatory control increases O2 consumtion to oxidation of NADH
- Ingestion of uncouplers = increases metabolic fuels but NO energy is made into ATP– RATHER the energy is released as heat
- DNP = in herbacides + fungociedes
DNP use
Some people take DNP as weight loss uspplement + Russian soldiers were gievn DNP to keep warm in the winter
Drugs + uncouplers
Drugs are bieng made to function. asmild uncouplers –> to be used for obesity + related pathologeniss
Example – Xanthomoal = prenylated CVhalcone –> shows promise
- Works by scavenging free radicals
- Is used for treatment of cancer
- Found. inBeer
Inhibition of ATP trasnport
ADP-ATP trasnlocase is inhibited by decrease. inconcentration of atfraftloside + Bonkrekic Acid
***WHen inhibited –> Oxidative phosphorylation stops – shows that ATP/ADP translocase is essntial. formanintingenough ADP to accept energy accoasited with the PMF
Atraftolosde
Binds to translocase when its nucleotide site faces the intermemebrane soace = inhibits ADP/ATP trasnlocase
Bankrekic Acid
Binds to ADP/ATP trasnlocase when the nucleotode site faces the moitocndrial matrix – inhibits ATP exort/inhibits ATP/ADP translocase
MOst common cause of mitocondrial disease
Disruption of complex I
Defects in compoenet of ETC
- reduce ATP synthsis
- Increase the amount of reactive oxygen species formed = increases mitocndrial damage
Number of diseases that are asociated with mitocondrial mutations
Increasing number as we grow our understanding of biuochemistry + genes
- Prevelance. =10-15 per 100,000 people
First mitocoindrial disease to be understood
Leber Hei Optic Neropathy – (LHON) –> Form of blindness that strikes midline. asa result of mutation in Complex 1
LHON
Form of blindness that is a result of mutation in complex 1 –> mutaion inpairs NADH utilization + some block of e- transfer to Q
Egg Mittocondrial DNA vs. Sperm DNA
Egg – has Mitocdnrial DNA BUT the sperm has much less
- because materninity inherited mitodndial disease – is large numbers + not all mutocndria may be affects
What Process is the mitocondria also associate with
Apoptosis – Mitocondria can control the process of apoptosis
- Mitocondria act as control centers regulating process
Apoptosis
Progrgrammed cell death –> results in selective cell death
Use of programmed cell death
Critical for tissue remodneling during development + for the removal of damaged cells
What occurs during apoptosis
Outter mitocondrial memebrane because highly permable = “mitocondria outter memebrane permabilization”
What activates the death pathway
Cytochrom C leaving the mitocondria – one of the most potent activators. =Cty C
- Cyc C = exits the mitocondrial and interacts with APAF1 –> leade to formation of Apsome – Aposome recruits Casphase 9 = protease –> Activased the cascdae of other casphases –: Eachh caspahse tyoe destroys a target
What makes the Outter memebrane highly permable durong apoptosis
Bcl family of proteins (initailly discivered bevcaise of role in cancer)
DNAase
Frees CAD to cleave DNA –> used in apotpsos
Nickname for apoptosis
Cascade of proteolitoc enzymes = “detah by 1000 cuts”
LHON (overall)
Form. ofvision loss due. todeath in optic nuerons
hat neuron is affected in LHON
Nerve 2 = optic nerve –> it is defected = blind
Mutation in LHON
Due to mutations in mitocondrial genes that encode Complex 1
Exampple mutaion – Pro –> Lys (lys = positive AA)
LHON mice
A mouse with mutations was made to allow the phenotype to be sutidies
- Mitocondria in the optic nerve of these mice were dound yo. bean abnormal shape + in higher numbver –> matches what is observed in LHON pateints
- Mitcondria from LHON mice were isolated. ad studies to determine where the LHON phenotuoes results from a decrease in ATP proudctiom or increase in ROS damage
Curing LHON
It s a metabolism disorder = no cure
Cures for mitocindria disoerders
NO cures
