Mitochondria Flashcards
Origin of Mitochondria
1886 Altman - granules that looked like bacteria “bioblasts” 1897 Benda - “mitochondria” (saw them as threadlike granules connected by threads) Michaelis (of michaelis mentin) - realized they were involved in redox reactions using Janus B redox dye - 1912 Kingbury suggests they are site of respiration (cell oxidation) 1949 Kennedy & Lehninger confirm kingbury
Evidence for bacterial origin
Symbiosis, symbionts, & endosymbionts They look like bacteria in light microscope Base pair distribution (A-T and G-C pairs equal in bacteria whereas in eukaryotes A-T > G-C) Related to obligate parasites Rickettsia (No histones, bacterial ribosomes) Realized they had more similarities to bacteria like similar ribosomes, DNA more similiar to bacteria
Endo Theory
Final product = eukaryotes protobacterium engulfed by archea and then nuclues was introduced nucleus forming was evolutionarily beneficial because mixing of archea and bacteria was DNA was common and bad. •Archaea do the engulfing because they have an internal skeleton as opposed to bacteria’s exoskeleton (cell wall). •Phagocytosis is the defining feature that set the eukaryotes apart from the bacteria as is the nucleus. •Stress of loss of cell wall. •Internal cytoskeleton.
Mainstream View
•α-proteobacteria •Guest an aerobic cell, host an anaerobic cell (Ox-Tox hypothesis) •Archaean methanogen engulfs rickettsia-like α-proteobacteria
Hydrogen Hypothesis
•“Archezoan” hydrogenosomes •Nitrate preferring anaerobic mitochondria The hydrogen hypothesis is a model proposed by William F. Martin and Miklós Müller in 1998 that describes a possible way in which the mitochondrion arose as an endosymbiont within a prokaryote (an archaeon), giving rise to a symbiotic association of two cells from which the first eukaryotic cell could have arisen (symbiogenesis). According to the hydrogen hypothesis: The host that acquired the mitochondrion was a prokaryote, a hydrogen-dependent archaeon, possibly similar in physiology to a modern methanogenic archaea, which use hydrogen and carbon dioxide to produce methane; The future mitochondrion was a facultatively anaerobic eubacterium which produced hydrogen and carbon dioxide as byproducts of anaerobic respiration; A symbiotic relationship between the two started, based on the host’s hydrogen dependence (anaerobic syntrophy).
Purple bacteria
•Proteobacteria that are phototropic and can produce energy through photosynthesis. •They are pigmented with bacteriochlorophyll a or b, together with various carotenoids (good antioxidants), which give them colors ranging between purple, red, brown, and orange. •No oxygen released, usually some kind of sulfur compounds involved. •End product of photosynthesis is sulfur instead of oxygen.
Hydrothermal vents (black smokers)
•Chemosynthetic bacteria, archaea, “extremophiles” •“Pioneer organism” “iron-sulfur world theory” •Archaea - methanogens get energy from CO2 and H2, but H2 scarce •Universal distribution? Autotrophic bacteria can live everywhere •Iron-sulfur minerals have an ability to catalyze organic reactions - as they still do today in the prosthetic groups of many enzymes, such as iron-sulfur proteins
Archea
Archaea survive today in extremely harsh environments, such as evaporative salt ponds on the edge of Great Salt Lake and the boiling hot springs of Yellowstone National Park. Methanogens i.e. Archaea have cell membrane consisting of polysaccharides (instead of lipid bilayer).
DNA evidence that mitochondria come from bacteria
•Each human cell has 2-3 meters of DNA, 1 copy total; in body 1013 m, (70+ round trips to moon) •25,000+ genes (nuclear) vs 13 genes (mitochondrial) •Mitochondrial DNA is in plasmid (ring) form ◦13 genes, 5-10 copies of genome/mito, hundreds per cell ◦Nuclear set: one copy per cell ◦Big difference is that every eukaryotic cell only has one copy of the genome while bacteria and mitochondria have multiple copies of their genome
Significance of mitochondrial DNA
•Advantage is local control •Maternal inheritance (Mitochondrial Eve 170,000 years ago) Proves human race originated in Africa. Can trace back to original mitochondria by looking at mutations •Control of apoptosis (cyt c) •Mitochondrial aging •Substrate level oxidation about 10% efficient, so no real food chains as energy falls below 1% after first iteration. •Oxidative is about 40% efficient so there can be six levels and predators can survive. •Predation favors growth in size of both predator and prey. •Why don’t bacteria get larger? •Why do bacteria have small genes? •Large survival value for dividing rapidly. Bacteria that divide faster take over the substrate in competition against those that divide more slowly. To divide fast, if you have fewer genes to divide, then you can divide faster. Bacterial physiology puts a constraint on developing larger sizes or more complicated cells.
Oxidative phosphorylation
•Protons are pushed out and e- are transferred down to chain to oxygen to make water •H+ gradient produces energy to make ATP •H+ + O2 = H2O •Krebs cycle produces H+
Claude Bernard 1813-1878
•Color of blood, artery vs vein ◦Vein blood dark due to CO2 made by combustion in RBC •Thought that respiration occurs in the blood and there is no exchange of gases with the tissue
Eduard Pfluger 1829-1910
•Established respiration occurs in tissues •First suggested by Spallanzani in 1807
Instrumentation and methodology I
•Hoppe (-Seyler) describes absorption spectrum of arterial blood i.e. oxyhemoglobin. 1862 •Stokes showed reversible combination with oxygen and described the absorption spectrum of venous blood i.e. deoxyhemoglobin. 1864
Instrumentation and methodology II
•“Histohaematin” •C.A. MacMunn, 1852-1911 •Other scientist insisted his finding of histohaeatin was artifact from improper set up
Instrumentation and methodology III
•David Keilin 1887-1963 ◦Veterinarian ◦Blowfly larvae ◦Spectroscope: color appeared on blowfly larvae slides with lid, disappeared when lid was removed ◦Cytochrome = color (air enzyme) ◾A, B, C (left to right order of absorption spectrum) ◾B, C, A physiological order ◦Cytochrome oxidase
Otto Warburg 1883-1970
◦Warburg effect: increased glycolysis in the presence of oxygen ◾Cancer ◦“Action spectra” → perturb biological sample and look at light between activated and non-activated forms ◦1931 Nobel Prize ◦“Atmungsferment”
Mitchell hypothesis (chemiosmosis)
Proton motive force Using proton motive force to make ATP OXPHOS are coupled
Britton Chance 1913-2010
Descriptions of mitochondria in different states States 1-5
F.F. Jobsis-VanderVliet
NADH fluoroscopy LaManna Worked With Him - oxidation of cytochrome oxidase Metabolism in C2
Oxidation of Cyt Ox
603 nm Cyt A 550 nm Cyt C Shoulder Cyt B More reduced to less reduced (state 4 to 3 transition) CytOx is fully oxidized under almost all conditions Study indicated that brain isn’t under fully oxygenated conditions Cytochrome partially reduced under normoxic conditions Other studies: multiple wavelength, spectral scanning With activation of a small area of the tissue, State 4 (resting) → State 3 Instead of using up oxygen and becoming hypoxic, signal to increase blood supply which increases oxygen
BOLD fMRI
Deoxygenated hemoglobin has a magnetic signal; oxygenated hemoglobin does not More oxygen in tissue when its metabolism is higher
Conclusions
Cytochrome a, a3 is partly reduced under normal physiological conditions in brain (in vivo) NO raises effective Km? Competes with oxygen for cytochrome oxidase Cytochrome a, a3 becomes more oxidized with neuronal activation Recall: regulated to minimize brain exposure to oxygen Excess oxygen competes with NO? Brain oxygen delivery is regulated to minimize brain exposure to oxygen Protective mechanism against free radical damage?
Physiological concepts
Just sufficient oxygen Vascular, structural, and functional compensation Oxygen avoidance
Issues with mitochondria usually present when
young
Energy expenditure:
mitochondria work to generate ATP Indirect calorimetry VO2 = mL/kg.hr VCO2 = mL/kg/hr VO2 and VCO2 = used to calculate heat = kcal/kg/hr Ketogenic diet = high fat, contains protein, no carbs. Burning mostly fat. (similar to Adkins) Less efficient in burning substrate Obese person burns fewer calories In animal experiment: VO2 is up, VCO2 is down = using more oxygen to make same amount of oxygen as wt or control We produce CO2 at the level of the TCA Faster TCA = faster generation of reducing equivalents Relative to wild type, energy expenditure in these animals is lower From indirect calorimetry, we can calculate RQ (respiratory quotient) [CO2 production/O2 consumption] When burning only fat, RQ is much lower than when burning carbohydrate-fat mixed diet CHO (all carb) = 1.0, mix diet =< 0.7 and >1.0, fat = 0.7
Fate of food
Oxidation-reduction reactions Transduction to chemical energy “ATP equivalents” Production of water, heat, protons as a result of electron transfer
Respiratory Chain - What drives it
Combination of oxygen + carbon-based substrate = ATP What drives this process (carbon → TCA cycle → ETC)? Energy (ATP) demand Cell says “I need ATP”
Structure
Inner membrane generates proton gradient
Mitochondrial energetics
ETC = 1, 3, 5
Wallace: mitochondrial diseases in man and mouse:
“mitochondrial defects implicated in a wide variety of degenerative diseases, aging, and cancer”
Mitochondrial genome
LHON, MELAS, MERRF Sites of mitochondrial defect
History
1950s Eugene Kennedy and Albert Lehninger CAC, FA enzymes (beta oxidation) Respiratory assembly (2x0.5 mm) Independent genome and similarity to bacteria These organelles had a symbiotic relationship with eukaryotic cells three billion years ago Mito genome: genetic info for 13 proteins and some RNA DNA size similar to bacterial chromosomes (16, 569 base pairs) Maternal transmission - evaluating variation in mtDNA - global movement of humans mtDNA (10%) - circular double stranded DNA Genes encoding 13 proteins (ETC) 7 subunits of NADH: ubiquinone oxidoreductase (CI) 1 subunit (cyt b) ubiquinol:cytochrome c oxidoreductase (CIII) 3 subunits of cytochrome c oxidase (CIV) 2 subunits of ATP synthase (CV) Complexes I and III in particular generate ROS Complex V isn’t really part of the ETC
Oxidative phosphorylation
Oxidative side of the ETC are the first four complexes (really only three if you eliminate Complex II) Complexes I-IV + V = oxidative phosphorylation Complex V does phosphorylating If uncoupled, will you make ATP? NO If you have a defect in Complex I, will you make ATP? YES, if substrate is entering at Complex III You make less ATP as you move down the chain Succinate dehydrogenase (Complex II) Scheme is incorrect because Complex I DOES NOT go through Complex II (do not pass through from 1 → 2 → 3). Passes from 1 → Q → 3. Succinate dehydrogenase passes from Q → 3.
Chemiosmotic hypothesis
Peter Mitchell, 1961 - ATP synthesis occurs as a “primary energy conserving event”
1st phase
3 e- driven proton pumps - contain oxidation reduction centers (CI, CIII, CIV): NADH - reductase, cytochrome reductase, cytochrome oxidase (flavins, quinines, iron sulfur clusters, hemes, copper irons)
2nd phase
ATP synthase (ATP; e- flow back into matrix)
ETC-linked oxidation-reduction
1st phase 3 e- driven proton pumps - contain oxidation reduction centers (CI, CIII, CIV): NADH - reductase, cytochrome reductase, cytochrome oxidase (flavins, quinines, iron sulfur clusters, hemes, copper irons) 2nd phase ATP synthase (ATP; e- flow back into matrix)
Co Q
Complexes I and II will not be able to pass e- without Co Q Oxidized and reduced forms Q=CoQ10 Ubiqinol is reduced coenzyme Q
Coupling of oxidation and phosphorylation
Coupling of a proton gradient across the inner mitochondrial membrane to the pumping of protons out of the mitochondrial matrix ATP synthesized when protons flow back to the mitochondrial matrix through enzyme complex (complex V)
Oxidative phosphorylation (OXPHOS)
A process in which ATP is synthesized as a result of the transfer of e- from NADH and FADH2 to O2 by a series of e- carriers NADH, FADH2 - reducing equivalents generated from the oxidation of food stuffs Complete oxidation: 1.5, 2.5 ATP mol/reducing equiv mol transferred to O2
OXPHOS currency
Proton motive force (pmf) generated (consists of pH gradient and transmembrane potential) e-mf ≈ pmf ≈ phosphoryl potential (Δ GO′)
Equation for life:
“ liberation of energy vs consumption of energy = redox” 2H+ + O2 → 2H2O + energy ½ O2 + NADH + H+ ⇄ H2O + NAD+ ΔE’o = 1.14 V = ΔG’o = -52.6 kcal/molH+ ADP +Pi + H+ ⇄ ATP + H2O ΔG’o = +7.3 kcal/molH Nernst Potential Energy vs Gibbs free energy
Majority of mutations observed in the human genome are in
Complex I. Some mutations located on proteins outside of the ETC.
Enzyme complexes tested to see
Enzyme complexes tested to see if they function well individually. Can assess whether there is a metabolic block in any one of these components.
Coupling of oxidation and phosphorylation
Coupling of a proton gradient across the inner mitochondrial membrane to the pumping of protons out of the mitochondrial matrix ATP synthesized when protons flow back to the mitochondrial matrix through enzyme complex (complex V) What does it mean to couple a proton gradient across a membrane? As the gradient builds as a result of passing electrons across each of the complexes, protons build up a gradient in the inner membrane. The coupling to complex V as a result of I-IV, you then can make ATP. Protons flow back in through complex V to make ATP.
Oxidative phosphorylation
Complexes I-IV → oxidative portion, pumping H+ as a result of passing e- along the chain. Quinone → small molecule located between complexes I, II, and III. Recall: succinate does not span the membrane (error in image) Complexes I and II communicate through Q on to complex III Defects associated with lack of making quinone CoQ10 is a quinone If we can’t make ubiquinone, we won’t efficiently pass e- from I to III Other defect: quinone stays in its reduced form and cannot be oxidized back to its oxidized form to accept e- Other intermediate outside of complex: cytochromes Take on e- that go from III to IV Defect in V: can pass e- along but can’t make ATP Defects associated with uncoupling protein If there’s a buildup of H+, then UCP allows protons to leak out and the gradient collapses “Poke holes in the membrane” to allow e- to pass but collapse the H+ gradient. Idea to reduce weight gain. Make a lot of heat, pass a lot of e-, but you won’t make ATP and you’ll burn a lot of energy → won’t be obese. Too much UCP → you’ll make a lot of heat, you won’t maintain your body weight, you’ll be fatigued.
ETC-linked oxidation-reduction
State 1 Defect in complex II won’t affect the gradient, but you may affect ATP production Defect in II also linked to fatigue. Complex II connected to TCA cycle via succinate dehydrogenase. Defect associated with lack of reducing equivalents generated. State 2
States of respiratory control
Conditions limiting rate of respiration State 1: availability of ADP and substrate State 2: availability of substrate State 3: integrated functional capacity of ETC (all substrates in saturating amounts) State 4: availability of ADP only State 5: availability of oxygen State 3 is the working state of the mitochondria. What limits the mitochondria is the actual electron transport system. If there’s any block anywhere along the transport system, that’s going to show up in State 3. What’s limiting here is the ETC itself. State 4 is resting state. You’ve used up all your ADP so you can’t make ATP, but you’re just resting/idling. Cytochrome oxidase burns some oxygen to make water. Substrate i.e. glutamate, malate, pyruvate, fumarate, succinate. What’s limiting of the whole thing is either oxygen or ADP. State 1 is the slow state. When coupled, every complex is limiting. When uncoupled, complex V is removed from the equation.
Electrochemical concept
pmf = pH gradient + transmembrane potential
Physiological forces vs chemical driving forces
= fx {available ADP “ATP demand” vs work (ΔG) What drives the Weibel diagram? ATP demand, not substrate availability. We pull food through, we don’t push.
Thermodynamic expression
quation 1 ΔE = ΔE’o + 2.3RTnF log [oxidant / reductant] Ex. reducing agent → NAD+/NADH Equation 2 ΔG = ΔG’o + RT ln { [ADP][Pi] / [ATP] } “REDOX pairs” Keep in mind: if you become more reduced, your bioenergetics dies.
Cunningham steel ball: oxygen toxicity?
Elevated production of ROS Aging Stroke: ischemic reperfusion injury Ischemic conditions: no oxygen and no substrate (e-), would you make ROS? NO Reperfusion causes oxidative injury. Vessels dilated due to nitric oxide. Huge flow of oxygen and substrate in the blood. Overload of reducing equivalents. Neurodegenerative disease Ex. Alzheimer’s
Aging process?
The mitochondrial ETC is the primary source for the production of ROS The localization of ROS production in the brain has been described to occur at Complex I and III…their relative contribution to net release remains to be discerned
Treatment strategies?
Cells are normally able to defend themselves against ROS damage with enzymes such as SODs, catalases, lactoperoxidase, glutathione peroxidases, and peroxiredoxins. Small molecule antioxidants such as ascorbic acid (vit C), tocopherol (vit E), uric acid, and glutathione also play important roles as cellular antioxidants. Similarly, polyphenol antioxidants assist in preventing ROS damage by scavenging free radicals. Don’t want to eliminate all ROS- some, like NO, important for vasodilation If we shift the redox too far (more into reduced state), antioxidants will make things worse.
Compartmentation
Catalase, which is concentrated in peroxisomes located next to mitochondria, reacts with H2O2 to catalyze the formation of water and O2. Glutathione peroxidase reduces H2O2 by transferring the energy of the reactive peroxidases to a very small sulfur-containing protein called glutathione. The selenium contained in these enzymes acts as the reactive center, carrying reactive electrons from the peroxide to the glutathione. Peroxiredoxins also degrade H2O2 within the mitochondria, cytosol, and nucleus. 2 H2O2 → 2 H2O + O2 (catalase) 2 GSH + H2O2 → GS-SG + H2O (glutathione peroxidase)
Upregulating antioxidant systems is a
prevention strategy
Paper: prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes
How does high glucose in the blood generate ROS? Increased [glucose] → increased reducing equivalents Increased proton electrochemical potential gradients ?? More ROS production → increased UCP → leak at the expense of making ATP Release pressure gradient and reduce oxidative production (reducing damage at the expense of making ATP) Increased ROS with high glucose and diabetes
Paper: pathobiology of diabetic complications
Glucose make toxic aldehydes that become inactive alcohols Increased glucose the system is overwhelmed so you make more toxic aldehydes but cannot detox them so you shift the redox and make toxic fructose and use up oxidized glutathione so we r reduced at glut level and NADH level ROS → toxic aldehydes (more made under diabetic conditions). With huge increase in glucose, normal system cannot effectively detoxify aldehydes. Also generate toxic fructose. Oxidized GSSG → reduced GSH. Overall shift in redox → bad. Uncoupler and glu reduced ROS Enzyme and glu reduced ROS Ox phos and mito intact needed for ROS production C1 and 3 produce ros
Mitochondrial metabolic blocks
Fats and lipids used to make reducing equivalents Theory that fewer ROS made under these conditions Associated diseases: Parkinson’s, Alzheimer’s PD attack C1 exacerbated by MTPT - part of LSD affect C1 looks like PD Cells can’t use glu in alzheimer’s pyruvate dehydrogenase not work shift redox TCA not function provide exogenous source via ketone bodies to make ketogluterate - bypass blocks by offering different substrates
Oxidation rates: polarography
X axis: time (minutes) In humans state 4 is more of a horizontal line Y axis: % oxygen First: a little oxygen, no substrate. Some activity: cytochrome oxidase taking oxygen to water. No ADP yet so not ATP generated Adding ADP next, then the reaction goes very rapidly (state 3) Mitochondria rest (state 4). Horizontal line is humans. Slope: % oxygen used over time New state 3 that is very active under the presence of unlimited ADP DNP (uncoupler): pass lots of e- here but not make ATP. This assesses the ability of the ETC to pass e- or to undergo oxidation-reduction reactions If you had a metabolic block in the ETC, this state 3 uncoupled state would be low because you’re not able to pass electrons but state 3 on top will also be low
If you wanted to test a metabolic block:
Present substrate at Complex I and then through OXPHOS measure the rates of State 3 and State 4 If you wanted to test complex I, you’d measure the rates with substrate If you wanted to test complex II, you’d measure the rates with succinate If you wanted to test complex IV, add TMPD and ascorbate
OXPHOS analysis - Oxidation Rates with a Block
Notice state 3 is not as fast. State 4 in this case is normal but in some cases it has a sharp slope like complex III, which is not good because this means there is constant running through of e- and thus generation of ROS. All rates are low with I, II, III, and CoQ. Once you bypass the block and the rates are normal, you know it’s a Complex III problem. No rest you generate ROS Everytime you are at state 3 you generate ROS
Assess for block by
Assess for block by adding substrate and checking complex and assessing the rates of state 3 and 4. All the rates will be low until you bypass the block. So if the block is at 1 and you add substrates for 1 the rates will be low but they will be normal when you add substrates for 2-4 So if the block is at 2 and you add substrates for 1 or 2 the rates will be low but they will be normal when you add substrates for 3-4 So if the block is at 3 and you add substrates for 1 or 2 or 3 the rates will be low but they will be normal when you add substrates for 4 So if the block is at 4 the rates will always be low
Mitochondrial function: brain
Recovery from ischemic insult? Cardiac arrest and resuscitation (CAR) Focal stroke (MCAO) Aging?
State 3: CAR recovery
Evaluated functionality of mitochondria in brain following cardiac arrest Pre, post ischemic insult 1 hour, 24 hours, 48 hours State 3 (nA O/mg/min) was around 300 Increases ROS generated 0-48 hours after recover from CA
Endosymiobtic Theory Origins
Ubiquinol
Anaerobic Metabolism
Mitochondrial Genome
Metabolic States
Reaction Spectrum 1
Oxidation of Cytochrome
Mito In Vitro
Response to Cytochrome
Reflectance Spectra
Cytochrome Wavelength Differentials
Difference Spectra
in Vivo Reflectance Spectra
Prevention of ROS damage
SOD system
Gibbs Free Energy
Components of the Respiratory Chain
OxPhos Food
Transport of Reducing Equivalents
Diff Spectra 2
Diff Spectra 3
Ox Phos Analysis
States of Resp Control
Catalase
Electrical Stimulation of the Brain
Compartmentation
ETC Analysis
ETC linked Redox
ETC Flow
Respiration and ROS production
Sources of ROS
Oxidation States Polarography
Figure 2
Fig 2 Part 2
C 1 and 3 and ROS
Block - Ox Rates
Mito Function Brain
Pathobiology of Daibetic Complications
Scientic Approach
Figure 1
Ox damage Diabetes
E flow trhough resp chain
Electrochemical Concept
Blocks Strategy
REDOX carriers and proton pumps
Graphs
Facts
Resp Chain Pic
Energy Expenditure
Kieblers Law
SOD
