Mitochondria Flashcards
(167 cards)
1
Q
Origin of Mitochondria
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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
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Q
Evidence for bacterial origin
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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
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Q
Endo Theory
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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.
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Q
Mainstream View
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•α-proteobacteria •Guest an aerobic cell, host an anaerobic cell (Ox-Tox hypothesis) •Archaean methanogen engulfs rickettsia-like α-proteobacteria
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Q
Hydrogen Hypothesis
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•“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).
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Q
Purple bacteria
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•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.
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Q
Hydrothermal vents (black smokers)
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•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
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Q
Archea
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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).
9
Q
DNA evidence that mitochondria come from bacteria
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•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
10
Q
Significance of mitochondrial DNA
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•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.
11
Q
Oxidative phosphorylation
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•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+
12
Q
Claude Bernard 1813-1878
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•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
13
Q
Eduard Pfluger 1829-1910
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•Established respiration occurs in tissues •First suggested by Spallanzani in 1807
14
Q
Instrumentation and methodology I
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•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
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Q
Instrumentation and methodology II
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•“Histohaematin” •C.A. MacMunn, 1852-1911 •Other scientist insisted his finding of histohaeatin was artifact from improper set up
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Q
Instrumentation and methodology III
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•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
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Q
Otto Warburg 1883-1970
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◦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”
18
Q
Mitchell hypothesis (chemiosmosis)
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Proton motive force Using proton motive force to make ATP OXPHOS are coupled
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Q
Britton Chance 1913-2010
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Descriptions of mitochondria in different states States 1-5
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Q
F.F. Jobsis-VanderVliet
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NADH fluoroscopy LaManna Worked With Him - oxidation of cytochrome oxidase Metabolism in C2
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Q
Oxidation of Cyt Ox
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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
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Q
BOLD fMRI
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Deoxygenated hemoglobin has a magnetic signal; oxygenated hemoglobin does not More oxygen in tissue when its metabolism is higher
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Q
Conclusions
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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?
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Q
Physiological concepts
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Just sufficient oxygen Vascular, structural, and functional compensation Oxygen avoidance
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Issues with mitochondria usually present when
young
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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
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Fate of food
Oxidation-reduction reactions Transduction to chemical energy “ATP equivalents” Production of water, heat, protons as a result of electron transfer
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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”
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Structure
Inner membrane generates proton gradient
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Mitochondrial energetics
ETC = 1, 3, 5
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Wallace: mitochondrial diseases in man and mouse:
“mitochondrial defects implicated in a wide variety of degenerative diseases, aging, and cancer”
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Mitochondrial genome
LHON, MELAS, MERRF Sites of mitochondrial defect
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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
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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.
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Chemiosmotic hypothesis
Peter Mitchell, 1961 - ATP synthesis occurs as a “primary energy conserving event”
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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)
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2nd phase
ATP synthase (ATP; e- flow back into matrix)
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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)
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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
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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)
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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
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OXPHOS currency
Proton motive force (pmf) generated (consists of pH gradient and transmembrane potential) e-mf ≈ pmf ≈ phosphoryl potential (Δ GO′)
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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
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Majority of mutations observed in the human genome are in
Complex I. Some mutations located on proteins outside of the ETC.
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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.
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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.
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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.
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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
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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.
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Electrochemical concept
pmf = pH gradient + transmembrane potential
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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.
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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.
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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
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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
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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.
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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)
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Upregulating antioxidant systems is a
prevention strategy
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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
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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
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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
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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
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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
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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
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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
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Mitochondrial function: brain
Recovery from ischemic insult? Cardiac arrest and resuscitation (CAR) Focal stroke (MCAO) Aging?
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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
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ADP/O: CAR recovery
Mitochondria are able to efficiently make ATP
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RCR: CAR recovery
Respiratory control rate State 3/State 4 Low during 1 hour and 24 hours in all regions of the brain (treatment window) Due to high state 4 (too many e- flowing through, you’re generating ROS) Appears to recover after 48 hours Brain undergo further insult later - not from initial insult or if it is it is from an unknown signaling pathway
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Paper: aging skeletal muscle mitochondria in the rat: decreased UCP3 content
Hypothesis: can’t make ATP like normal Found that in the aged: state 3 is normal but decrease in state 4 Can’t uncouple enough → building up ROS RCR higher No difference in mitochondrial efficiency (ADP/O) One stage of aging too much UCP second stage not enough Aging process due to lack of UCP
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major site of ROS production
Found that Complex III is the major site of ROS production
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ROS facts
Coupling - protons flow back in via complex 5 to make ATP metal components also make electrons majority of mutations lead to a malfunction in C1 antiport transport enzyme components - reducing equivalents assess whether or not there is a block. REOX - first 4 complexes Quinone - small mol located btw comp 1,2,3 Succcinate does not span the membrane
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General Facts
I and II complex onto quinone to pass electrons to 3 If q stays reduced it can not be oxidized back Cytochromes - take on electrons that go from 3 to four - prob w redox through metal charging Complex 5 - synthase system Defect here is often due to uncoupling Uncoupling can stop obesity 2 phase redox - proton pumping Complex 2 - decreased reducing equivalents and ATP production but not a gradient problem because it does not pump protons States: 3 - working state - limits mito any block shows up in state 3 - ETC is limiitng here State 4: used up all ADP just make water cant make ATP so resting ADP or oxygen are limiting - state 3 runs out of ADP rests Running out of O2 - anoxia Can only offer substrates that directly enter the mito in vitro State 3: working state coupled c1-5 are limiting Uncoupled state 3 is very fast - now complex 5 is not limiting State 4 resting state ‘ Forces based on ATP demand Pull food and redox through tca Oxygen toxic - radicals ROS - cause of aging Reperfusion after ischemia causes injury by causing ROS damage Shift redox - abundance of antioxidants - problems Vit c is a prooxidant Too much glu make toxic aldehyde switch redox ROS produced in face of high ROS Measure ROS production Complex I - Glutamate +/- malate and rotonone - no state 3 but state 4 Inhibitior no state 3 rate
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The Origin of the Eukaryotic Cell
“The first eukaryote—one cell engulfed another to form an extraordinary chimera two billion years ago. The evolution of these complex cells is shrouded in mystery, and may have been one of the most unlikely events in the entire history of life. The critical moment was not the formation of a nucleus, but rather the union of two cells, in which one cell physically engulfed another, giving rise to a chimeric cell containing mitochondria. Yet one cell engulfing another is commonplace; what was so special about the eukaryotic merger that it happened only once?”
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Mito Origin
Phagocytosis along with the nucleus is the defining feature that set the eukaryotes apart from the bacteria. Stress of loss of cell wall Internal cytoskeleton Archea wall can do engulfing and change shape because its wall is made of polysaccharides
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Purple Bacteria
Proteobacteria that are phototrophic & can produce energy through photosynthesis. They are pigmented with bacteriochlorophyll a or b, together with various carotenoids, which give them colors ranging between purple, red, brown, and orange. No oxygen released, usually some kind of sulfur compounds involved
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Black Smoker
Black smoker at a mid-ocean ridge hydrothermal vent – where volcanoes are - no oxygen – first cell types developed A black smoker community comprised of giant red tubeworms and hundreds of squat lobsters. This vent is located in Strawberry Fields of the Main Endeavour hydrothermal field on the Juan de Fuca Ridge. Vibrant colonies of tube worms with red gills thrive on this vent which is predominantly composed of iron- and sulfur-bearing minerals.
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Black Smoker Facts
Driven by volcanic activity, near magma Composed of Sulfides, so black color Discovered in 1977 Hot acidic superheated water (250-400°C) meets cold (2 °C) alkaline water – gives energy for chemical reactions Carbon in form of CO2 Source of Fe2+ and Ni2+ Home to chemosynthetic bacteria
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Hydrothermal Vents (black smokers)
Chemosynthetic bacteria, archaea, “extremophiles” “Pioneer organism” “iron-sulfur world theory – iron sulfur minerals participated in energy forming reactions for life” Archaea – Methanogens get energy from CO2 and H2, but H2 scarce. Universal distribution? Autotrophic bacteria can live everywhere. Cell membranes consist of polysaccharides Iron-sulphur minerals have an ability to catalyse organic reactions—as indeed they still do today in the prosthetic groups of many enzymes, such as iron-sulphur proteins – still seen in humans
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White Smokers
White flocculent mats in and around the extremely gassy, high-temperature (\>100°C, 212°F) white smokers at Champagne Vent. White smoker further away from the hottest part of the vent alkalin rather than acidic
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Hydrothermal Deep Sea Vents: “White Smoker”
Discovered in 2000, 10 miles from mid atlantic ridge The Lost City alkaline hydrothermal vent field features a collection of about 30 carbonate chimneys each between 30– 60 metres tall. This picture shows a five-foot-wide ledge on the side of a chimney which is topped with dendritic carbonate growths.
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White Smoker (or “alkaline vents”)
Not volcanic Emit minerals rich in barium, calcium and silicon Labyrinth of interconnected micropores makes small channels in rock which mimics the hydrogen concentration gradient seen in mito membranes Reacts with mantle peridotite to make serpentinite “Warm” strongly alkaline water at 40-90°C
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Archaea in Extreme Environments
Grand Prismatic Spring, Yellowstone National Park. Steam rising from hot and sterile deep azure blue in the center surrounded by huge mats of brilliant orange algae, bacteria and archaea This photo shows steam rising from hot and sterile deep azure blue water (owing to the light absorbing overtone of an OH stretch which is shifted to 698 nm by hydrogen bonding [1]) in the center surrounded by huge mats of brilliant orange algae, bacteria and archaea. The color of which is due to the ratio of chlorophyll to carotenoid molecules produced by the organisms. During summertime the chlorophyll content of the organisms is low and thus the mats appear orange, red, or yellow. However during the winter, the mats are usually dark green, because sunlight is more scarce and the microbes produce more chlorophyll to compensate, thereby masking the carotenoid colors.
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Archaea Today
Archaea survive today in extremely harsh environments, such as evaporative salt ponds on the edge of Great Salt Lake (above) and the boiling hot springs of Yellowstone National Park (right). (Univ Utah website) – Live in our guts
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Methanogens
Methanogens, ie, Archaea have cell membrane consisting of polysaccharides
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Anaerobic Metabolism
Take carb and turn it into endpoint like pyruvate – similar to glycolysis Archea in gut make methane Bacteria make sulfur – used to produce sulfate
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DNA Evidence for Bacterial Origin of Mitochondria
Each human cell has 2-3 meters DNA, 1 copy total; in body 1013 m, (70+ round trips to moon), 25,000 (probably more) genes Mitochondria: 13 genes/ 5-10 copies/mito, 100’s/cell Eukaryotes have more A-T pairs than G-C pairs Mitochondria have equal amounts of A-T and G-C pairs Bacteria only have about 500 genes (e.coli only have about 4300 genes) DNA evidence – one copy of th genome in every cell – each person has enough DNA to to stretch to the moon Eukaryotes have more AT pairs than GC pairs Bacteria has 500 or more genes Mito – bac ribosomes - no histones Mito genes are beneficial because it allows them to maintain control
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Significance of Mitochondrial DNA
Advantage is local control Loss of mitochondrial genome nuclear dependence Maternal inheritance (Mitochondrial Eve ~170,000 yrs ago) 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 6 levels and predators can survive. Predation favors growth in size of both predator and prey Why don’t bacteria get larger?? (S/V constraint) Why do bacteria have small genes? (reproductive speed advantage) Pretty much all metabolic evolution accomplished by prokaryotes
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krebs cycle
Takes sugar and makes CO2 and hydrogen
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Mito similar to
hydrogen fuel cell
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Energy Advantage
Sugar Lactic Acid = 2 ATP Sugar + Oxygen Water + Carbon Dioxide = 38 ATP !!
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\_\_\_\_ percent of cardiac muscle is mitochondria
40 percent of cardiac muscle is mitochondria
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\_\_\_\_ percent of cardiac muscle is mitochondria
40 percent of cardiac muscle is mitochondria
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Nucleus develops to
Nucleus develops to keep genomes of engulfed cell separate from host cell genome
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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. – RBC use oxygen make energy pass to tissue Bernard came up with the term homeostasis
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Hoppe (-Seyler)
describes absorption spectrum of arterial blood, i.e. oxyhemoglobin. 1862
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Stokes showed
reversible combination with oxygen and described the absorption spectrum of venous blood, i.e. deoxyhemoglobin. 1864
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MacMunn
Spectroscopic – light on muscle absorption spectrum bands pf color – called histohematin bec colors were similar for other iron contatining compounds. Sylar decided MacMunn was wrong and his measurements were flawed by hemeglobin being broken down and artifact
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Klein
Klein – blowfly larvae – cover slip on them – saw bands of absorption – realized it had to be part of the O2 process – and called it cytochrome Warburg was trying to find air enzyme – Kline discovered cytochrome oxidase Major battles btw the two Warburg effect Rat brain cytochrome spectrum from Monge et al Mol Cell Biochem 2008 Use prisim to get wavelength absorption – get negative – dark band – absorption o cyt – prisim goes from blue to red – 600 nm and then another band – not in order in mito bec not discovered in a functional way Blue bands – max absorption in this range He took the spectra for this for a lot of org and they all had these bands in the same way – order B -) C -) A
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Warburg
Warburg Effect Increased glycolysis in the presence of oxygen “Action Spectra” 1931 Nobel “Atmungsferment – enzyme He was looking for” CO2 in blood change color and absorption spectrum Bind hemeglobin and change wavelength – define action spectrum
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Mitchell Hypothesis (Chemiosmosis)
Mitchell – chemiosmotic hypothesis – h ions going down concentration gradient from high to low spin atp synthase backwards which makes ATP Matrix alkalinic compared to intermembrance space – intermembrane space is in equilibrium with cyto and at like 7.2 while matrix is about 8 Number of protons sitting in 1/10 micron – 100 or 200 free protons in mitochondria
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Britton Chance
Made photomultiplier tubes Post code modification of radars Was Lamanas scientific grandfather Mito in vitro Chance and williams states of mitochondria 4-3-4 transition No met w/o oxygen Method of looking at flourscence of NADH Came up with states
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F.F. Jöbsis-VanderVliet
La Manas boss In vivo flourscent measurements Bumps – 577 and 585
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Bold fmri –
initial decrease and then ox of field of measurements Ratio deoxy up and oxy down – curve is the same
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Conclusions
Cytochrome a,a3 is partly reduced under normal physiological conditions in brain NO raises effective Km and causes the partial reduction why you see in vivo but not in vitro? Cytochrome a,a3 becomes more oxidized with neuronal activation Excess Oxygen competes with NO? Brain oxygen delivery is regulated to minimize brain exposure to oxygen Protective mechanism against free radical damage?
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Physiological Concepts
Just sufficient oxygen Vascular structural and functional compensation to keep o2 level where it is necessary w/o too much LaManna, 1992 Oxygen avoidance
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Physiological Concepts
Just sufficient oxygen Vascular structural and functional compensation to keep o2 level where it is necessary w/o too much LaManna, 1992 Oxygen avoidance
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Coupling of Oxidation & Phosphorylation
Coupling of a proton gradient 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)
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OXPHOS Currency:
ated (consists of pH gradient proton motive force (and transmembrane potential) emf ≈ pmf ≈ phosphoryl potential (Δ G′)
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GIBBs Free Energy
...thermodynamic potential that can be used to calculate the max of reversible work ~ POTENTIAL ENERGY
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GIBBs Free Energy
During a reversible electrochemical reaction at constant temperature and pressure, the following equations involving the Gibbs free energy hold: ∘ Δ rG Δ + R T ln Qr = = Gibbs free energy change per mole of reaction rG rG Δ Δ products at standard conditions R = gas constant T = absolute temperature (in K) ln = natural logarithm Qr rG = Gibbs free energy change per mole of reaction for unmixed reactants and °
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Nernst
ΔE = ΔEo' +2.3 RT F log [Oxidant] / [Reductant]
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Elevated production of ROS:
Aging, Neurodegenerative disease, Stroke: ischemia-reprefusion injury
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Primary source of ROS
The mitochondrial electron transport chain (ETC) is the primary source for the production of reactive oxygen species (ROS).
114
The localization of ROS production in brain has been described to occur at
The localization of ROS production in brain has been described to occur at Complex I and III....their relative contribution to net release remains to be discerned
115
Diabetes Paper
FIG. 1. Mitochondrial oxidative damage. The mitochondrial respiratory chain (top) passes electrons from the electron carriers NADH and FADH2 through the respiratory chain to oxygen. This leads to the pumping of protons across the mitochondrial inner membrane to establish a proton electrochemical potential gradient (H), negative inside: only the membrane potential (m) component of H is shown. The H is used to drive ATP synthesis by the F0F1ATP synthase. The exchange of ATP and ADP across the inner membrane is catalyzed by the adenine nucleotide transporter (ANT) and the movement of inorganic phosphate (Pi) is catalyzed by the phosphate carrier (PC) (top left). There are also proton leak pathways that dissipate H without formation of ATP (top right). The respiratory chain also produces superoxide (O2), which can react with and damage iron sulfur proteins such as aconitase, thereby ejecting ferrous iron. Superoxide also reacts with nitric oxide (NO) to form peroxynitrite (ONOO). In the presence of ferrous iron, hydrogen peroxide forms the very reactive hydroxyl radical (OH). Both peroxynitrite and hydroxyl radical can cause extensive oxidative damage (bottom right). The defenses against oxidative damage (bottom left) include MnSOD, and the hydrogen peroxide it produces is degraded by glutathione peroxidase (GPX) and peroxiredoxin III (PRX III). Glutathione (GSH) is regenerated from glutathione disulfide (GSSG) by the action of glutathione reductase (GR), and the NADPH for this is in part supplied by a transhydrogenase (TH)
116
Endosymiobtic Theory Origins
117
Ubiquinol
118
Anaerobic Metabolism
119
Mitochondrial Genome
120
Metabolic States
121
Reaction Spectrum 1
122
Oxidation of Cytochrome
123
Mito In Vitro
124
Response to Cytochrome
125
Reflectance Spectra
126
Cytochrome Wavelength Differentials
127
Difference Spectra
128
in Vivo Reflectance Spectra
129
Prevention of ROS damage
130
SOD system
131
Gibbs Free Energy
132
Components of the Respiratory Chain
133
OxPhos Food
134
Transport of Reducing Equivalents
135
Diff Spectra 2
136
Diff Spectra 3
137
Ox Phos Analysis
138
States of Resp Control
139
Catalase
140
Electrical Stimulation of the Brain
141
Compartmentation
142
ETC Analysis
143
ETC linked Redox
144
ETC Flow
145
Respiration and ROS production
146
Sources of ROS
147
Oxidation States Polarography
148
Figure 2
149
Fig 2 Part 2
150
C 1 and 3 and ROS
151
Block - Ox Rates
152
Mito Function Brain
153
Pathobiology of Daibetic Complications
154
Scientic Approach
155
Figure 1
156
Ox damage Diabetes
157
E flow trhough resp chain
158
Electrochemical Concept
159
Blocks Strategy
160
REDOX carriers and proton pumps
161
Graphs
162
Facts
163
Resp Chain Pic
164
Energy Expenditure
165
Kieblers Law
166
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SOD
