Thermodynamics, Directions of Reactions, Thermodynamic (Thermo = „heat‟; Dynamics = „force‟) - every reaction is associated with energy transformation - will apply to photosynthesis and respiration - direction of a reaction is determined by laws of thermodynamics Energy – Capacity to do work; to move an object against an opposing force Work – the actual process of moving an object against a force (e.g. biological reactions) - Potential energy: energy available for use in the future - coal; ball at rest - chemical bonds store potential energy - Kinetic energy: energy in current use First Law of Thermodynamics: Total amount of energy stays constant in any process. - can change form (this is what we‟ll be talking about in photosynthesis and respiration) Second Law of Thermodynamics: amount of energy available to do work is constantly decreasing - rock tumbles; work is finished; rock will not go back to top Heat is “disordered energy” and not available for biological work; heat is a final “degraded” form of energy. 2nd law states that entropy will increase - reactions have directionality - since heat is lost, the amount of energy (in an isolated system) can only decrease (random molecules don‟t become orderly; ATP does not spontaneously form from ADP + P; cold cups of coffee do not spontaneously heat up…) The “Loophole”…“isolated” vs. “open” systems (2 nd Law applies to isolated systems…) - isolated systems: regions that don‟t exchange matter/energy with surroundings - open systems: DO exchange matter/energy with surroundings (local order can be achieved, but overall entropy will always increase) How does this apply to biology? - Photosynthesis (order from chaos); Respiration Laws help us predict which way a biological reaction will go…SO: when potential energy is converted to kinetic energy, reactions tend to go in direction which releases largest amount of heat. To predict the direction of a reaction: - we need a measure of “available” or “potential” energy - “free energy” (“G”)…a measure of available energy under conditions of a biochemical reaction
“G” and “ΔG” Our concern: changes in energy values before and after a reaction… Every chemical reaction has a measurable change in free energy - designated as ΔG; a change in amount of energy available to do work - figured as: ΔG = G-products minus G-reactants In a closed system, for a reaction to proceed spontaneously, the energy (G) of the products must be less than that of the reactants ( ΔG is negative) - that is: less energy remains for work after the reaction - means products (low energy) are more stable than reactants (initial high energy). EXERGONIC – energy is released - products more stable than reactants - has negative ΔG (goes from higher to lower free energy…) - SO: 2nd law states that – without input of energy, processes tend to go downhill (“spontaneous”) ENDERGONIC: Reactions that require energy input. - has positive ΔG - does NOT occur spontaneously - results in more high energy bonds (energy comes from the sun or from some exergonic reaction) ATP and Coupled Reactions - to store energy takes energy (“coupled reaction”) - exergonic reactions can drive endergonic reactions - 2nd Law: stipulates that combined process is exergonic - reaction with a + ΔG is coupled to a reaction that has a - ΔG of a greater magnitude First Law of Thermodynamics tells us: - chemical energy released from ATP hydrolysis is exactly equal to mechanical work + heat. Second Law of Thermodynamics tells us: - chemical energy released is greater than the amount of work performed. The rest (the difference) is released as heat (since no reaction is 100% efficient) Changes in free energy are influenced by: temperature; pH; pressure ΔG 0‟ for ATP – ADP + Pi = -7.3 kcal/mol ATP - 3 negative phosphates working against an electrical force - release of a PO 4 can do work - “hydrolysis” of terminal releases PO 4 (7.3 kcals/mole) Differences in concentration can also drive work: - 2nd law states: any process that converts an orderly arrangeme nt to a less orderly arrangement can perform work (will have a - ΔG)
Enzymes - shape/architecture of proteins bestows ability to increase rates of reactions - “thermodynamics” says nothing about rates of reactions Every reaction, no matter how thermodynamically favorable, needs a “push” - ball needs a push, coal needs to be lit - materials can be kinetically stable, but thermodynamically unstable “Activation energy” - lowering of activation energy will make reactions occur more easily - heat can make reactions go faster, but can be harmful to cells. Catalysts: lower activation energy: - needed in small amounts - not used up in reaction - doesn‟t change thermodynamics of reaction (doesn‟t add energy) In cells, catalysts are called “enzymes.” They speed up reactions without having to add heat. How enzymes work… - enzymes bind a reacting molecule called a “substrate” - can speed up by million/billion times - enzyme-substrate complex is a non-covalent bond …but the reaction breaks a covalent bond “Contact” AAs vs “Catalytic” AAs - “active site” is specific complementary surface on enzyme (“lock and key” model) - induced fit: enzyme slightly changes shape to accommodate substrate
pH and Temperature: effect enzyme activity
Enzyme concentration also effects enzyme activity: Michelis-Menton Kinetics: - an analysis of the rate of an enzymatic reaction - maximum rate of reaction is proportional to amount of enzyme present - i.e. a relationship exists between enzyme and substrate
How do enzymes lower activation energy?? - enzymes orientate the substrates in a non-random way 1) orientate to increase chance of interaction (combining molecules) [endergonic] 2) orientate to strain covalent bonds of substrate [exergonic] - “transition state” occurs as substrate binds; this is what lowers activation energy 3) Enzyme itself participates in the reaction
- chymotrypsin (a “protease”): orientates substrate, strains bond, but: involves a covalent bond (i.e. enzyme itself directly participates in reaction) - “R” group from 3 AAs are located in different parts of enzyme - catalytic and contact AAs 1) serine 195 forms a covalent bond with peptide bond C atom 2) proton donated by His 57 cleaves peptide C-N bond 3) bond between remaining peptide fragment and Ser 195 is broken; enzyme returns to original state Metabolism - regulated by enzyme activity - involves “pathways”, or a network of chemical reactions 1) Anabolism (build-up of large molecules from small) - endergonic; require coupled reactions 2) Catabolism (breakdown of food to smaller molecules) - exergonic
Regulation of enzymes: 1) steric or “competitive” inhibition – similar shaped molecule binds to active site - can be overcome by increasing substrate concentration 2) non-competitive inhibitor: binds to enzyme at non-active site - lead; heavy metals 3) feedback inhibition: product (isoleucine) can inhibit its own pathway - isoleucine used for protein, so process continues. When need decreases, isoleucine builds up and inhibits first enzyme involved in its own synthesis. 4) allosteric inhibition (“other shape”) has a role in feedback inhibition - multiple subunits; two binding sites: - “active” or catalytic site (on catalytic subunit) - “allosteric” or regulatory site (on regulatory subunit) -“effector” molecule can change shape of enzyme - allosteric activators: enhances activity (high affinity) - allosteric inhibitors: reduces affinity for substrate (low affinity) 5) Irreversible inhibition: forms stable covalent bonds - inactivates enzyme (e.g. nerve gas) RNA as enzymes: “ribozymes”
Glycolysis Technically, we don‟t get energy from glucose – we get it from ATP. The whole point of the process of glycolysis and respiration, therefore, is to generate ATP. - Anaerobic – metabolism in the absence of oxygen - Aerobic – metabolism in the presence of oxygen - Aerobic processes can extract much more energy than anaerobic processes. Almost all
eukaryotes are aerobic. - Respiration refers to the extraction of energy from glucose in the presence of oxygen. - Respiration will transfer 24 electrons from glucose to O 2 Remember that oxygen is highly electronegative - it tends to draw electrons to itself. The presence of oxygen, therefore, drives the whole glycolytic/respiratory process. Oxygen in a sense “pulls” electrons (derived from glucose) through the glycolytic/respiratory process, and is the final “electron acceptor” at the end of the Electron Transport Chain (“ETC”).
Oxidizing agents (electron acceptors): -NAD+ and FAD+ (these are co-enzymes, not proteins) (they are derived from niacin and riboflavin, which are B vitamins) - eventually, about 90% of energy from glucose will be stored in reduced forms of these molecules (which will then be converted to ATP in the ETC which occurs in the mitochondria) - Overall reaction: C 6 H12 O6 + 6O2 6CO2 + H2O - G is –686 Kcal/mole (notice that this is the exact opposite of photosynthesis)
Three parts to energy extraction process: 1) Glycolysis, 2) Krebs, 3) Electron Transport Glycolysis: - primitive (i.e. evolved before aerobic respiration) - occurs in cytosol - operates in the absence of oxygen - all organisms do it – in exactly the same way (evidence for common ancestry). - overall G for glycolysis = -18.3 kcal/mole
Glycolysis proceeds in 10 small steps: Part I - initial investment of 2 ATP - is uphill - coupled to exergonic expenditure of ATP - end product = 2 x 3-carbon molecules or G3P (also found in the Calvin- Benson cycle)
Part II (occurs in duplicate) - Oxidation of G-3-P: generates NADH and ATP - synthesis of ATP here is called “substrate level phosphorylation” because the P for ATP comes from a substrate; not from electron transport (which is called “oxidative phosphorylation”) - 2 NADP are generated and are used (eventually) to generate ATP in the ETC
Part III - converts phosphoglycerate to pyruvate End product of glycolysis is TWO molecules of pyruvate - Pyruvate still has lots of energy!!
Three fates of pyruvate: - glycolysis requires presence of NAD and inorganic phosphate - phosophate supply is not a problem; there is lots around (ATP always being split) - however: NADH is fairly limited, because: regeneration of NAD from NADH requires that NADH give up electrons to Electron Transport (in the presence of oxygen) - this can occur only when oxygen is present to accept the final electron at the end of ETC - SO - if no oxygen is present, NAD will be depleted (by its conversion to NADH) - cells, however, can re- generate NAD in absence of oxygen by: 1) producing lactate (lactic acid) (pyruvate is reduced to lactate) 2) fermentation: (extraction of energy w/o oxygen) NADH is oxidized to NAD, which reduces pyruvate to ethanol and CO 2 3) the third fate of pyruvate is that it is converted to Acetyl CoA in the presence of oxygen (which then enters the Krebs cycle)
Regulation of glycolysis: Major site of regulation: Step 3 of glycolysis - abundance of ATP inhibits enzyme (phosphofructokinase) that mediates step 3 - is allosteric inhibition: when little ATP is around, the enzyme at this step has a high affinity for the substrate (fructose-6-phosphate). When ATP accumulates, it can bind at the allosteric site of the enzyme and slow down the glycolytic pathway.
Krebs/Electron Transport Review: - glycolysis occurred in cytosol - pyruvate: still has energy - Krebs cycle is an aerobic process An intermediate step between glycolysis and Krebs (this is the 3 rd fate of pyruvic acid): - Pyruvate is converted to “Acetyl Coenzyme A” - one CO 2 is given off - 2 electrons/2 H+ are removed from pyruvate - one NADH is formed
- Mitochondrial compartments (membrane as “workbench”) - Acetyl CoA enters Krebs cycle - Krebs occurs in mitochondrial matrix - remember: there are two Krebs cycles for every glucose Note the “decarboxylation” reaction where CO 2 is formed… - the Krebs cycle gives back CO 2 Part I: Acetyl CoA combines with oxaloacetate to form citric acid Part II: isocitrate to succinate - production of: - 2 CO2 - 2 NADH - 1 GTP (is equal to ATP) Part III: regeneration of oxaloacetate - production of one NADH; one FADH2 Balance for Krebs Cycle: (2 CO 2 formed from conversion of pyruvate to Acetyl CoA) - 4 (2 x 2) CO 2 from “part II” (above) (Total CO 2 = 6 per glucose) - 6 x NADH (2 x 3 in Krebs) - 2 x FADH2 - 2 x GTP Krebs cycle has a central role in metabolism: - Acetyl CoA is also breakdown product of fatty acids - each fatty acid carbon provides more electrons for ATP production than does CHO carbon
Electron Trans port - it is in electron transport where most of the ATP is generated - 90% of glucose energy is now tied up in NADH and FADH2 - (there are no more glucose intermediates. All (well, most) energy is tied up in NADH and FADH2 . All carbon from glucose is now back in CO 2 ) - NAD accepts 2 electrons and one H+ - FAD accepts 2 electrons and TWO H+ - have high negative redox potentials (are strong electron donors) - H+ is split off from NADH and FADH2 (and goes to matrix) - electrons get pulled through electron transport (steeply downhill) - At end of electron transport: - 2 electrons from each co-enzyme will reduce one oxygen; - H+ comes in from surrounding water to form water Electron Carriers: - “Cytochromes” (“color”) - each has a heme group that is oxidized and/or reduced
- heme (like NAD and FAD) accepts/donates electrons Process of electron transport: - NADH and FADH2 deliver their electron load to top of electron transport - FMN and CoQ are intermediates - cytochrome chain: electrons move to cytochromes of ever increasing electron affinity (each is more positive than one before; more negative than one ahead) - “Oxidative phosphorylation” vs. substrate- level phosphorylation - NADH and FADH2 is now in mitochondrial matrix - electron transport occurs on inner membrane - protons are pumped FROM matrix TO intermembrane space - a 3-protein complex are proton pumpers - protein complex has a fixed orientation; protons are pumped in one direction - proton (“electrochemical) gradient is established (difference across membrane of one pH unit) - only one way for protons to get back into matrix – that is through a protein channel: - F0F1 complex: - F0 is protein channel - F1 is ATP-synthase - “chemiosmotic hypothesis” of ATP production End: - 2 electrons from each co-enzyme; - plus H+ - plus oxygen = water
Photosynthesis I Carbon bonds in biomolecules store energy – where does that energy come from? - all biological energy originates with photosynthesis - earliest view of photosynthesis: plant bulk was extracted from soil by roots Origin of oxygen from photosynthesis: 1) early hypothesis: a) sunlight splits CO 2 and releases oxygen; b) water then combines with carbon to make carbohydrate 2) sulfur-reducing bacteria (H2 S) release of sulfur; this was a hint that H2 O was source of oxygen
Oxidation/Reduction: Energy-producing pathways are “redox” reactions - oxidation = loss of an electron - reduction = gain of an electron - reducing agent – DONATES electrons - oxidizing agent – ACCEPTS electrons Redox Reactions - energy transfer (redox) molecules: NAD-NADH, NADP-NADPH, FAD-FADH - Electron Transport Chain – many redox pairs in a row = affinity for electrons is based on “electronegativity” (the tendency of an atom to gain electrons) - movement of electrons = movement of energy - redox potentials can be thought of in terms of G - SO: “Direction” of electron movement to an oxidizing agent has a negative G (and occurs spontaneously) LIGHT - visible light = 400-750nm (= different colors) - “photon” is wave- like and particle- like - each photon has an energy based on “wavelength” Photosynthesis is driven by light in the blue and red spectrum; green is reflected Pigments: - chlorophyll a – occurs in all eukaryotes; chlorophyll b – occurs in all vascular plants - carotenoids – occurs in all plants - absorb in blue-violet; reflects reds and oranges - also absorb photons and pass them on to chlorophyll Chloroplast - granum; thylakoid; - thylakoid membrane – site of light dependent reaction - thylakoid “lumen” is inside membrane - “stroma” is outside of membrane – site of light independent reaction Antenna complex: - several hundred chlorophylls; about 50 carotenoids - most don‟t do real photosynthesis - only one “special” chlorophyll can process the light energy - antenna complex absorbs photons and passes them on to the “reaction center” Reaction center - a pair of special chlorophyll a molecules that carry out photosynthetic reactions Light travels until: - it bounces (= reflection) - it passing through (= transmission) - it is ABSORBED = photon disappears, but energy does not
Process of photon absorption: - photon raises energy of an electron, boosting it to next orbital - electron is unstable (2nd law!) and falls back (emits some energy as heat) Photosynthesis, however, uses that energy: - pigments absorb photons and make use of the energy - the light captured increases free energy of the absorbing molecule - electron is boosted into an energy-carrying molecule There are TWO photosystems; they absorb sunlight at different wavelengths Photosystem I (“PS I”) absorbs at 700 nm (and is called P700) Photosystem II (PS II”) absorbs at 680 nm (and is called P680) Summary of Photosystem Reactions 1) pigments absorb light (twice) 2) transfer of energy (electrons) via Electron Transport to make ATP and NADPH 3) pigments get replacement electrons Photosystem I: Cyclic mode 1)…chlorophyll becomes an electron donor (reducing agent) 2) electron enters Electron Transport Chain… 3) electron arrives at ferredoxin (a protein) 3) electron arrives back to chlorophyll and reduces (adds an electron back to) P700 Electron transport results in a “proton gradient” - components of Electron Transport Chain pump H+ FROM stroma INTO thylakoid lumen - accumulation of charge represents POTENTIAL ENERGY (4 pH units!) - protons build up and are pumped back to stroma through ATP-ase - this catalyzes the addition of a phosphate group onto ADP (called “phosphorylation”) This process is called “cyclic photophosphorylation” - ATP accumulates in stroma - (Cyclic route does not produce NADPH) PS I in the non-cyclic route forms NADPH, not ATP… Electron reaches ferrodoxin and then: - reduces NADP to form NADPH (- NADPH accumulates in stroma) HOWEVER: now, P700 remains in an oxidized state; - it wants an electron back, which will come from Photosystem II: Photosystem II (“PS-II”) - photon triggers release of electron (- PS-II is now oxidized; wants an E back…)
- proton gradient is established by electron flow to PS-I - protons accumulate in lumen - ATP is produced (accumulates is stroma) - electron reduces P700 in PS I But now: P680 is oxidized and wants an electron back - P680 is most powerful oxidizing agent in biological systems! - it can grab an electron from water Photolysis of water: - water is split into: 2 protons go to thylakoid lumen; - two electrons goes to chlorophyll (P680) …and OXYGEN is the waste product! Photosynthesis II – the Light Independent Reaction - Light- independent reaction = “synthesis” - synthesis of carbohydrate occurs in chloroplast stroma To make glucose, we have (from light dependent reaction): - ATP provides the energy for synthesis (endergonic) - NADPH provides the “reducing power” (ability to contribute electrons) - NADPH also provides the hydrogens for synthesis of glucose - CO2 provides the carbon Synthesis of glucose in the Calvin-Benson cycle (broken into big three steps): Step One: a) CO 2 diffuses in through stomata and into stroma b) CO 2 enters the Calvin-Benson cycle: attaches to RuBP c) Rubisco (“ribulose 1,5-bisphosphate carboxylase”) is the enzyme that attaches CO 2 to RuBP - This is the instant of carbon fixation! (3 molecules CO 2 per second) d) Forms a 6-carbon sugar, which is very unstable, which immediately breaks down… e) …breaks down to 3-phosphoglycerate – “3PG” Step Two: a) 3PG is converted to glyceraldehyde-3-phosphate (“G3P”) …this requires use of ATP and NADPH (leftover ADP and NADP diffuses back to stroma for regeneration) b) some of the G3P leaves stroma and diffuses to cytosol c) two moleucles (of G3P) are converted to glucose d) other G3P intermediates form: - lipids, amino acids, nucleic acids Step Three: a) the remainder of the G3P is used to re-generate ribulose 1,5-bisphosphate (proportion: of 12 molecules of G3P: 2 go to glucose, 10 go to regeneration of RuBP)
6 CO 2 + 6 H2 0 C6 H12 O6 + 6 O2 ………………….G0‟ = +686 Kcal/mole Overall efficiency of photosynthesis: about 20% (!) Photorespiration: Stomata must regulate 1) water 2) carbon dioxide 3) oxygen In warm/hot weather, stomata closes to save water this restricts movement of oxygen OUT of cell and of carbon dioxide INTO cell. Oxygen (from photolysis) accumulates in cell. - Rubisco: will bind one O 2 for every 3 CO 2. When this occurs, Calvin cycle cannot occur. C3 plants – are those that form 3-carbon intermediates C4 plants – are those that posses a cycle that forms 4-carbon intermediates In C4 plants, CO 2 is fixed by another process: - enzyme found in mesophyll cells binds only CO 2 - forms 4-carbon intermediates (oxaloacetate) - reduced to malate - malate enters cell, breaks down - and delivers pure CO 2 to Calvin-Benson cycle - carbon is fixed twice This process is advantageous but costs energy: 2 more ATP than in C3 plants