22 1

2

2 x C 3 2 x C 2 Cytosol Glucose pyruvate 2 x CO 2 4 x CO 2 glycolysis 1 x C 6 Mitochondrion pyruvate 3 CO 2 CAC 3

4 The Citric Acid Cycle 8 A series of 8 reactions in the mitochondrial matrix. oxidizes the C 2 acetyl component of acetyl-CoA to 2 molecules of CO 2 & energy in the form of reduced coenzymes, 3NADH and FADH 2. Krebs cycle The cycle carries his name, the Krebs cycle. Citric Acid Cycle Because the 1st intermediate in the cycle is citric acid, it is also referred to as the Citric Acid Cycle. TCA cycle Because citric acid is a tricarboxylic acid, it is also referred to as TCA cycle. Hans Adolf Krebs ( )

At the pyruvate position: The enzyme that initiates it, pyruvate dehydrogenase(PDH) is inhibited by ATP and NADH because these are abundant when energy is readily available. It is activated by ADP, which is abundant when the cell needs energy. At the CAC position: Rxn #3 - isocitrate dehydrogenase Activated by ADP and NAD + inhibited by ATP & NADH Rxn #4 – ketoglutarate dehydrogenase ADP activates NADH & succinyl-CoA inhibits CAC Regulation of CAC 5

One turn of CAC 3 NADHFADH 2 GTP acetyl CoA + 2 H 2 O + 3 NAD+ + FAD + GDP + Pi  3 NADH + 3H + + FADH 2 + GTP + HS-CoA + 2 CO 2 The main function of CAC is to oxidize pyruvate and reduce NAD + and FAD in order to produce high E compounds (NADH and FADH 2 ) required for ATP synthesis. With pyruvate NADH Pyruvate + HS-CoA + NAD+  acetyl CoA + NADH + H + + CO 2 Overall 4 NADHFADH 2 GTP Pyruvate + 4 NAD+ + FAD + GDP + Pi + 2 H 2 O  4 NADH + 4H + + FADH 2 + GTP + 3 CO 2 6 If we take into account glycolysis and the pyruvate dehydrogenase reaction, and the fact that one molecule of glucose generates two molecules of pyruvate, we can write an equation for the catabolism of glucose through glycolysis and the CAC as: 10 NADHFADH 2 ATP Glucose + 2 H 2 O + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi  10 NADH + 10 H FADH ATP + 6 CO 2

7 Most of the ATP generated during glucose oxidation is not formed directly from reactions of glycolysis and CAC, but rather from the reoxidation of reduced electron carriers in the respiratory chain. These electron carriers, NADH and FADH 2, have a high redox potential, in the sense that their oxidation is highly exergonic. As electrons are transferred from the reduced carriers to molecular oxygen, in a stepwise fashion, the energy released is used to drive the synthesis of ATP from ADP, producing about 2.5 moles of ATP per mole of NADH and 1.5 moles of ATP per mole of FADH 2. NADH is a stronger reducing agent than FADH 2 and therefore can produce ~1 more ATP. NADH and FADH 2 are reoxidized by electron transport proteins bound to the inner mitochondrial membrane. A series of linked oxidation reduction reactions occurs, with electrons being passed along a series of electron carriers known as the electron transport chain, or respiratory chain.

electron transport chain The reduced coenzymes formed in the citric acid cycle enter the electron transport chain and the electrons they carry are transferred from one molecule to another by a series of oxidation–reduction reactions. Each reaction releases energy until electrons and protons react with oxygen to form water. Electron Transport 2 H + +2 e - + ½ O 2  H 2 O oxidative phosphorylation The energy released during electron transport is used to synthesize ATP from ADP and P i in a process called oxidative phosphorylation. 8

There are four types of electron carriers: Flavins:FMN  FMNH 2 oxidized reduced Iron-sulfur :Fe 3+ + e -  Fe 2+ clustersoxidized reduced Coenzyme Q;Q  QH 2 oxidized reduced Cytochromes: Fe 3+ + e-  Fe 2+ oxidized reduced Flavin mononucleotide 9

Coenzyme Q Fe-S cluster = nonheme iron protein 10

The different types of cytochromes are designated a lowercase letter (a, b, c); further distinctions are done with subscripts, as in C 1. Cytochromes identified as: a, a 3, b, c, c 1 Cytochromes proteins that contain heme groups in which the iron cycles between Fe +2 and Fe +3 and proteins with iron–sulfur groups in which the iron also cycles between Fe +2 and Fe

12 Electron transport Electron transport is a sequence of reactions that facilitate the flow of electrons from NADH and FADH 2 all the way to the reduction of oxygen to form water: complex is a multienzyme system The membrane has four distinct protein complexes: complex I, II, III, IV. Each complex is a multienzyme system and contain electron carriers needed for electron transport.

Complex IV – Cytochrome c oxidase. Complex IV – Cytochrome c oxidase. Contains about 10 subunits Here electrons are transferred from cytochrome c to cytochrome a, and then to cytochrome a 3, the last cytochrome. In the final step of electron transport, electrons and H+ combine with oxygen to form water: 2 H e - + ½ O 2  H 2 O 13

Complex I and II Complex I and II receive electrons from the oxidation of NADH and FADH 2, respectively, and pass them along to a lipid soluble electron carrier, coenzyme Q, which moves freely in the membrane. Complex III Complex III catalyzes the transfer of electrons from the reduced form of coenzyme Q to cytochrome c, a soluble protein electron carrier that is mobile within the intermembrane space. Complex IV Complex IV catalyzes the oxidation of cytochrome c, reducing O 2 to water. The energy released by these exergonic reactions is used to pump protons from the matrix into the inner membrane space, creating a proton gradient across the inner membrane. Complex I, III, and IV create a proton gradient across the inner mitochondrial membrane, and the energy of this gradient is utilized by complex V to synthesize ATP from ADP.

15 Oxidative Phosphorylation The energy-releasing oxidation reactions give rise to a pH gradient across the inter mitochondrial membrane. traversing the entire chain For a pair of electrons entering the respiratory chain as NADH and traversing the entire chain to O 2, the energy change is about 218 kJ/mol ( ): NADH + H + + ½ O 2 NAD + + H 2 OΔG˚̛ = -218 kJ/mol ΔG for ATP hydrolysis under intracellular conditions is estimated to be -50 kJ/mol, so the synthesis of 2.5 mol ATP requires at least 2.5 x 50 = 125 kJ. Therefore, enough E is available to drive ATP synthesis.

oxidative phosphorylation The process where the energy generated through the electron transport is coupled with the synthesis of ATP is called oxidative phosphorylation. This pH gradient generates an electrochemical potential difference, a voltage drop, across the membrane. The energy possessed by the voltage difference is coupled to the synthesis of ATP. 16

Chemiosmotic coupling chemiosmotic model To explain how the H+ gradient is used by complex V to synthesize ATP, Peter Mitchell proposed the chemiosmotic model in “The free energy from electron transport drives an active transport system (transport against a conc. gradient), which pumps protons out of the matrix into the intermembrane space. This action generates an electrochemical gradient for protons. The protons on the outside have a thermodynamic tendency to flow back in, down their electrochemical gradient, and this flow provides the energy for ATP synthesis”. ATP synthase To equalize the H + concentrations, these use a protein complex called ATP synthase, which allows them to return to the matrix. As the protons flow, energy is generated and used to drive ATP synthesis: ADP + Pi + energy  ATP 17

18 Therefore, proton pumping results in the conversion of the energy of the electron transport to osmotic energy in the form of an electrochemical gradient. This is a concentration gradient that also establishes an electrical potential. The energy released from discharging this gradient can be coupled with phosphorylation of ADP to ATP by means of complex V, ATP synthase. The proton gradient leads to ATP production by means of ion channels present in ATP synthase. The F 0 part of the protein is the proton channel and permits the passage of H+ The F 1 part of the protein catalyzes the phosphorylation reaction

Conformational coupling The effect of the proton flux is to cause a conformational change that leads to the release of already formed ATP from ATP synthase. 19

ADP + Pi enter (O) It converts into (L) and ADP & Pi react Then (L) turns into (T) which has little affinity for ATP and releases it. The activity of electron transport & oxidative phosphorylation depends on ADP and ATP levels. ↑↓ ↑ ADP activates↓ ATP deactivates In essence, the chemical energy of the proton gradient is converted to mechanical energy in the form of rotating proteins. This mechanical energy is then converted to the chemical energy stored in the high- energy phosphate bond of ATP. 20

21 Cytoplasmic NADH is not permeable to the mitochondria. In order to extract the energy from this NADH, the reducing equivalents must be transferred into the mitochondrion. Therefore, the reducing equivalents must be shuttled to the inner mitochondrial membrane without physical movement of NADH. This process involves the reduction of a substrate by NADH in the cytoplasm, passage of the reduced substrate into the mitochondrial matrix, and passage of the oxidized substrate back to the cytoplasm, where it can undergo the same cycle again. There are two shuttle systems: Dihydroxyacetone phosphate/glycerol-3-phosphate 1. Dihydroxyacetone phosphate/glycerol-3-phosphate shuttle is active in brain and skeletal muscle. Malate/aspartate 2. Malate/aspartate shuttle is active in liver, kidney and heart.

22 Dihydroxyacetone phosphate/glycerol-3-phosphate 1. Dihydroxyacetone phosphate/glycerol-3-phosphate FAD This process involves the reduction of FAD by G3P followed by transfer of an electron pair from FADH2 to coenzyme Q, just like intramitochondrial NADH transfers electrons to CoQ. G/P shuttle NADH (cytosol)    FADH 2 (mitochondrion) 2 ATP

23 Malate/aspartate 2. Malate/aspartate NAD + This process involves the reduction of NAD + by malate followed by transfer of an electron pair from NADH to complex I, just like intramitochondrial NADH transfers electrons to complex I. M/A shuttle NADH (cytosol)    NADH (mitochondrion) 3 ATP

Considers NADH from glycolysis using the M/A shuttle Where 2 NADH  2 FADH 2 Based on your textbook 24

25