Electron Transport & Oxidative Phosphorylation; Aerobic Respiration

Transcription

1 Electron Transport & Oxidative Phosphorylation; Aerobic Respiration

2 The mitochondria is a double membrane structure with and intermembraneous space and a matrix. The bulk of the mito proteins are bound or associated with the inner membrane or found in the matrix. These are the major pathways of the mito. that will be covered this course, but there are many other that will not be cover.

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4 The outer mitochondrial membranethe mitochondrial outer membrane can associate with the ER membrane, in a structure called MAM (mitochondria- associated ER- membrane). This is important in the ER- mitochondria calcium signaling and involved in the transfer of lipids between the ER and mitochondria. The intermembrane spacebecause the outer membrane is freely permeable to small molecules, the concentrabons of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. Inner mitochondrial membrane contains more than 151 different polypepbdes, and has a very high protein- to- phospholipid rabo (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addibon, the inner membrane is rich in an unusual phospholipid, cardiolipin The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the producbon of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, trna, and several copies of the mitochondrial DNA genome. Of the enzymes, the major funcbons include oxidabon of pyruvate and fary acids, and the TCA cycle.

5 EukaryoBc cells evolved segregabon of funcbons into separate organelles. CompartmentalizaBon increases the efficiency of biochemical reacbons by creabng tailored chemical microenvironments, but also creates a need for communicabon and routes of metabolite exchange. Membrane lipids, for example, are primarily synthesized in the endoplasmic rebculum (ER) and distributed to other organelles. Many organelles exchange phospholipids with the ER via vesicular transport. In contrast, mitochondria are not connected to vesicular trafficking pathways, and many lipids of the inner and outer mitochondrial membranes (IMM and OMM) cannot be synthesized within mitochondria but are imported by unclear mechanisms. Phospholipids may transfer from the ER to the OMM at spabally restricted sites

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7 Protein entry is dependent on membrane potential. This is set up by the electron transport complexes. This membrane potential actually helps pulls the protein into the matrix. The protein then enters the matrix where the cleavable pre-protein is clipped off by a protease and a chaperone protein ( mt-hsp70) in the matrix works with Tim44 to complete the full transfer to the matrix. This chaperone mthsp70 and Tim 44 actually "pull" the protein into the matrix by a process that requires ATP. It also requires the membrane potential set up by the electron transport chain. Negative charges in the matrix, set up by the pumping of hydrogen ions to the inter-cristae space, attract the protein which has positive charges on the end that enters the GIP. Some mitochondrial proteins destined for the inner membrane have a cleavable signal peptide followed by one or more membrane-spanning segments that serve to insert the polypeptide into the inner membrane after it gets in the matrix. This is how the electron transport proteins get into the inner membrane.

8 It was recently asserted that the voltage-dependent anion channel (VDAC) serves as a global regulator, or governor, of mitochondrial function. This large channel plays the role of a "switch" that defines in which direction mitochondria will go: to normal respiration or to suppression of mitochondria metabolism that leads to apoptosis and cell death. As the most abundant protein in the mitochondrial outer membrane (MOM), VDAC is known to be responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM.

9 Mitochondrial Genome Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin, with the mtdna being derived from the circular genomes of the bacteria that were engulfed by the early ancestors of today's eukaryotic cells. This theory is called the endosymbiotic theory. Each mitochondrion is estimated to contain 2-10 mtdna copies. In the cells of extant organisms, the vast majority of the proteins present in the mitochondria (numbering approximately 1500 different types in mammals) are coded for by nuclear DNA, but the genes for some of them, if not most, are thought to have originally been of bacterial origin, having since been transferred to the eukaryotic nucleus during evolution. In humans cardiac mitochondria contain 615 proteins, it is 940 in rat.

10 Mitochondrial DNA is replicated by the DNA polymerase gamma complex which is composed of a 140 kda catalytic DNA polymerase encoded by the POLG gene and a 55 kda accessory subunit encoded by the POLG2 gene The replisome machinery is formed by DNA polymerase, TWINKLE and mitochondrial SSB proteins. TWINKLE is a helicase, which unwinds short stretches of dsdna in the 5 to 3 direction. What is the origin of Mitochondrial genome? This now is an open question. Definitely it is through endosymbiosis but what gave rise to the mitochondira? Is there and anaerobic mitochondria?

11 These are major trans-membranous proteins of the inner mito membrane. One of the most unique transporters only found in the mito is the ADP/ ATP antiporter. The mitochondrial permeability transition pore, which opens in response to increased mitochondrial Ca 2+ load and oxidative stress. The mitochondrial uniporter which transports Ca +2 from the cytosol of the cell into the mitochondrial matrix. The mitochondrial sodium/calcium exchanger, which carries Ca 2+ ions out of the matrix in exchange for Na + ions. These transport proteins serve to maintain the proper electrical and chemical gradients in mitochondria by keeping ions and other factors in the right balance between the inside and outside of mitochondria.

12 There are two mitochondrial Ca 2+ transport systems. The efflux operates at a constant rate independent of [Ca] Activity of influx is dependent on [Ca] in cytosol At set pt. two pathways are equal- no net Ca flux An increase in cytosolic [Ca] results in net mito influx A decrease in cytosolic [Ca], net mito efflux The regulation of cytosolic & mito [Ca 2+ ] around a set point.

13 ½ mol O 2 + NADH 2 H 2 O + NAD + ΔE O =0.82-(-0.32)=1.14V ΔG O = -218kJ/mol The oxidation of 1mol of NADH is associated with the release of 218kJ of free energy The formation of ATP by the rxn ; ADP + P i ATP requires 30.5 kj/mol of free energy

14 The ETS complexes are laterally mobile in the inner mitochondrial membrane, they do not appear to form any stable higher structure. The ETS complexes are not present in equimolar ratios. But, this is impossible given the data accumulated over decades. There must be a topography that puts within angstroms several of these proteins.

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16 The free energy necessary to generate ATP is extracted from the oxidation of NADH & FADH 2 by the ETS, a series of 4 complexes thru which e - pass from higher to lower standard reduction potentials and the release of free energy.!

17 But only 3 of the complexes generates enough free energy to synthesis an ATP. So, although Complex 2 generates e- it can not generate an ATP.

18 Determination of the stoichiometry of coupled oxidation and phosphorylation (the P/O ratio) with different electron donors.!

19 This does not take into account the H + formed from the shunts & shuttle pathways into the mitochondria, these include glycerol-3-po 4 dh, Malate- Aspartate shuttles, pyrimidine biosynthesis, urea cycle, and first reaction of β-oxidation of fatty acids. All of these pathways generate ATP through the ETS & ox-phos pathways.

20 The changes in std. reduction potential of an e - pr. as it successively transverses complexes I, III, & IV, correspond at each stage to sufficient free energy to power the synthesis of an ATP.! Complex I ΔE oʹ = 0.36V ΔG oʹ = kj/mol! Complex III ΔE oʹ = 0.19 V ΔG oʹ = kj/mol! Complex IV ΔE oʹ = 0.58 V ΔG oʹ = kj/mol!

21 Also known as! NADH oxidoreductase. Portions! of the Complex I! protein are present! in numerous proteins found in anaerobic! (microaerophilic)! Eukaryotes, that generate H2.! 7 of the 43 subunits of Complex I are coded for by mito genes. Although NADH can only transfer 2 e -, both FMN & CoQ can accept & donate 1 or 2 e - because of their semiquinone forms are stable. These then are conduits betw the 2e- donation of NADH & the 1e- acceptors, the cytochromes.

22 Complex II contains succinate dh & 3 other subunits all coded for by nuclear genes. It passes e- from succinate to CoQ with the participation of covalently bound FAD. For this reason they are called flavoproteins. Three other proteins reduce CoQ, to power ox-phos thru complex III & IV; these are glycerol-3-po4 dh, ETF-ubiquinone oxireductase of βfa oxidation & oratadate dh of pyrimidine synthesis in eukaryotes. The structure of complex II is not known, but the structure of quinol-fumerate reductase of E.coli is known. It functions in the opposite direction, it reduces fumerate to succunate in anaerobes. Size of both are similar, QFR is 121kD hetrotetramer. The 6 cofactors are organized in a near linear chain with a squence of; FAD [2Fe-2S] [4Fe-4S] [3Fe-4S] Q P Q D. These cofactors are separated by 7 to 11 Α.

23 But, E.coli, which can also grow in aerobic conditions also has a separate complex II structure independent of QFR.

24 QFR does produce 25 times more ROS (superoxide & H 2 O 2 ). Structure of fumarate reductase. (A) Stereoview of a fumarate reductase monomer. The flavoprotein is in blue, the iron protein is in red, and the membrane anchors are ingreen (FrdC) and purple (FrdD). The [Fe:S] clusters are shown as purple (Fe atoms) and yellow (S atoms), while the menaquinones and FAD are shown in yellow. (B) Space-filling model of fumarate reductase showing the crystal contacts between the two complexes in the membrane-spanning portion. In the fumarate reductase complex, oxygen atomsare shown in red, nitrogen atoms are shown in blue, sulfur atoms are shown in yellow. Menaquinone molecules are shown in magenta (see right-hand monomer). The location of the membrane-spanning region can be inferred from the coloring of the atoms in this representation. The more hydrophilic (soluble) region contains many polar oxygen and nitrogen atoms (red and blue) while the hydrophobic (membrane-spanning) region contain mostly apolar carbon atoms (gray). (C) Crystal packing of fumarate reductase is through the transmembrane regions of the protein (green and purple) and forms a continuous membrane-spanning portion in the crystal. In this representation, the FAD and menaquinone are shown in gray.

25 Proteins of the fumarate reductase complex. (A) The flavoprotein. Stereoview of the C trace of the flavoprotein (blue) with the Rossmann fold highlighted in dark blue. The view is looking down onto the plane of the membrane and rotated 90 Å from the view in Fig. 1A. (B) The iron protein. Stereoview of the C trace of the iron protein (red) aligned with the 8Fe ferredoxin from Peptococcus aerogenes (light green) and the 2Fe ferredoxin from Spirulina platensis (cyan). The rmsd for the C atoms in these alignments is 0.8 and 1.6 Å, respectively. (C) The membrane anchor proteins. The view is down the center of the four-helix bundle, approximately normal to the plane of the membrane. FrdC (green) consists of helices I to III, and FrdD (purple) consists of helices IV to VI.

26 Quinone binding pockets and active site residues. (A) Stereoview of the QP binding site shows QP is bound in a polar pocket likely positioned just above the membrane bilayer. (B) Stereoview of the QD site shows QD is in a relatively apolar pocket within the membrane bilayer. (C) Binding site for the physiological inhibitor oxaloacetate adjacent to the FAD. Oxaloacetate lies beneath the isoalloxazine ring of the flavin. The flavin ring and inhibitor are shown superimposed onto a 2 Fo Fc map contoured at 1. The adenine has been omitted for clarity. Side chains that appear to interact directly with the inhibitor are labeled.

27 Ubiquinone s binding site, is located in a gap composed of SdhB, SdhC, and SdhD. Ubiquinone is stabilized by the side chains of His207 of subunit B, Ser27 and Arg31 of subunit C, and Tyr83 of subunit D. The quinone ring is surrounded by Ile28 of subunit C and Pro160 of subunit B. These residues, along with Il209, Trp163, and Trp164 of subunit B, and Ser27 (C atom) of subunit C, form the hydrophobic environment of the quinone-binding pocket. SdhA provides the binding site for the oxidabon of succinate. The side chains Thr254, His354, and Arg399 of subunit A stabilize the molecule while FAD oxidizes and carries the e- to the first of the clusters, [2Fe- 2S].

28 SdhA provides the binding site for the oxidabon of succinate. The side chains Thr254, His354, and Arg399 of subunit A stabilize the molecule while FAD oxidizes and carries the electrons to the first of the iron- sulfur clusters, [2Fe- 2S]. After the e- are derived from succinate oxidation via FAD, they tunnel along the [Fe-S] relay until they reach the [3Fe-4S] cluster. These e- are subsequently transferred to an awaiting ubiquinone molecule within the active site.

29 Mammalian, mitochondrial, and many bacterial monomer SQRs are composed of four subunits: two hydrophilic and two hydrophobic. The first two subunits, a flavoprotein (SdhA) and an iron-sulfur protein (SdhB), are hydrophilic. SdhA contains a covalently attached flavin adenine dinucleotide (FAD) cofactor and the succinate binding site and SdhB contains three iron-sulfur clusters: [2Fe-2S], [4Fe-4S], and [3Fe-4S]. The second two subunits are hydrophobic membrane anchor subunits, SdhC and SdhD. Human mitochondria contain two distinct isoforms of SdhA (Fp subunits type I and type II). The subunits form a membrane-bound cytochrome b complex with six transmembrane helices containing one heme b group and a ubiquinone-binding site, which can be seen in Image 4. Two phospholipid molecules, one cardiolipin and one phosphatidylethanolamine, are also found in the SdhC and SdhD subunits (not shown in the image). They serve to occupy the hydrophobic space below the heme b. These subunits are displayed in image 3. SdhA is green, SdhB is teal, SdhC is fuchsia, and SdhD is yellow. Around SdhC and SdhD is a phospholipid membrane with the intermembrane space at the top of the image.

30 Electrons are feed into! The Q cycle from both! Complex I and II.! Although complex I sends H + and e - from! only NADH, complex II sends them from several sources, both from the! cytosol in origin and the! mitochondria.! The Q cycle is then accepting more e - s than any other portion of the ETS and sends them to complex III at a specific rate. This is the importance of the semiquinone nature of these inner mito membrane constituents.!

31 Glycerol 3-phosphate shuttle. This alternative means of moving reducing equivalents from the cytosol to the mitochondrial matrix operates in skeletal muscle and the brain. In the cytosol, dihydroxyacetone phosphate accepts two reducing equivalents from NADH in a reaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase. An isozyme of glycerol 3-phosphate dehydrogenase bound to the outer face of the inner membrane then transfers two reducing equivalents from glycerol 3- phosphate in the intermembrane space to ubiquinone. Note that this shuttle does not involve membrane transport systems.!

32 Complex II catalyzes the oxidation of FADH 2 by CoQ,! FADH 2 +CoQ FAD + CoQH 2! ΔE Oʹ V ΔG O -16.4kJ/mol! This redox rxn. does not release sufficient free energy to synthesize an ATP, it functions only to bring e - from FADH 2 into the ETS!

33 The function of the ETS is to pump H + (protons) into the intermembranous space and to move e - perpendicular to the H + pumping thru the membrane. If the membrane is leaky, then the H + will leak back across the membrane. This is classically how ETS was separated from Ox-Phos. ATP synthesis requires the H + gradient between the 2 compartments. The Q cycle is that CoQH 2 undergoes 2 cycle reoxidation in which the semiquinone CoQ - is a stable intermediate. ISP is a Rieske Fe-S that bind Fe by both S and His. The circuitous route of e - transfer in complex III, is tied to the ability of CoQ to diffuse within the hydrophobic portion of the membr to bind to both Q o and Q i sites. This is facilitated by the surface of cyt b transmembr region. When CoQH 2 is oxidised two reduced cyt c s and 4 protons are on the outer side of the membr.

34 Cytochrome c alternately binds to cyt c1 of complex III and to Cytochrome oxidase (complex IV), it functions to shuttle e - between them.

35 COX is the terminal acceptor of ETS, catalyzing a 1 e - oxidations of 4 consecutive cyt c molecules & the concomitant 4 e - reduction of 1 O 2 to H 2 O. COX is made up of 8 to 13 subunits, 3 of which are coded for by mito genes. These are the largest & most hydrophobic of the subunits.

36 ΔE Oʹ = 0.58 ΔG O -112 kj/mol: 4cyt c 2+(red) + 4H + + O 2 4cyt c 3+(ox) + H 2 O This is a 200kD complex. It is formed of 8 to 13 peptides The largest three are coded mito DNA. Subunit I has 12 transmembr helices, subunit II has 2 and Subunit III has 7. The reduction of O 2 to H 2 O occurs an the binuclear center of a 3 -Cu B.

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38 In all mitochondria, chloroplasts and in many bacteria, chemiosmosis involves the proton-motive force (PMF) in some step. This can be described as the storing of energy as a combination of proton and voltage gradients across a membrane. The chemical potential energy refers to the difference in concentration of the protons on each side of the membrane and the electrical potential energy as a consequence of the charge separation across the membrane (when the protons move without a counter-ion, such as an electron). In most cases the proton motive force is generated by an electron transport chain which acts as a proton pump, using the energy in electrons from an electron carrier to pump protons (hydrogen ions) out across the membrane, creating a separation of charge across the membrane. In mitochondria, free energy released from electrons by the electron transport chain is used to move protons from the mitochondrial matrix to the intermembrane space of the mitochondrion. Moving the protons out of the mitochondrion creates a lower concentration of positively charged protons inside it, resulting in a slight negative on the inside of the membrane: The electrical potential gradient is about -200 mv, inside negative. This charge difference and the proton concentration difference create a combined electrochemical gradient across the membrane. This electrochemical gradient for protons is both a concentration and charge difference and is often called the proton motive force (PMF). In mitochondria, the PMF is almost entirely made up of the electrical component but in chloroplasts the PMF is made up mostly of the ph gradient. In either case, the PMF needs to be about 30 kj/mol for the ATP synthase to be able to make ATP.

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40 How is ATP generated in he mitochondria by the H + ATP synthase? Can ATP synthesis be uncoupled from the ETS? Can the H + ATP synthase be reversed and hydrolyse ATP Does it then become a H + pump? Are there similarities between the v-type H + ATPase and the H + ATP synthase?

41 The uncouplers act to separate the ETS from Ox-Phos. This was the indication that there where 2 pathways (emf) and connected by H + gradient (pmf).

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