1. Lecture #6.2 ETS & Ox- Phos; Aerobic Respiration

2. ½ mol O 2 + NADH 2  H 2 O + NAD +  E O ´=0.82-(-0.32)=1.14V  G 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

3. 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.

5. 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.

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

7. 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.

8. 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  -oxidation of fatty acids. All of these pathways generate ATP through the ETS & ox-phos pathways.

9. 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  E o = 0.36V  G o = -69.5 kJ/mol Complex III  E o = 0.19 V  G o = -36.7 kJ/mol Complex IV  E o = 0.58 V  G o = -112.0 kJ/mol

11. 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. Also known as NADH oxidoreductase. Portions of the Complex I protein are present in numerous proteins found in anaerobic prokaryotes and (microaerophilic) Eukaryotes, that generate H 2.

12. 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 .

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

14. QFR does produce 25 times more ROS (superoxide & H 2 O 2 ). Structure of fumarate reductase. (A) Stereo-view 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.

15. 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.

16. 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.

17. 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 oxidation 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].

19. 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 semi- quinone nature of these inner mito membrane constituents.

20. 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.

21. Complex II catalyzes the oxidation of FADH 2 by CoQ, FADH 2 +CoQ  FAD + CoQH 2  E O 0.085 V  G 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

22. 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 semi-quinone 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.

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

24. 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.

25.  E O = 0.58  G 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.

30. 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).