Department of Chemistry and Biochemistry Biochemistry 3300 University of Lethbridge III. Metabolism Oxidative Phosphorylation Biochemistry 3300 Slide 1 Biochemical Anatomy of Mitochondria Transmembrane channels allow small molecules (< 5 kD) and ions to pass through the outer membrane. Convolutions of the inner membrane provides large surface area. → depending on the tissue they are more or less profuse Specific transporters carry pyruvate, fatty acids and amino acids into the matrix for access to the citric acid cycle. Biochemistry 3300 Slide 2 Universal Electron Acceptors Collect Electrons Dehydrogenases (catabolism) transfer electrons to universal electron carriers which funnel electrons into the respiratory chain M mitochondria C cytosol Biochemistry 3300 Slide 3 Electron Carriers I. Nicotinamide Adenine Dinucleotide Optical Test NAD+ / NADP+ accept a hydride (H-) and a proton is released Biochemistry 3300 Slide 4 Electron Carriers II. Flavins FMN (Flavin Mononucleotide) is a prosthetic group of some flavoproteins. Similar in structure to FAD (Flavin Adenine Dinucleotide), but lacking the adenine nucleotide. When free in solution, FMN (like FAD) accepts - + 2 e + 2 H to form FMNH2. Biochemistry 3300 Slide 5 Electron Carriers II. Flavins When bound as a prosthetic, FMN (and FAD) can accept 1 e- to form the 'half-reduced' semiquinone radical. The semiquinone can then accept a 2nd e- to yield FMNH2. FMN (and FAD) mediating e- transfer between carriers that transfer 2e- (e.g., NADH) & those that can accept only 1e- (e.g., Fe+++). Biochemistry 3300 Slide 6 Electron Carriers III. Ubiquinone Coenzyme Q (CoQ, Q, ubiquinone) is very hydrophobic. Located in the hydrocarbon core of membranes. CoQ contains a long isoprenoid tail, with multiple units (typically n = 10) having a carbon skeleton comparable to that of isoprene. The isoprene tail of Q10 is longer than the width of a lipid bilayer (likely folded into compact shape) Biochemistry 3300 Slide 7 Electron Carriers – Ubiquinone The quinone ring of coenzyme Q can be reduced to the quinol in a 2e- reaction: + Q + 2 e + 2 H QH2. When bound to special sites in respiratory complexes, CoQ can accept 1 e− to form a semiquinone radical (Q·−). Thus CoQ, like FMN (& FAD), can mediate between 1 e− & 2 e− donors/acceptors. Coenzyme Q functions as a mobile e- carrier within the mitochondrial inner membrane. Biochemistry 3300 Slide 8 Electron Carriers IV. Cytochromes Cytochromes contain a Heme prosthetic group. Heme contains an iron atom in a porphyrin ring system. The Fe is bonded to 4 N atoms of the porphyrin ring and is the redox center. Hemes in the 3 classes of cytochrome (a, b, c) have different porphyrin ring substituents (eg. proprionate) Only heme c is covalently linked to the protein cytochrome c via thioether bonds to cysteine residues. Biochemistry 3300 Slide 9 Electron Carriers - Cytochromes Cytochrome c Heme iron undergoes 1 e- transition between ferric and ferrous states: Fe+++ + e- ↔ Fe++ Met80 Heme Fe interacts with: - 4 N of polyporphyrin ring and - 2 axial ligands above & below heme X His18 N N Fe N N PDBid 5CYT Y Axial heme Fe ligands: His18 and Met80 Axial ligands alter reduction potential of heme Fe Biochemistry 3300 Slide 10 Electron Carriers IV. Cytochromes Heme prosthetic group absorbs light at characteristic wavelengths Absorbance spectra can follow the redox state of the heme (same as for all other electron carriers) Many cytochromes are subunits of large integral membrane complexes containing multiple electron carriers - located within mitochondrial inner membrane Cytochrome c is a small, water-soluble protein with a single heme group Biochemistry 3300 Slide 11 Electron Carriers V. Iron-sulfur Centers Iron-sulfur centers (Fe-S) are prosthetic groups containing 1-4 iron atoms complexed to elemental & cysteine S atoms. Electron transfer proteins may contain multiple Fe-S centers. 4-Fe centers have a tetrahedral structure, with Fe & S atoms alternating as vertices of a cube. Biochemistry 3300 Slide 12 Electron Carriers V. Iron-sulfur Centers Iron-sulfur centers transfer only one electron!! (even when they have more than one Fe) Eg., a 4-Fe center might cycle between redox states described as: 3Fe+++, 1Fe++ (oxidized) + 1 e- 2Fe+++, 2Fe++ (reduced) Iron-sulfur proteins where one Fe atom is coordinated by two His residues are named Rieske iron-sulfur proteins. Biochemistry 3300 Slide 13 Electron Carriers Electron carriers that are organic compounds have lower standard reduction potentials than heme iron electron carriers Note: Fe:S electron carriers tend to have intermediate standard reduction potentials Biochemistry 3300 Slide 14 Respiratory Chain Most respiratory chain proteins are embedded in the inner mitochondrial membrane (or in the cytoplasmic membrane of aerobic bacteria). Biochemistry 3300 Slide 15 Respiratory Chain Complexes Protein components of the electron-transfer chain are primarily organized as large, transmembrane (or membrane associated) protein complexes Biochemistry 3300 Slide 16 Respiratory Chain 4H+ Electron transfer from NADH to O2 involves multi-subunit inner membrane complexes I, III & IV, plus CoQ & cyt c. Within each complex, electrons pass sequentially through a series of electron carriers. CoQ is located in the lipid core of the membrane. There are also binding sites for CoQ within protein complexes. Cytochrome c resides in the intermembrane space. It alternately binds to complex III or IV during e- transfer. Biochemistry 3300 Slide 17 Respiratory Chain 4H+ The standard reduction potentials of constituent e- carriers are consistent with the e- transfers observed. Biochemistry 3300 Slide 18 Inhibitors of Electron Transport Respiratory chain inhibitors include: Rotenone (a rat poison) & Amytal Complex I Antimycin A Complex III - CN & CO Complex IV - Any of these sites will block e transfer from NADH to O2. Experimental setup? How do we measure 'Electron Transfer Chain' activity Biochemistry 3300 Slide 19 Effect of Inhibitors on Electron Transport Oxygen electrode: O2 selective membrane permits measurement of [O2] O2 produced in sample chamber is reduced by anode generating a measurable current Biochemistry 3300 Slide 20 Electron Transport Inhibitors Experiment (sample chamber of O2 electrode): Buffered mitochondria solution with excess ADP + Pi are equilibrated Reagents added and [O2] is monitored over time Example experiment: 1 - Hydroxybutyrate is substrate that allows TCA cycle to function; NADH is source of electrons - O2 levels will decrease as e are transferred to complex IV where O2 is reduced 2 - Rotenone or amytal inhibit Complex I stopping the electron transfer reactions O2 levels remain constant as electrons do not reach complex IV where O2 is reduced 3 - Succinate provides electrons via Complex II - O2 levels will decrease as e are transferred from complex II to complex IV where O2 is reduced 4 - Antimycin inhibits complex III O2 levels remain constant as electrons do not reach complex IV where O2 is reduced 5 - TMPD/Ascorbate provide electrons to cyctochrome C - O2 levels will decrease as e are transferred from cytochrome C to complex IV where O2 is reduced 6 - CN- (or CO) inhibit complex IV O2 levels remain constant as O2 is not reduced Biochemistry 3300 Slide 21 Complex I Bovine complex I at 17 Å resolution. Complex I catalyzes oxidation of NADH, with reduction of coenzyme Q: + → + NADH + H + Q NAD + QH2 And the transfer of 4 H+ across the membrane: Grigorieff, N. (1998). J. Mol. Biol., 277, 1033-1046 Overall: + → + + NADH + 5H N + Q NAD + QH2 + 4H P Complex I is a proton pump that uses the energy of electron transfer for the vectorial movement of protons across the membrane. Complex I : L-shaped and contains six iron sulfur centers and a FMN-containing protein. No high-resolution crystal structure of mammalian complex which includes > 46 proteins. Biochemistry 3300 Slide 22 Complex I NADH interacts with a solvent exposed domain of the mitochondrial matrix. Coenzyme Q binds within the membrane domain. Fe-S centers are in the NADH-binding domain & in a connecting domain closer to the membrane segment. The initial electron transfers are: + + NADH + H + FMN ↔ NAD + FMNH2 + FMNH2 + (Fe-S)ox ↔ FMNH· + (Fe-S)red + H Biochemistry 3300 Slide 23 Complex I After Fe-S is reoxidized by transfer of the electron to the next iron-sulfur center in the pathway: + FMNH· + (Fe-S)ox FMN + (Fe-S)red + H Electrons pass through a series of iron- sulfur centers in complex I, eventually to coenzyme Q. − + Coenzyme Q accepts 2 e and picks up 2 H to yield the fully reduced QH2. Biochemistry 3300 Slide 24 Complex II Succinate Dehydrogenase of the TCA Cycle is also called complex II or Succinate-CoQ Reductase. FAD is the initial electron receptor. FAD is reduced to FADH2 during oxidation of succinate to fumarate. FADH2 is then reoxidized by transfer of electrons through a series of iron-sulfur centers to Coenzyme Q, yielding QH2. Biochemistry 3300 Slide 25 Complex II X-ray crystallographic analysis of E. coli complex II indicates a linear arrangement of electron carriers within complex II, consistent with the predicted sequence of electron transfers: → → → → FAD FeS1 FeS 2 FeS 3 CoQ In this crystal structure oxaloacetate (OAA) is bound in place of succinate. PDBid 1NEK Biochemistry 3300 Slide 26 Path of Electrons to Ubiquinone Other substrates for mitochondrial dehydrogenases pass their e- into the respiratory chain at the level of ubiquinon, but not through complex II. Example: Fatty acyl-CoA electrons via Acyl-CoA dehydrogenase (β oxidation) via