Electron Transport

Electron Transport Andy HowardBiochemistry Lectures, Spring 2019Thursday 11 April 2019

Oxidative phosphorylation • The energetics in oxidative phosphorylation all depend on the electron transport systems and the proton pump Electron Transport

What we’ll discuss • Electron transport • Overview • Complexes I – V • Mitochondria • Chemiosmotic theory • Energetics & Control • Electron Transport II • Uncouplers • Transporters • Shuttles • Terminal acceptors and donors • Oxidative damage Electron Transport

Overall role of electron transport • Last 3 lectures: we discussed carbohydrate metabolism and the TCA cycle, each of which produced some high-energy phosphate energy directly. • In both of those systems much of the energy generated took the form of reduced cofactors—NADH in both systems, and FADH2 (or QH) in the TCA cycle. • Now we’ll see what happens to those! Electron Transport

Overall reactions (CF&M Ch. 20) • NADH + H+ + (1/2)O2 + 2.5 ADP + 2.5 Pi NAD+ + H2O + 2.5 ATP • ETS also catalyzes transformations of the flavin coenzyme FAD:FADH2 + (1/2)O2 + 1.5 ADP + 1.5 Pi FAD + H2O + 1.5 ATP • These are mediated through other cofactors: Q, cytochromes, and Fe-S proteins • Proton translocation is crucial Electron Transport

Chemiosmotic theory:What it says • Protons are translocated from outside of mitochondrial inner membrane into its interior • That passage actually generates both chemical and electrical energy. • This is because they are moving down a concentration and electrical-potential gradient:Remember: Gtrans = RTln[Cin]/[Cout] + zF Electron Transport

How it works • This energy is used to drive the synthesis of ATP from ADP and Pi within an enzyme called ATP synthase, which is (big surprise!) anchored on the inside of the inner mitochondrial membrane. • The structure of two components of this enzyme system were determined in 1999 by Andrew Leslie and others. Electron Transport

Oxidation state and energy • We typically measure oxidation states in volts. • We can relate the energy associated with an oxidation-reduction reaction—the so-called change in redox potential—with the change in the oxidation state of the molecules involved in the reaction. Electron Transport

What is a volt? • A volt is actually a measure of energy per unit charge; in fact, a volt is one joule per coulomb. • When we say that a double-A battery has a voltage of 1.5 V, we mean that it can (under optimal conditions) deliver 1.5 joules of energy( = 0.359 cal, or 3.59*10-4 kcal) per coulomb of charge. Electron Transport

Charge and energy • One electron carries a charge of1.602 * 10 -19 coulomb • If change in redox potential in a reaction is 0.32 V and all of that change is delivered to a single electron:then energy imparted to that electron is • eΔE = (1.602 * 10-19 coulomb / e-) *(0.32 J/coulomb)= 0.513*10-19J / e- = 1.23* 10 -23 kcal / e- Electron Transport

… in biochemical units … • That doesn't sound like much, but if we look at that on a per mole basis, 0.32V applied to a single electron is (1.23 * 10-23 kcal/e-) *6.022 * 1023 e -/mole= 30.87 kJ/mol = 7.38 kcal/mol • which is a reasonable amount of energy on the scale we're accustomed to examining(~1 ATP hydrolysis). Electron Transport

So what can we get? • There is enough energy bound up in the reduced state of NAD relative to the oxidized state to drive the net creation of 2.5 molecules of ATP from ADP and phosphate, as indicated in the equations shown above. • Since there are NADH molecules created in several steps in glycolysis and the TCA cycle, there are numerous net ATP molecules that arise from the overall process. Electron Transport

Results from TCA cycle • 3 NADH produce 7.5 ATP • 1 FADH2 produces 1.5 ATP • 1 substrate-level phosphorylation(from succinyl CoA hydrolysis) • Total: 10 ATP per round, if we don’t get interrupted! • Since we formally shove 2 molecules of acetyl CoA into the system per molecule of glucose input into glycolysis, we get 20 ATPs out of the TCA cycle per glucose. Electron Transport

ETS: The big picture • 5 membrane-associated, multi-enzyme complexes in mitochondrial inner membrane • Complexes I-IV associated with electron transport and proton translocation:Complexes I, III, IV move protons • Complex V uses that proton gradient to produce ATP from ADP and Pi Electron Transport

Complexes I-IV • There are several multi-enzyme complexes involved in converting the reductive energy in NADH to its final products. # Name I NADH-Ubiquinone oxidoreductase II Succinate-ubiquinone oxidoreductase III Ubiquinol-cytochrome c oxidoreductase IV Cytochrome c oxidase Electron Transport

Overview of Oxidative Steps Chart courtesyMichael King,Indiana State Electron Transport

Reduced cofactors to ATP • We will discuss how the energy latent in these reduced cofactors can be turned into energy in the form of high-energy phosphate bonds in nucleoside triphosphates--the standard currency of energy. Electron Transport

Role of ETS (CF&M §20.3) • The electron transport system (ETS) is responsible for these transformations. • Like the TCA cycle or glycolysis, the electron transport chain is a series of chemical transformations facilitated by proteins. Electron Transport

Roles of these systems • Some of these proteins are enzymes in the conventional sense • others are not—they're electron transport proteins only: • so they can only be regarded as enzymes if we allow that the entire ETS is a large, multi-polypeptide transformation system--a multi-component enzyme, analogous to the pyruvate dehydrogenase complex or the fatty acid synthase complex Electron Transport

Flavin cofactors Flavin adenine dinucleotide • Participants in ETS • Sometimes depicted as starting points, but it’s often better to think of them as intermediaries Electron Transport

Reduced flavins FADH2 FMNH2 Electron Transport

Complex I Plasmodium Complex I115 kDa dimerPDB 5JWB, 2.7Å • NADH:Ubiquinoneoxidoreductase • Embedded in inner mitochondrial membrane • Passes electrons from NADH to ubiquinone ubiquinone Electron Transport

Properties of ubiquinone • The quinone moiety is where the oxidation-reduction takes place (1e- or 2e- at a time) • The hydrophobic tail (15C = 3 isoprene units) anchors this cofactor to the mitochondrial membrane Electron Transport

Protons in Complex I • Complex I picks up a pair of protons from the matrix and passes them up through to the other side of the membrane • Other 2 protons derived from reoxidation of QH2 • Energy for this translocation (against the concentration gradient) supplied by oxidation of NADH Diagram courtesy Rice University Electron Transport

Complex II • Succinate-ubiquinone oxidoreductase • We’ve looked at this already as succinate dehydrogenase • Succinate + Q  Fumarate + QH2 • No protons translocated in this step:Reaction is close to isoergic • FAD and Fe-S proteins involved Chicken SDH246 kDadimer of hetero-tetramersPDB 2FBW, 2.1Å Electron Transport

Complex III • Ubiquinol:cytochrome c oxidoreductase • Transfers electrons from reduced ubiquinol to iron atoms in cytochrome c • One electron per cytochrome c • Enzyme contains three main subunits: cytochrome c1, cytochrome b,and an iron-sulfur (“Rieske”) protein Electron Transport

Reactions in Complex III • Net reactions: • QH2 + cyto c–Fe3+ QH• + cyto c–Fe2++ H+ • QH• + cyto c–Fe3+ Q + cyto c–Fe2+ + H+ • Each of those sub-steps releases 2 protons across the membrane Electron Transport

Complex III structures • Cytochrome bc1 complexChicken mitochondrionEC 1.10.2.2, 481kDa dimer; monomer shownPDB 3H1J, 3Å • Rieske Fe-S proteinRhodobacterSoluble portion: 15.5kDaPDB 2NWF, 1.1Å Electron Transport

Cytochrome-dependent steps • Cytochromes are, in general, proteins involved in electron transport. • Cytochrome c is a mobile, soluble compound • Others are generally membrane-associated and are typically much larger • What they have in common:covalently bound heme Electron Transport

Cytochromes • The name derives from the fact that many of them are colored(–chrome) due to possession of heme; and found in cells (cyto–). • Most have covalently bound hemes, unlike globins, where the heme is noncovalently bound • Covalent connection is typically through vinyl sidechains to cys-X-X-cys-his in the polypeptide Electron Transport

Where do the individual names come from? • Early analyses of cellular or mitochondrial extracts showed several peaks in the absorption spectra • Cytochromes were named as those spectral peaks became associated with specific spectral peaks • Sometimes two different species (e.g. cytochrome c and cytochrome c1) have very similar spectra Electron Transport

Cytochrome c: special case • In particular, cytochrome c, which is a significant intermediary in the ETS, is a water-soluble, relatively small, heme-containing protein • Cytochrome c received substantial attention in the early years of biochemistry both because of its inherent importance and because it's easy to study. Electron Transport

Cytochrome c and evolution Cytochrome c is highly conserved • rate of mutation across the billions of years of evolution is remarkably slow, as compared to other proteins. • This is generally a sign that its function is sufficiently irreplaceable that even a modest modification in the protein renders the cell unviable. Electron Transport

Cytochrome c structure • Monomer is only 12kDa • Heme group appears between helices • Covalent linkage to heme visible here • Highly soluble aqueous protein, unlike the larger, membrane bound cytochromes Yeast cytochrome cPDB 1YCC, 12.8 kDa1.23Å; dimer shown Electron Transport

Cytochrome c and apoptosis • Cytochrome c plays a role in apoptosis: • When a cell receives (and pays attention to) an external apoptotic signal, typically via IP3 signaling pathway, Ca2+ is released from the ER • One of the first responses is release of cytochrome c from the mitochondrion into the cytosol • This triggers lytic events that eventually lead to shrinkage and absorption of the cell fragment Electron Transport

Complex III: schematic CourtesyU.Texas Electron Transport

Complex IV • Cytochrome c oxidase • Transfers electrons from (soluble) cytochrome c to molecular oxygen:Product is water • 2Cyto c–Fe2+ + (1/2)O2 + 2H+ 2Cyto c–Fe3+ + H2O Electron Transport

Cytochrome oxidase • 2 functional units; • Up to 13 subunits containing membrane-spanning helices • 4 protons produced per oxidation of two molecules of QH2 at the Q0 site RhodobacterCatalytic core of cytochrome oxidase86 kDa heterodimerPDB 3OMI, 2.15Å Electron Transport

Cytochrome oxidase mechanism • Depends on two Cu+ ions www.steve.gb.com Electron Transport

iClicker question #1 Of Complexes I though IV, which does not translocate protons? • (a) complex I • (b) complex II • (c) complex III • (d) complex IV • (e) they all translocate protons Electron Transport

Mitochondrial sequestration • Mitochondrion is a fairly complex organelle, found in all eukaryotes. • Some simple algae have one mitochondrion per cell, whereas some protozoa have a half-million per cell. • A mammalian liver cell contains about 5000 mitochondria. These organelles resemble bacteria in size and complexity. Electron Transport

Mitochondrial genetics • Vertebrate mitochondrion has its own chromosome, but it does not code for many proteins • Human mitochondrion codes for 17 proteins, plus two dozen specialized tRNAs and (presumably) some control elements. Electron Transport

Where is the rest of the mitochondrion’s function specified? • Those 17 proteins are ~2% of the functioning proteins in the mitochondrion • Everything else (~1000 proteins) is specified in the nucleus Electron Transport

Nuclear genes for mitochondrial proteins • Most of the ~1000 proteins that function in the mitochondrion are coded for in the host genome and are translocated, sometimes with some amount of proteolytic processing, from the ribosomal protein-synthesis mechanisms of the cytosol into the interior of the mitochondrion. Electron Transport

Mitochondrial structure • The mitochondrion has asmooth outer membraneand a highly convoluted innermembrane • Intermembrane space between them • Outer membrane is permeable to small molecules, so functionally the intermembrane space is equivalent to the cytosol. Electron Transport

Mitochondrial localization • In eukaryotes, ETS and TCA cycle reactions take place in the mitochondrion. • Many reactions occur on the inner membrane in its folded surfaces called cristae. • Localization to the membrane provides for orderly passage of substrates or electrons from one protein to the next, helping to defeat old man entropy. Electron Transport

Mitochondrial matrix enzymes Some proteins do function in the matrix, the aqueous compartment of the mitochondrion interior to the inner membrane. • Pyruvate dehydrogenase complex • TCA-cycle enzymes (except succinate dehydrogenase, which is embedded in the inner membrane) • Some enzymes involved in fatty acid oxidation. Electron Transport

What’s the matrix like? • This matrix is has such a high overall protein concentration that it is not really an aqueous medium; it's a gel. • Think of reactions that occur in the mitochondrial matrix as occuring in Karo syrup (except the syrup is made of protein + H2O, not sugar + H2O) Electron Transport

ATP synthase (Complex V) • Crucial example of a molecular motor, i.e.,a machine that translates between chemical energy and mechanical work • Ultimately its job is to pull protons across the membrane, using the energy associated with that favorable translocation to drive the synthesis of ATP from ADP and Pi • The motor is what rotates the protein through various positions to enable its reactions Electron Transport

Chemiosmotic theory: history • How are these reactions actually employed to drive ATP synthesis? • This was hotly debated for many years. • Problem was finally solved in 1960's: chemiosmotic theory, which links ATP synthesis to proton translocation across the membrane • Oxidations in complexes I, III, and IV is what drove the protons against a concentration gradient in the first place Electron Transport

Electron Transport