CMB

Lecture on electron transport

D. Nelson, last modified Dec. 3, 2004

Reading: Berg, Tymoczko and Stryer 5th edition, Chapter 18, pp. 491-507 

Pyruvate oxidation in the mitochondrion proceeds through the pyruvate 
dehydrogenase complex and then into the TCA cycle.  During transit through the TCA cycle 
there are four steps where NADH is produced and one step that forms FADH2.

1. pyruvate dehydrogenase (NADH)
2. isocitrate dehydrogenase (NADH)
3. alpha-ketoglutarate dehydrogenase (NADH)
4. succinate dehydrogenase (FADH2)
5. malate dehydrogenase (NADH)
(see Fig 17.15, p 478) There are limited amounts of these coenzymes in the mitochondria and they must be continuously recycled between the oxidized and reduced states. The reduced forms generated in the TCA cycle must be reoxidized or the cycle will stop for lack of a needed substrate, even if plenty of pyruvate is present. In this lecture we are going to talk about how these coenzymes are recycled and what happens to the protons and electrons they carry. (For the definition of a coenzyme and some examples see p. 191-192) The short version of this story is the following: NADH and FADH2 give up their protons and electrons to the electron transport chain. This chain is a series of four protein complexes that transport the electrons to oxygen and at the same time pump protons to create a proton gradient across the mitochondrial inner membrane. (see p. 491 top right) The proton gradient is used as a source of energy to generate ATP by the ATP synthase. The final step in the process is export of the newly formed ATP out of the mitochondrion to the cytosol where it can be used for synthesis, transport, etc. (Figure 18.39 p. 516). We have started with the reduced coenzymes NADH and FADH2 and ended with ATP, yet the ATP was not made directly from the NADH or the FADH2. We understand that ATP is an energy rich chemical compound and we have made it from NADH and FADH2, so these must also be energy rich compounds. How can this be? The answer lies in the reduction potential of NADH and FADH2. The reduction potential is a value that shows how electrons will flow when two compounds are brought together. Remember that chemical reactions involve a change in free energy, and there is a preferred direction for any given reaction. When electrons move spontaneously between two compounds, they will move in the direction that releases free energy, like water going downhill. The electrons in NADH and FADH2 are at a very high negative reduction potential. Their tendency will be to move to more positive reduction potentials and release free energy as they move. NADH is like a snowball at the top of Mt. Everest. FADH2 is like a snowball at the top of Mt. Fuji. Oxygen is at the bottom of the mountain and is the final electron acceptor. You get more energy out of NADH as it gives its electrons to oxygen than you do from FADH2 because more free energy is released on the way down. The electron transport chain supplies a series of electron acceptors or redox active centers that can receive the electrons from NADH and FADH2. These acceptors are of more positive reduction potential, so the electrons flow downhill and free energy is released. The spectacular feature of these protein complexes is their ability to capture the free energy released and convert it into motion of protein side chains and perhaps more long range motions of peptide backbones and larger structures to effect proton pumping against a gradient. This process is not understood at the molecular level yet, but much progress is being made in determining the 3-dimensional structures of the complexes. By pumping the protons out of the mitochondrion, the free energy of the electrons is conserved in a chemical and electrical gradient. These two are not the same thing. It is possible to eliminate the proton gradient and maintain the membrane potential or vice versa. We will talk about this later.

The complexes of the electron transport chain

 Complex I is the NADH Dehydrogenase complex. The mammalian complex is composed of at least 42 subunits. The complex molecular weight is about one million Daltons. Seven of its subunits are coded in the mitochondrial genome. This is highly significant, since only 13 protein coding genes are found in the mitochondrial genome and more than half of them are for complex I. The complex is composed of two domains, the membrane arm and the peripheral arm. These seem to be assembled separately. The peripheral arm contains most of the redox active centers. The membrane arm contains all of the mitochondrially encoded subunits. For a schematic diagram see Fig 18.11 p. 499 and your handout. The whole May 6, 1998 issue of BBA Bioenergetics is about complex I. In this issue, I learned to my surprise that yeast do not have complex I. Instead, they have a 57kDa protein that is an NADH-ubiquinone oxidoreductase like complex I, only smaller. I have received an email about this. Apparently not all yeast are missing complex I, but Bakers yeast is. The E. coli equivalent of this complex has only 14 subunits. Of these 14, seven are homologous to the 7 subunits coded in the mitochondrion. If this bacterial complex is cleaved by proteolysis, three fragments are produced: a membrane fragment with all seven subunits equivalent to the mitochondrially encoded subunits of complex I; a soluble fragment with 3 subunits that contains 5 iron-sulfur centers and one FMN; a connecting fragment of 4 subunits with one iron-sulfur center. A soluble piece with 5 subunits has been over expressed and crystallized, but the solution of the structure has not been completed. When it is done, we will have a good idea of how the iron-sulfur centers are laid out and how the FMN is situated in the protein. The inhibitor rotenone binds to complex I and competes at one of the ubiquinone binding sites. There appear to be two ubiquinone sites in complex I. One is tightly bound and does not seem to come off in purified preparations. One is loose and presumably is the one that can transfer electrons from complex I to complex III. When rotenone binds, electron transfer from complex I is blocked. The stoichiometry of protons pumped to electrons transferred is 4 protons/2 electrons. Two electrons are passed for each NADH oxidized. We have introduced three new and important players in the function of electron transport, and they each need to be discussed. 1. FMN 2. iron-sulfur centers 3. ubiquinone FMN is a cofactor tightly bound to the peripheral arm of complex I. FMN stands for flavin mononucleotide. See page 384 for details on flavin structure and reduction by NADH. The fact that FMN is bound to Complex I makes this a flavoprotein. Please note that there are four flavoproteins that feed electrons to ubiquinone. Flavoproteins that reduce ubiquinone in the mammalian electron transport pathway 1. Complex I 2. Complex II 3. Electron transfer flavoprotein dehydrogenase (ETF-QO) 4. sn-glycerophosphate dehydrogenase Complex II is a tetramer of non-identical subunits. It contains FAD and three iron sulfur clusters. Succinate dehydrogenase is part of the TCA cycle. It is the only TCA cycle enzyme to be an integral membrane protein. None of its subunits are coded in the mitochondrial genome, and it cannot pump protons across the inner membrane, so it does not contribute to the proton gradient like complexes I, III and IV do. This is being challenged now by examining the crystal structure and the location of critical amino acids (FEBS Lett. 504, 133 2001). The crystal structure of the bacterial succinate dehydrogenase is being solved now. A homologous enzyme fumarate reductase from E. coli has been solved (Science 284 June 18, 1999 Pages 1941-1942 for commentary and pages 1961-1966 for the structure. Fumarate reductase catlayzes the reverse reaction. We talked about it last time as a step in running the TCA cycle backwards. The structure of succinate dehydrogenase can be modeled on the fumarate reductase structure. The protein is modular. An FAD containing subunit is in contact with an iron sulfur containing subunit and this is in contact with two membrane bound subunits. The Iron sulfur subunit has three different iron sulfur centers, a 2Fe-2S, a 3Fe-4S and a 4Fe-4S cluster. Complex II has two hydrophobic membrane subunits that are fairly small. In E.coli, There is a heme sandwiched between these subunits. This heme is liganded by two histidine residues, one from each subunit. Mutagenesis studies in Robert Gennis' lab have shown that the histidines can be changed to other side chains. This causes the loss of heme, but the enzyme still assembles and works nearly as well as wild type, so the heme is not needed structurally or enzymatically it is probably an evolutionary relic. The fumarate reductase has no hemes, but it does have two quinones that are tightly bound at opposite sides of the membrane. Electrons flow from the quinone at the matrix side to the iron sulfur clusters and on to the FAD at the active site. This is the direction in fumarate reductase. In succinate dehydrogenase the electrons flow from succinate to FAD then to the iron sulfur clusters and on to the quinones. ETF-QO is a membrane protein with one FAD and one 4Fe4S center. It has one subunit of 67 kDa with two predicted transmembrane segments, so it is not a large complex like complex I, but it is important. At least nine mitochondrial matrix dehydrogenases feed electrons into ubiquinone by way of this intermediate electron carrier. Dehydrogenases that funnel electrons to ubiquinone by ETF-QO 1-4. 4 different, chain length specific acyl-CoA dehydrogenases 5. isovaleryl CoA dehydrogenase 6. 2-methylbutyryl CoA dehydrogenase 7. glutaryl CoA dehydrogenase 8. sarcosine dehydrogenase 9. dimethylglycine dehydrogenase You should not memorize this list. It is for your curiosity not for an EXAM. Defects in the ETF-QO cause the substrates of these dehydrogenases to build up. This can be fatal in infancy if the defect is severe. The disease is called glutaric acidemia type II. A paper in the Aug. 21 2001 Biochemistry 9758-9769 presents evidence that mitochondrial dehydrogenases in yeast are part of a supramolecular complex. At least eight dehydrogenases and four TCA cycle enzymes plus other proteins (up to 38 different proteins) were identified in a single complex. This supports the metabolon idea of Srere and also suggests that NADH may be channeled from several sources directly to the membrane bound dehydrogenases. The mitochondrial sn-glycerophosphate dehydrogenase is part of the glycerophosphate shuttle for moving electrons from cytosolic NADH into the electron transfer pathway. Remember that NADH does not cross the mitochondrial inner membrane, so the electrons are fed in from the periplasmic space in the form of glycerol 3 phosphate. This compound becomes oxidized to dihydroxyacetone phosphate by the mitochondrial sn-glycerophosphate dehydrogenase (see figure 18.37 p. 514) located on the outside of the inner membrane. The human cDNA was just sequenced in 1994 and the gene structure determined in 1996. There are 17 exons over 83 kb of DNA. The protein has 727 amino acids and contains FAD. There is a cytosolic glycerophosphate dehydrogenase, but it does not resemble the membrane bound form. It actually performs the reverse reaction, converting dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, but it is named for the forward reaction. The soluble and the membrane bound glycerophosphate dehydrogenases together form the glycerophosphate shuttle shown in fig 18.37 Electrons from cytosolic NADH are transferred to DHAP to form glycerophosphate and then back to the membrane bound glycerophosphate dehydrogenase FAD coenzyme and then on to ubiquinone in the membrane. Yeast have two additional genes NDE1 and NDE2 that are external faqcing NADH dehydrogenases. These can oxidize cytosolic NADH directly, but they do not pump protons like complex I, so they return less energy than complex I. Please note that progress in cloning and sequencing these electron transport components is current research not old history. John Walker and Paul Boyer were just awarded the Nobel Prize October 15, 1997 for work done on the X-ray crystal structure and mechanism of ATP synthase. We will talk about that in our next lecture. John Walker has also been a major contributor to understanding all the mammalian complexes at the level of their genes. The last eight human genes were just sequenced at the end of 1998. (Biochem. Biophys. Res. Commun. 1998 Dec. 18;253(2):415-22). Levels of the mitochondrial form of the glycerophosphate dehydrogenase are high in pancreatic islet cells. In patients and in rats with non-insulin dependent diabetes mellitus(NIDDM), the levels are quite low, suggesting that this protein might be a candidate gene involved in this disease. We began this discussion talking about three important players in electron transfer. The first was FMN (and as we have seen FAD) and their flavoproteins. The second was iron- sulfur clusters. These are a second type of redox active center that can accept and then donate electrons in the electron transfer pathway. They come in several varieties including a 1 Fe center coordinated with four cysteines, a 2Fe-2S cluster (Fig. 18.13 p. 500) or the 4Fe-4S cluster (also in Fig. 17.12 p. 474) The 3Fe-4S cluster is not shown. The first three flavoproteins we talked about all have these iron-sulfur clusters. Complex I from E. coli has six (three of each type), the mammalian Complex I has 6-7. Complex II has three, and we already saw that the electron transfer flavoprotein dehydrogenase had one. The glycerophosphate dehydrogenase has non-heme iron, but it is not clear if it is an iron-sulfur cluster. There is a disease in humans that is caused by a defect in iron sulfur cluster formation in proteins. As you can expect, complex I, complex II and other iron-sulfur containing proteins are pretty severely affected. These include aconitase in the TCA cycle. and to a lesser extent complex III. Animals have a ferredoxin protein in their mitochondria. This protein gives electrons to several cytochrome P450 enzymes that require electrons for their catalysis. A related protein is seen in yeast, but yeast do not have any p450s in their mitochondria, so it was not clear what this protein did. If the gene is knocked out in yeast it is fatal, so it is doing an essential function. It was shown recently that this function is assembly of iron sulfur clusters. One thing in common among these flavoproteins is this: the flavin gets the electrons first and then passes them on to the iron-sulfur centers. This is absolutely required since NADH is an obligatory 2 electron donor(see pages 384 and appendix B5) and Fe3+ is an obligatory one electron acceptor. NADH cannot donate its electrons directly to iron. There has to be an intermediate electron acceptor that can accept two electrons and give them to iron one electron at a time. That is why the flavin gets the electrons first. It cannot happen any other way. In our journey down the electron transfer pathway, so far all our electrons have been passed to ubiquinone. Ubiquinone is a lipid soluble electron carrier. It exists in the mitochondrial inner membrane and it carriers electrons between complexes in the electron transport pathway. Up until now we have not talked about how the electrons get between the complexes. They do this on carriers. Ubiquinone is one of these carriers. The other is cytochrome c, but we need to talk about ubiquinone first. Ubiquinone is like a flavin in that it can accept one electron at a time. When it has accepted only one electron it is called a semiquinone(Fig. 18.10 p. 499). When it has two electrons it is fully reduced and when it has none it is fully oxidized. Ubiquinone picks up electrons from our four different flavoproteins. As it exists in the membrane, it is either fully reduced or fully oxidized. The semiquinone is apparently stabilized by binding to protein sites, but does not float around freely in the membrane. The next electron acceptor in the path is complex III also called the bc1 complex. This structure has been solved in 1996 by Chang-An Yu of Oklahoma State University at Stillwater and Hans Deisenhofer who got the Nobel prize for the first crystal structure of a membrane protein complex (the light reaction center) He is in Dallas now. This structure is the largest protein structure ever solved, with 16 monomers of bc1 complex in the unit cell. The bc1 complex (complex III) has 11 subunits in mammals, (Figure 18.15 p. 502) but only three in bacteria. These three each contain important redox centers. The extra subunits in mammals have no known function and they are often called supernumerary subunits. One of these poses a curious problem of biogenesis that I will mention in a minute. The redox centers that you must know to understand complex III are two b type hemes that are contained in the same subunit and are directly above one another in the membrane. They are perpendicular to the membrane. At the cytosolic side of the membrane there is the Rieske iron-sulfur protein. Also on the cytosolic side of the membrane is cytochrome c1. In mammals there are 13 transmembrane segments, with 8 coming from cytochrome b that contains the two b type hemes. One of the transmembrane segments is actually the presequence of the rieske iron-sulfur protein. This presequence is cleaved off by a matrix metallo proteinase located at the matrix surface. The biogenesis problem I mentioned has to do with the orientation of this one transmembrane segment. The cleavage site of this peptide is found on the cytosolic side of the membrane, not at the matrix side where it had to be cleaved. This means that the transmembrane helix had to flip after it was cut. This is the only example I know of where a leader sequence of a protein is used as a subunit in a complex. Complex three operates in an unusual way. The mechanism is called the protonmotive Q cycle, or just the Q cycle. In this cycle, electrons from ubiquinone flow in two different pathways through the protein. Ulrich Brandt gives a detailed description of this process in a paper in Biochim. Biophys. Acta 1275, 41-46 (1996). This special issue of BBA is in my office if you want to read it. It is a collection of papers from an Aug. 1996 conference I attended on bioenergetics, and it covers many items we will discuss in this class. The Q cycle has a reputation for being difficult to understand and a bit abstract. It helps a lot to pay attention to the structure of the complex and where the electrons are flowing to understand the Q cycle. The electrons are donated to complex III from ubiquinone. Up until now we have talked about electrons, though protons are also being transferred and these are not equivalent. In the Q cycle it is important to keep in mind where the electrons and the protons are going, because they do not go to the same places. Note: your book is not completely up to date in its discussion of the Q cycle. It suggests that electrons move before protons at the P center, which is probably not true, and it suggests that an electron flows all the way to cytochrome c before the other electron moves to BL. See below for a more up to date account. The Figure 18.17 on p. 503 may be helpful in visualizing what is going on. At the start, fully reduced ubiquinone diffuses to the cytosolic side of the membrane where it binds to a site known as center P (P for positive side of the membrane) or the oxidation center, because the ubiquinone will become oxidized here. Its two electrons will be split into two different paths, one going to the Rieske iron sulfur center (reduction potential +290 mV) and the other going to the first b heme called BL (for its low potential -20 mV). As we discussed earlier, electrons will prefer to choose the pathway toward the most positive reduction potential so they would prefer to both go to the iron-sulfur center, one at a time rather than split up and go both ways. This bifurcation of electron flow is unique to the bc1 complex. It has a structure that forces this splitting to take place. Remember that iron can only accept one electron at a time so both the Rieske protein and the BL cytochrome can only take on one electron. The protein forces them to go different ways by requiring that both electrons are released from ubiquinone simultaneously. This way they have to go to two different acceptors because each acceptor can take only one electron. There is evidence that two ubiquinone molecules bind at the P center. One is ten times more tightly bound than the other and acts as a prosthetic group, never leaving the enzyme. This tightly bound ubiquinone is oxidized at the start of the cycle. In this paired arrangement, the major energy barrier to reaction is deprotonation of the loose ubiquinone. This occurs first before the electrons move. Then in a more speculative part of this model, one electron moves from the deprotonated ubiquinone to the tightly bound form and they both become semiquinones. It is these two semiquinones that simultaneously give up their electrons to the two different pathways. Only the loosely bound form is released as a fully oxidized ubiquinone This splitting creates a problem. One electron goes from the iron-sulfur protein to cytochrome c1 and is finally passed on to cytochrome c. This electron eventually is used to reduce oxygen in complex IV, cytochrome oxidase. What happens to the other electron? This non-productive electron passes on to the second b heme BH (for high reduction potential +50 mV). Finally this electron is given to a fully oxidized ubiquinone bound near the matrix side of the membrane at a site called the N center (N for negative) or the reduction center, because ubiquinone becomes reduced here. This is a different site than the P center and the ubiquinones are different. This electron transfer forms a semiquinone that stays tightly bound to this site. In a second reaction, another ubiquinone binds at center P and undergoes the same reactions as before. The low potential electron moves down through the two b hemes to the semiquinone and fully reduces it. This ubiquinone must pick up two protons from the matrix side of the membrane, then it is released. Note that two protons were released on the cytosolic side at the P center each time ubiquinone was oxidized. Two protons were picked up at the matrix side for each two electrons that were passed down the b heme branch of the pathway. The net movement of protons is 4 out at the cytosol and two in from the matrix for every two ubiquinones that get oxidized. Remember that the net movement of protons out of the mitochondrion has to be paid for by free energy release of the electrons flowing down their pathways. The majority must come from transfer to the Rieske iron-sulfur protein since it has by far the biggest drop in reduction potential The movement of protons in membrane proteins is being revealed by studies of bacteriorhodopsin. Crystal structures at high resolution have caught this proton pump in action (Science 286 8 Oct 1999 p.252-253 for commentary and p255-260 for article. The takehome message is that water molecules are very important, and only small side chain motions are required. These shift water networks and cause the pK of side chains to shift. Thus the proton flows as the pK of one group goes up and the pK of another goes down.

Inhibitors that bind to the bc1 complex.

 Antimycin binds very tightly to the N center at the matrix side of the membrane and prevents electrons from reaching ubiquinone from the b hemes. Therefore these two hemes get completely reduced, backed up with electrons that now have nowhere to go. Stigmatellin binds at the P center, at the interface between the iron-sulfur protein and cytochrome b. It prevents entry of the loosely bound ubiquinone and stops electron flow through the complex. The b hemes and the other redox centers become fully oxidized as their electrons leave and cannot be replaced.

Complex IV cytochrome c oxidase

 Cytochrome c oxidase has made many careers in biochemistry. In 1995 the structure of the bacterial form was reported (Nature 376, 660 (1995) and in May 1996 the mammalian form was published in Science 272, 1136-1144 (1996). These were landmark events. Cytochrome oxidase does the hard chemistry of reducing oxygen to water that is at the heart of all aerobic life on the planet. It is a proton pump like complex I and complex III. It also has numerous redox centers as we should expect for another member of the electron transport chain. Cytochrome c oxidase has 13 subunits in mammals and 28 transmembrane segments. Its molecular weight is about 210,000 Da. 3 subunits are from the mitochondrial genome and these contain all the redox centers. The 10 nuclear coded subunits seem to be embellishments, for regulatory purposes, or insulation. The protein is purified as a dimer, but this is probably not relevant to the function, since the bacterial enzyme is monomeric, and so are several eukaryotic forms. One surprising feature is the channel for oxygen to reach the active site. This channel appears to open in the lipid bilayer. This makes sense since oxygen is more soluble in the lipid than in water, but it was not suspected and it was not seen by the authors on the first paper. How do the electrons get to cytochrome oxidase? They come on a carrier. This time the carrier is cytochrome c. Cytochromes can only deliver one electron at a time, and it takes 4 electrons to reduce oxygen, so 4 cytochrome c proteins have to bind sequentially to one docking site on the periplasmic surface of the protein. Adding a charge to a protein interior, such as placing an electron into cytochrome c oxidase is thermodynamically expensive. One way to compensate for the cost is to take up a proton. This is called charge compensation. According to Peter Rich, cytochrome c oxidase is an extreme example of charge compensation. Four electrons are taken in and four protons are taken up. This is linked to the role of cytochrome oxidase as a proton pump. What are the redox centers? There are 2 copper centers called copper A and copper B. Please refer to Fig. 18.18 and 18.19 pp. 504-505. Copper A actually has two copper ions near one another. There are two hemes of the a type. These are called heme a and heme a3. There is also a magnesium ion and a zinc ion, though the role these play is not understood. The copper A site is the first place the electron goes upon leaving cytochrome c. Experiments have been done that modify this site so it binds only a single copper ion instead of two. The enzyme activity is nearly completely lost, so both coppers are required for function (Biochemistry 36, 3232-3236 1997). From the copper A site the electron migrates to heme a then to heme a3. Heme a3 and copper B form what is called the binuclear center. The heme a3 Fe is very close to the copper B ion. This is critical in the chemistry of the enzyme. There is no obvious reason to have two heme "a"s in the enzyme. Both hemes are about equal distance from the copper A site and theoretically, the electron from Copper A could go directly to heme a3, but it does not. The structure suggests a network that would favor electrons moving to heme a first. Peter Rich, who is an expert in the thermodymanics of this enzyme, proposes that the reason there are two hemes has to do with charge compensation. One site that takes up a proton during the entry of the four electrons is the copper B. When an electron moves from heme a to the heme a3, copper B binuclear center, the proton that was associated with copper B is forced to leave. This might be part of the driving force for proton pumping. Since 4 electrons have to come in to reduce oxygen, what is the order of addition? (see Figure 18.19 p. 504) Experiments with various types of spectroscopy show that 2 electrons enter the heme a copper B site, one on each metal ion, then oxygen binds between them and both electrons are added to the dioxygen molecule at the same time. This forms a peroxy bridge between the two metal ions Fe-O-O-Cu. Now two protons come in and one more electron and the O-O bond is broken. Another 2 protons and another electron finish the reduction to water. The two waters leave through a water channel. Note that while this is going on 4 more protons are being pumped across the membrane through the protein. There are some candidate proton conductance networks that are proposed and these are being tested by site directed mutagenesis. This is difficult to do since the three large subunits with all the channels are coded in the mitochondrial genome and there are no plasmid based systems for making these mutants in eukaryotes, so this work is being done on the bacterial system. It is also important to know that the protons that react with oxygen to form water move through the protein by different routes than the protons that get pumped. These are called chemical protons and pumped protons and they have to be kept separate. For a nice web site on oxphos, bioenergetics and ATP synthase try this link http://bmbpcu01.leeds.ac.uk/illingworth.oxphos/index.htm click here