An Overview of Mitochondrial Electron Transport
The main oxidizing agent used during aerobic metabolism is NAD+ (although FAD is used in one step) which get converted to NADH. Unless the NAD+ can be regenerated, glycolysis and the Kreb's cycle will grind to a halt. Luckily, under these conditions we are actually continually breathing one of the best oxidizing agents around, dioxygen. NADH is oxidized back to NAD+ not directly by dioxygen, but indirectly as electrons flow from NADH through a series of electron carriers to dioxygen, which gets reduced to water. This process is called electron transport. No atoms of oxygen are incorporated into NADH or any intermediary electron carrier. Hence the enzymes involved in the terminal electron transport step, in which electrons pass to dioxygen, is an oxidase. The enzymes of the Kreb's cycle and electron transport are localized in mitochondria.
By analogy to the coupling mechanism under anaerobic conditions, it would be useful from a biological perspective if this electron transport from NADH to dioxygen, a thermodynamically favorable reaction (as you calculated in the last study guide - a value of about -55 kcal/mol), were coupled to ATP synthesis. It is! For years scientist tried to find a high energy phosphorylated intermediate, similar to that formed by glyceraldehyde-3-phosphate dehydrogenase in glycolysis, which could drive ATP synthesis (which likewise occurs in the mitochondria). None could be found. A startling hypothesis was put forward by Peter Mitchell, which was proven correct and for which he was awarded the Nobel Prize in Chemistry in 1978. The immediate source of energy to drive ATP synthesis was shown to come not from a phosphorylated intermediate, but a proton gradient across the mitochondrial inner membrane. All the enzymes complexes in electron transport are in the inner membrane of the mitochondria, as opposed to the cytoplasmic enzymes of glycolysis. A pH gradient is formed across the inner membrane occurs in respiring mitochondria. In electron transport, electrons are passed from mobile electron carriers through membrane complexes back to another mobile carrier. Initially, NADH shuttles electrons (2 electron oxidation, characteristic of NAD+/NADH), to a flavin derivative, FMN, covalently attached to Complex I. The reduced form of FMN then passes electrons in single electron steps (characteristic of FAD-like molecules, which can undergo 1 or 2 electrons transfers) through the complex to the lipophilic electron carrier, ubiquinone, UQ.
This then passes electrons through Complex III to another mobile electron carrier, a small protein, cytochrome C. Then cytochrome C passes electrons through complex IV, cytochrome C oxidase, to dioxygen to form water. At each step electrons are passed to better and better oxidizing agents, as reflected in their increasing positive standard reduction potential. Hence the oxidation at each complex is thermodynamically favored.
Complex II (also called succinate:quinone oxidoreductase) is a Kreb cycle enzyme that catalyzes the oxidation of succinate to fumarate by bound FAD (hence its other name: succinate dehydrogenase). It is not involved in flow of electrons from NADH to dioxygen described above but passes electrons from the reduced succinate to ubiquinone to form fumarate and reduced ubiquone which then can transfer electrons to cytochrome C through Complex III. The crystal structure of this complex has recently been solved by Yankovskaya et al. who have shown that the arrangement of the redox-active sites in the complex minimizes potential oxidation of bound FADH2 by dioxygen, minimizing production of harmful reactive oxygen species like superoxide.
At each complex, the energy released by the oxidative event is used to drive protons through each complex from the matrix to the intermembrane space of the mitochondria, and is not used to form a high energy mixed anhydride as we saw in the glyceraldehyde-3-phosphate dehydrogenase reaction. The actual mechanism of proton transfer is unclear.
Complex I - NADH-quinone oxidoreductase
Now lets explore electron transport in greater detail by looking at the mechanisms of two specific complex, I and IV. Before that look at a detailed view of the entry pathway of electron transport and oxidative phosphorylation.
Complex I - NADH-quinone oxidoreductase
Complex I is located in the inner mitochondrial membrane in eukaryotes and in the plasma membrane of bacteria. Mammalian complex 1 consists of 45 subunits, 7 of which are encoded by mitochondrial gene. Bacteria have only 13-14 subunits. The significance of the extra mammalian subunits is still unclear. A cartoon model and the actual crystal structure of the Thermus thermophilus (bacterial) complex are shown below. The hydrophilic or peripheral domain catalyzes electrons transfer while the membrane domain (encoded by mitochondrial DNA) is involved in active transport of protons.
7/10/17: The following Jmol links contains multiple view of parts of the complex as well as the entire complex. It is repeated several times below.
Electron flow occurs from NADH to UQ through a series of one electron carriers in the hydrophilic or peripheral domain of complex 1. Initial handoff of electrons occurs to a flavin cofactor, FMN, and then through a series of Fe/S clusters. In the left figure below, these electron acceptors include a tetranuclear Fe/S cluster (SF4 shown yellow/red spacefill, a binuclear Fe/S cluster (FE2/S2) shown in blue, a FMN flavin mononucleotide shown in red, and a MN (II) ion, shown in purple N1a and N1b are binuclear clusters and N2, N3, N4, N5, and N6 are tetranuclear clusters.
The tetranuclear Fe/S cluster is based on the cubane structure with Fe and S occupying alternating corners of a square in a tetrahedral geometry. Each Fe is also coordinated to thiolate anions. The actual structure is a distorted cube as shown below, along with that of the binuclear cluster, whose bond angle also deviate from those in a tetrahedron.
Many possible micro-redox states with different standard reduction potential are possible for tetranuclear Fe/S clusters, much as a polyprotic acid has multiple pKa values. The two relevant for Complex I and other tetranuclear clusters are shown below:
a. FeIIFe3IIIS4(CysS)41- + e- ↔ Fe2IIFe2IIIS4(CysS)42- (lower standard reduction potentials)
b. Fe2IIFe2IIIS4(CysS)42- + e- ↔ Fe3IIFeIIIS4(CysS)43- (higher standard reduction potentials)
Electrons are passed singly to oxidized UQ in one electron steps to form UQH2.
Fe-S clusters are synthesized predominately in the mitochondria where they serve as redox cofactors in electron transport as described above. They are ubiquitous in all life forms and serve roles in addition to redox cofactors per se as they serve structural roles in proteins and are used in redox signaling within the cell as they change oxidation states. Many proteins that interact with DNA (repair enzymes, polymerases and helicases) contain an Fe-S cluster.
Evidence suggests that they placed critical roles in the abiotic evolution of life in the absence of oxygen as a a terminal electron acceptor in exergonic oxidation reactions. When oxidation became available, they became potentially became toxic to the cell as the Fe2+ could participate in reactions (such as the Fenton reaction) leading to the generation of deleterious reactive oxygen species (such as superoxide). To prevent toxicity, when delivered to the cytoplasm and nucleus they must carried and delivered by cytoplasmic iron-sulfur assembly (CIA) proteins.
Proton transport occurs in the membrane domain
Available evidence suggests that 4 protons move from the cytoplasm to the periplasmic space against a concentration gradient during a catalytic cycle of Complex I in bacteria. One appears to be associated with the reduction of UQ at the terminal tetranuclear Fe/S cluster N2. The other three protons move across the membrane domain.
Nqo4 (proximal to the membrane domain as seen in KEGG diagram) residues in chain D have been implicated in H+ flow to the N2 cluster. These include, starting from the N2 cluster, H169, H170, D86, R350, D401, H129, R279, H89, R125, E122, R249, Y257, Y254, Y260, R296 (conserved residues are in bold). The terminal N2 cluster is coordinated by two tandem Cys side chains (C45 and C46) that in their thiolate (deprotonated form) are ligated to the Fe in the cluster. What properties do these amino acids have that make them candidates for this H+ flow?
A model of electron and H+ flow is shown below (after Berrisford and Sazanov, JBC, 284, 29773, 2009). Iron-sulfur clusters N2 and N6b are depicted as O (for oxidized) or R (for reduced). C46 and C45 indicate the tandem cysteines from Nqo6 subunit (nearest the membrane domain). Q/QH2 indicate quinone/quinol. Tyr 87 (Y-O) and Glu 49 (D-O) are proton acceptors. H-path indicates proton delivery pathway from the cytosol to tandem cysteines. One proton from this cycle appears to be transported across the membrane. What happens to the other two protons shown in the diagram?
Additional proton are transported by the membrane domain. The NuoL, M, N, A/J/K and H transmembrane domains are shown below. The L, M and N domains have structures similar to proteins involve in the coupled movement of K+/H+ in opposite directions across a membrane (antiporter). There are discontinuous helices in each subunit. Possible residues at the discontinuity buried in the membrane helices are Glu 144 and Lys 234. Would you expect to find these buried in the membrane? If they are part of an unopened channel that is exposed on a concerted conformational change across 3 similar membrane protein domains, how would they participate in proton transfer.
Looking at the left three antiporter subunits L, M, and N, notice a large helix that runs horizontally across all of them. How might that helix function to couple movement of protons across all the antiporter subunits (L, M, and N)? (Hint: think mechanically)
Inhibition of Complex I
Complex I is inhibited by more than 60 different families of compounds. They include the classic Complex I inhibitor rotenone and many other synthetic insecticides/acaricides. The classes include: Class I/A (the prototype of which is Piericidin A), Class II/B (the prototype of which is Rotenone) and Class C (the prototype of which is Capsaicin). They appear to bind at the same site. From the structure of the 3 prototypes, what are the characteristics of the pharmacophore, the “ideal binding ligand”? Where do they likely bind? How “promiscuous” is the binding site?
Many devastating neurological diseases are associated with defects in Complex I. In addition to major problems with oxidative ATP production, reactive oxygen species (ROS) increase. The major sites for generation of ROS are Complex 1 and Complex III. Given the locations of the electron carriers at the periphery and internal within the protein complex, which electron carriers might most readily leak electrons to dioxygen? What ROS is likely to form in the process?
Inhibitors might block access of UQ or conformational changes necessary for final reduction of the ubiqinone free radical. Class A inhibitors dramatically increase ROS production. The actual site of ROS production in Complex is a bit controversial. One possible electron donor to dioxygen is FMN. Why is this a likely candidate? Mutants that lack N2 iron-sulfur cluster showed ROS production. Is this consistent with FMN site involvement in ROS production?
In submitochondrial preparations, normal Complex I activity occurs (which leads to formation of a sustained proton gradient). Also reverse electron transport, powered by an artificial proton gradient can occur, which leads to the reduction of NAD+ can occur (see diagram below).
A summary of finding on superoxide production by Complex I is given below:
- Superoxide production is inhibited flavin site inhibitors but not Q site inhibitors.
- Reverse electron transport leads to NAD+ and O2 reduction
- Reverse electron transport superoxide production is inhibited by both flavin and Q site inhibitors
Based on these findings, which site, the flavin or Q site, is involved in superoxide production?
Complex III is a complicated, multisubunit protein. The subunits involved in electron transfer are cytochrome b, cytochrome c1and the Rieske iron sulfur protein (ISP). Cytochrome b has two hemes. One is cyto b562 which is also called the low potential heme or cyto bL. The other is cyto b566 which is called the high potential heme or cyto bH. The cytochrome c1 subunit has one heme.
The following Jmol links contains multiple views of the complex. It is repeated several times below.
The Rieske iron sulfur protein has a Fe2S2 iron sulfur cluster which differs from other such clusters in that each Fe is also coordinated to two His side changes, as shown in the figure below. Alterations in H bonds to the histidines and to the sulfurs in the complex can dramatically affect the standard reduction potential of the cluster.
As with complex I and IV, proton and electron transfer are coupled processes. However, in contrast to Complex I, in which protons pass through protein domains that have homology to K+/H+ antiporters, and Complex IV, in which they pass through a combination of a water channel and the H-bond network, the protons in Complex III are carried across the inner membrane by ubiquinone itself. Two reduced ubiquinones (UQH2) from complex I pass their four matrix-derived protons into the inner membrane space. In the process four electrons are removed in a multiple step process called the Q cycle.
The two electrons from each UQH2 take different paths. One electron moves to a Fe/S Rieske cluster and the other to cytochrome bL. The electrons moved to the Rieske center then moves to cytochrome c1s and then to the mobile electron carrier cytochrome C which is bound to the complex in the intermolecular space. The electrons moved to cyto bLs are transferred to cytochrome bH in the complex. Though this latter path, two electrons (from two UQH2) are then moved to oxidized UQ, and two matrix protons are added to reform one UQH2. Hence, only one UQH2 participates in the net reaction shown as below.
QH2 + 2 cyto c3+ + 2H+matrix → Q + 2 cyto c 2+ + 4H+IMS
This net overall reaction, the Q cycle, is illustrated below. This net overall reaction, the Q cycle, is illustrated below.
Once again, there are no “proton” channels or H bonded networks in the protein for proton transfer across the inner membrane.
The figure below shows the relative position of the bound mobile electron carrier, cytochrome C, and the internal ones, the Rieske Fe/S cluster and cytochrome bL and bH. Note also the molecule stigmatellin A, which binds to the site where UQ becomes reduced (called the Qo site) and inhibits the complex. This shows that UQ/UQH2 are in position to react readily with the Rieske canter and cytochrome bL heme.
Another way to think about the electron transfer process from UQH2 to cytochrome C is that the 2 electrons from UQH2 take two different paths, one a high potential path to the Rieske center and on to cytochrome C, and another low potential path to the bL heme and on to the bH heme and then to UQ to reform UQH2 (see figure above).
Complex III, along with Complex I, can also produce unwanted reactive oxygen species (ROS). Only three of the protein subunits, cytochrome b (with the bL and bH hemes), cytochrome c1, and the Rieske iron sulfur protein (ISP) are involved in electron transfer, so one of those is mostly likely involved in ROS production. Experiments and mathematical models support a mechanism that involves a reduction of UQ by addition of one electron from cytochrome bL to form UQ. which then passes its electron on to dioxygen to form superoxide (O2-.).
As two ubiquinones must bind to the complex, there must be two proximal sites. One is the Qi site where oxidized UQ binds and receive an electron. The other is the Qo site where UQH2 binds.
From a kinetic perspective, the first UQH2 binds and transfers two electrons, one to the Rieske cluster (and on to cytochrome c1 and then to cytochrome C) and one to cytochrome bL (and on to heme bH) and then to an oxidized UQ bound at the Qi site. The UQ. radical is stabilized by the adjacent bH heme which has a lower affinity for electrons. Now a second UQH2 binds to the Qo site, and transfers two electrons, again one via the Rieske cluster and the second through cytochrome bL and bH to the UQ. radical present at the Qi site to form UQH2 after two protons are transferred to it from the matrix.
Now a second UQH2 binds to the Qo site, and transfers two electrons, again one via the Rieske cluster and the second through cytochrome bL and bH to the UQ. radical present at the Qi site to form UQH2 after two protons are transferred to it from the matrix.
Antimycin A, an extremely toxic drug, binds to the UQ Qi site and hence blocks electron transfer from cytochrome bL to bH at the Qi site. Heme bL can then pass its electron to dioxygen to produce superoxide.
Complex IV - Cytochrome C oxidase (CCOx)
The structure of complex IV is shown in the left figure and to the right in a diagram taken from the KEGG pathways (with permission).
Cytochrome C, the initial “substrate” of this complex, delivers electrons from its heme cofactor to a dinuclear copper cluster, CuA. From there electrons flow to an adjacent heme a (low spin) which transfers them to another heme a3 (high spin) and then finally to dioxygen which is coordinated to the Fe in heme a3 and to an adjacent CuB. The heme a3 Fe:Cu dinuclear cluster is unique among all hemes. Which of the hemes is mostly likely to have two His side chains coordinated to the iron heme? One?
7/13/17: The following Jmol links contains multiple views of the complex. It is repeated several times below.
First let’s consider the transfer of electrons from heme a to heme a3 to dioxygen (we will consider entry of electrons into the complex later). What would be the consequence if dioxygen, a substrate for the reaction, dissociated from the heme a3 Fe before it were completely reduced? Suggest a reason for evolution of this key enzyme to have produced the unique heme a3 Fe:Cu dinuclear cluster. Heme a and a3 vary from the heme in hemoglobin as they both have a formyl group replacing a methyl and a hydroxyethylfarnesyl group added to a vinyl substituent. Its structure is shown below. What is its overall charge of the heme in its reduced state? In its oxidized state?
The key challenge has been to understand the redox coupling to H+ transport. How is this done? The formyl group on the heme is coplanar with the heme in both oxidation states. How might this coplanarity effect the charge in the heme?
From the figure above, what type of interaction would likely occur between Arg 38 (R38) and the formyl group? What might occur to the protonation state of adjacent protein side chains, specifically Arg 38 (R38) on reduction of the heme? How would this link electron and proton transfer?
How might an electron be “transported” the 20 ï¿½ distance from the dinuclear CuA cluster to the Fe in heme a and ultimately on to dioxygen? H+ transfer does not occur by physical movement of an individual proton through a “proton pore”. What is the most likely mechanism for transfer? Consider the ratio of the mass of a proton and an electron. Which is more likely to display wavelike behavior?
Crystal structures of oxidized and reduced CCOx show water channels and small “cavities” which calculations show can hold 1-3 water molecules. Hence groups around the heme, including R38 are assessable to water. What might be the function/role of the waters in the channel? Some of the amino acid residues associated with the water channels are shown in the figure below and include R38, S34, T 424, S461, S382, H413. How might these amino acids be involved in proton transfer? Draw an arrow on the diagram showing the direction of proton flow.
Another consequence of electron transfer to heme a involves the interaction of S382 and the farnesyl OH group, which are close in space and proximal to a water cavity. How might they interact? On reduction of the heme, a conformation change occurs which increases the S382-farnesyl OH group. What effect would this have on the interaction of the two and the S382-L381-Val380 localized conformation? A new water cavity appears to emerge on reduction in this region. How might this impact proton transfer from the matrix?
Now let’s consider the entry site of electrons into the complex and how they might influence proton transport. Structural and functional studies show a key role for Asp 51 (D51) (see figure above). In the oxidized state, D51 interactions with two OH side chains and amide NH backbone groups but is not exposed to water. On reduction, D51 lies on the surface in an aqueous environment. Draw both side chains in their likely protonation state in both the oxidized and reduced complex. What are the consequence of these structures for proton transport?
Near D51 is Y440-S441. The backbone carbonyl group between 440 and 441 forms an “indirect” interaction with R38 which we showed earlier is affected by the redox state of heme a. They are too far apart to form H bonds. Show by adding two waters how an interaction could occur between the carbonyl O and the side chain R38 via Y371. Also show how the water that interacts with Y371 also forms a H bond with the heme a proprionate.
How could these groups participate in a proton transfer mechanism coupled to electron transfer? Would you expect a backbone carbonyl to be involved in proton transfer? Draw a reaction mechanism which shows how protonation of the carbonyl O could lead to formation of an imidic acid which leads to proton transfer from the backbone N. Also show reformation of the normal amide link through a tautomerization reaction. Why would this occur?
How does D51 connect to the this H bond network? In the oxidized state D51 is exposed on the surface. How might it change on reduction? Site specific mutagenesis has been used to change D51. What amino acid replacement might be optimal to affect activity but not protein folding? The mutation uncouples electron and proton transport. Which is likely to be affected?
A final pathway for coupling electron and proton transport has been proposed. Use the information above to complete the following statements: When heme a oxidized, Arg-38 is mainly ____________ (protonated/deprotonated) since _______ is available from the matrix. Asp 51 is ______________ (buried or exposed) and is ______________ (protonated/deprotonated). On reduction of heme a the net charge on heme a _________________ This leads to _________ (increased exposure/decreased exposure) of Asp-51 to the ___________ (intermembrane space, matrix, membrane) and _______ increased/decreased size of the water channel. Hence water molecules are _____________ (taken up/released from) the matrix. Coupled to this, protons on Asp-51 are ___________ (released to or taken up from) the intermembrane space On reoxidaiton of heme a, Asp-51 moves back to the ___________ (interior/exterior) of the protein and the net positive charge on heme a ___________ (increases or decreases) This leads to a _________ (increased or decreased) affinity of the heme formyl group for Arg 38. This leads to proton transfer __________ (to/from) Arg 38 toward Asp 51. Arg-38 then _____________ (gives up/take on) protons from water molecules in the water channel.
7/19/17 Mechanisms from higher resolution structures
A more nuanced understanding of the mechanism and linkage between H+ and e- movement derives from high resolution structures determined by Yano et al (2016). In their model (shown in the figures below based on the oxidized form of the protein, pdb 5b1a), protons from the negative (matrix) N side of the complex enter through a water channel and proceed to the positve (intermembrane side) through a H bond network (as described above and depicted below). These comprise the H Pathway.
Directional movement is mediated by proton:proton repulsion aided by an increase in + charge on heme a when it transfers an electron to heme a3. Of course, proton:proton repulsion would move protons in both directions. Reverse flow back through the water channel is prevented by a conformational change on oxygen binding that closes the channel.
Ultimately 4 electrons are transferred from cytochrome Cs (in single electron steps) to the dicopper cluster, CuA, and then sequentially to heme a to heme a3 (near the copper B ion) to dioxygen to form water. The motion of electrons and protons are coupled electrostatically.
The figure below gives an overview of these movements. The small red dots are the oxygen atoms of internal water molecules (the rest have rest have been removed using Pymol). It should be apparent, given the number and location of the internal water molecules, that many would be involved in the proton translocation pathways.
What's so interesting about this model is the detailed description of two types of protons, the ones that add to dioxygen and end up in water, and those that are vectorially transported to the IMS. In their model, the H+s that end up being transported move through the water and H bond network through a connecting H bond link region to a Mg2+/water cluster. Since binding of oxygen leads to structural changes that closes off the water channel, all protons to be transported to the IMS must be bound in the cluster before dioxygen binding.
The figure below show that initially, 4 H+s move through the H system to the Mg2+/H2O cluster. Oxygen binding the closes the water channels. This buildup of positive charges would certainly lead to a enhanced electrostatic attractions for the next phase of the reaction, the movement of electrons into the heme cofactors. Additionally, the 4 H+s in the cluster are probably prevented from leaking to the P side through water that are proximal (see above figure) by proline cluster, which presumably restricts the dynamical motion of the protein in that region necessary for proton movement. The figure does not show charge changes in the electron carriers.
The figure below breaks downs the mechanism to show the addition of the first electron to the CuA (dicopper cluster), delivered from cytochrome C, and the subsequent transport of one proton from the fully proton-loaded Mg2+/water cluster after dioxygen binding. This figure does show the stepwise redox changes in the electron carriers.
After CuA receives an electron from cytochrome C, it donates it to heme a and not to heme a3, even though both are close. The extra negative on heme a facilitates proton pumping thought the H pathways shown.