How many protons are transferred from the matrix to the intermembrane space during the transport of two electrons through each of the complexes?

Abstract

During apoptosis, mitochondria release proteins from the intermembrane space, leading to the activation of cell death proteases (caspases). This unique and critical process—mitochondrial outer membrane permeabilization, or MOMP—is regulated by the Bcl-2 family proteins. The molecular mechanisms regulating MOMP have long been a focus of intensive studies aiming to find therapeutic targets for diseases such as cancer. We have developed faithful vesicle systems to study MOMP and have employed cryo-electron microscopy (cryo-EM) to visualize the pores in the membrane. I describe here the rationale behind our development of these in vitro vesicle systems, the adaptation of these systems to cryo-EM investigation, and how our findings contribute to understanding the core molecular mechanisms of MOMP.

Oxidative Phosphorylation Couples Inward H+ Flux to ATP Synthesis

The inner mitochondrial membrane contains many copies of a protein called the F0F1ATPase. This is also called ATP synthase. It consists of two parts: the F0 component spans the membrane and provides a channel for protons to move into the matrix from the intermembrane space. The F1 component is a complex of five proteins with the composition α3β3γδε, with a molecular weight of about 360,000. This remarkable complex couples movement of H+ to the synthesis of ATP.

The ETC pumps H+ ions out of the matrix into the intermembrane space. Because the H+ ions move without counter ions, this movement is an outward current that separates charge, and therefore there is a potential developed across the inner mitochondrial membrane. This potential varies depending on the state of mitochondrial activity, but a typical value is about 160 mV, negative inside. In addition to the potential, there is a concentration difference in H+ established across the membrane. The pH of the intermembrane space is about 7.0, whereas the pH of the matrix is about 8.0. Recall that pH=−log [H+], so that pH=7.0 implies that [H+]=10−7 M and pH=8 means [H+]=10−8 M. Thus, there is a 10-fold difference in the [H+] established by the ETC. When H+ ions travel from the intermembrane space to the matrix, they release the free energy stored in the electrochemical gradient for H+, enabling the F1 subunit to synthesize ATP from ADP and Pi.

The coupling of the electrochemical gradient of H+ across the inner mitochondrial membrane with ATP synthesis is called chemiosmotic coupling (because there is a concentration difference across the membrane and an electric potential). It was first proposed by Peter Mitchell in 1961, who was awarded the Nobel Prize for the work in 1978. Figure 2.10.5 schematically illustrates the chemiosmotic hypothesis.

The Proton Electrochemical Gradient Provides the Energy for ATP Synthesis

The ETC pumps H+ ions out of the matrix into the intermembrane space. The stoichiometry is about 10H+ ions per 2e− when they originate from NADH, and about 6H+ when the 2e− originate from FADH2 (see Figure 2.10.7). Because the H+ ions move without counter ions, this movement is an outward current that separates charge, and therefore there is a potential developed across the inner mitochondrial membrane. This potential varies depending on the state of mitochondrial activity, but a typical value is about 160 mV, negative inside. In addition to the potential, there is a concentration difference in H+ established across the membrane. The pH of the intermembrane space is about 7.0, whereas the pH of the matrix is about 8.0. Recall that pH=−log [H+], so that pH=7.0 implies that [H+]=10−7 M and pH=8 means [H+]=10−8 M. Thus there is a 10-fold difference in the [H+] established by the ETC. When H+ ions travel from the intermembrane space to the matrix, they release the free energy stored in the electrochemical gradient for H+, enabling the F1 subunit to synthesize ATP from ADP and Pi. This proton electrochemical gradient is sometimes called the proton motive force. The free energy for H+ transfer from the intermembrane space to the mitochondrial matrix is calculated as

[2.10.10]Δμout→in=μin−μout=μ0+RTln[H+]in +ℑψin−μ0−R Tln[H+]out−ℑψout=RTln[H +]in[H+]out+ℑ(ψin−ψout)

The free energy change for ATP synthesis under the conditions of the cell varies from cell to cell and from place to place within the cell because the local concentrations of ADP, Pi, ATP, and ions that bind to them (H+, Ca2+, and Mg2+) also vary from place to place. Nevertheless, we have already calculated an approximate free energy change for ATP hydrolysis under conditions of the cell to be −57.1 kJ mol−1. The free energy of ATP synthesis should be the opposite of this, +57.1 kJ mol−1.

According to the result in Example 2.10.1, there is not enough energy in one H+ transport to synthesize ATP. If we assume integral stoichiometry, we need at least three of them. The free energy for the reaction

Example 2.10.1

Calculate the Free Energy in the Mitochondrial H+ Electrochemical Gradient

The free energy per mole of H+ is given by Eqn [2.10.1] as

ΔμHout→Hin=RTln([H+]in[H+]out)+ℑ(ψin–ψout)

Inserting values of R=8.314 J mol−1 K−1, T=310 K, [H+]in=10−8 M, [H+]out=10−7 M, we calculate the chemical part as

RTln([H+]in[H +]out)=8.314Jmol −1K−1×310K×ln( 10−8M10−7M )=−5.93kJmol−1

Using ℑ=9.649×104 C mol−1 and ψin=−0.16 V (with ψout=0), the electrical part of the free energy change is

ℑ(ψin–ψout)=9.649×104Cmol−1×(−0.16V–0)=−15.44kJmol −1

Thus the total free energy change for H+ transfer from the intermembrane space to the matrix, for this condition given here, is −21.37 kJ mol−1.

[2.10.11]ADP+Pi+3Hout+→ATP+3Hin+

is the sum of the free energy of two processes:

[2.10.12]ADP+Pi→ATP3Hout+→3Hin+

We add the two to get

[2.10.13]Δμ=ΔμADP+Pi→ATP+3Δμ Hout+→Hin+ =57.1kJmol−1+3(−21.37kJmol−1) =−7.01kJmol−1

The negative free energy change for this coupled reaction indicates that this process will proceed spontaneously. That is, there is enough energy in the electrochemical gradient of H+ across the inner mitochondrial membrane to synthesize 1 ATP for every 3H+ ions transported. As it turns out, the stoichiometry is not integral.

1.36.3.1 Intrinsic Pathway

Mitochondria are comprised of five distinct compartments (Figure 3): the outer membrane, the intermembrane space, the inner membrane, the cristae space, and the matrix [59, 65]. Cristae are formed by folds in the inner mitochondrial membrane, where it is estimated that 85–90% of cytochrome c stores are housed with the remainder found in the intermembrane space [19, 116]. The gatekeepers for the cristae are the cristae junctions composed of optic atrophy type I (OPAI) oligomers formed from OPAI monomers located on the inner mitochondrial membrane and truncated OPAI isoforms found in the inner membrane space. The average opening of a cristae junction, in healthy mitochondria, is 15–30 nm, which is wide enough to allow the passage of cytochrome c. However, OPAI oligomers at the junctions, which can be several 100 kDa in size, restrict the movement of cytochrome c into the inner membrane space. During apoptosis, the OPAI oligomer complex is disassociated by truncated Bid (tBid), which is generated by the cleaving of Bid by caspase-8 and [32], therefore, allows for the passage of cytochrome c into the matrix.

Cytochrome c plays two diverse yet vitally important roles within the mitochondria. The first role is to aid in the maintenance of cell viability. In a healthy cell, cytochrome c is sequestered in the intermembrane space and operates by transferring electrons between Complex III (cytochrome bc1) and Complex IV (cytochrome c oxidase). However, in the presence of an apoptotic-inducing stress signal such as nutrient deprivation or chemical toxin exposure, cytochrome c is released into the cytosol, where it functions as an apoptotic catalyst. The release of cytochrome c occurs through the formation of a mitochondrial outer membrane pore (MOMP). Permeabilization of the outer mitochondrial membrane (OMM) occurs in a sequential manner and involves several members of the pro-apoptotic Bcl-2 family, namely Bid, Bax, and Bak. Upon activation, tBid binds to the OMM where it then binds Bax/Bak. Bax is primarily found in the cytoplasm and incorporates into the OMM following an apoptotic stimulus. Bak, on the other hand, resides primarily at the OMM. Once tBid is bound to Bax/Bak, Bax/Bak auto-oligomerizes to create the pore that allows for the release of cytochrome c from the mitochondria [60, 93, 111]. The anti-apoptotic protein, Bcl-2, can bind tBid to suppress the interaction of tBid with the OMM to prevent Bax translocation, and attenuate Bax and Bak auto-oligomerization [93, 120]. Bcl-xL, another anti-apoptotic protein, has the ability to bind membrane-bound tBid and Bax to competitively inhibit Bax oligomerization and suppress MOMP formation [6]. By contrast, the pro-apoptotic protein, Bad, has the ability to break the tBid–Bcl-xL interaction in order to promote MOMP formation [98].

Once cytochrome c has migrated to the cytosol, it interacts with the apoptotic-protease-activating factor-1 (Apaf-1) in the presence of adenosine triphosphate (ATP) and oligomerizes into an Apaf-1/cytochrome c octamer. Concurrently, pro-caspase-9 binds Apaf-1 resulting in its autocatalytic activation. The resulting cytochrome c/Apaf-1/caspase-9 multimer is referred to as the apoptosome. Activated caspase-9, in its bound or unbound form, then activates effector caspases, which generate a positive feedback loop to further activate caspase-9 and accelerate the cell’s demise.

In addition to cytochrome c, the mitochondria contain a warehouse of other pro-apoptotic molecules such as second mitochondrial activator of caspases (Smac)/direct IAP-binding protein with low pl (DIABLO), HtrA2/Omi, EndoG, AIF, and CAD, which are also released from the confinement of the mitochondria following apoptotic stimuli. The translocation of Smac/DIABLO and HtrA2/Omi is a caspase-catalyzed event and, therefore, occurs after the release of cytochrome c. Once in the cytosol, Smac/DIABLO and Htr2/Omi bind IAP family members, thus eliminating the inhibitive effects of IAP on caspases. EndoG, AIF, and CAD are released from the mitochondria, only after the commitment to apoptosis has been made, and translocate to the nucleus where they are involved with various aspects of DNA fragmentation.

1 INTRODUCTION

The following methods for the localization of enzymes within mitochondria are used: (a) separation of inner and outer membranes; (b) separation of the enzymes of the intermembrane space, the matrix space, and the membranes; (c) latency measurements; (d) the use of translocator inhibitors; (e) the use of proteolytic enzymes; and (f) the use of histochemical methods in electron microscopy.

Most localization studies have been done with rat liver mitochondria (for review, see Ernster and Kuylenstierna, 1970). In this section, reference will only be made to work that has been done with heart mitochondria. When the results are compared with the localization in liver mitochondria, the similarity in localization is striking. This leads us to propose the following general rule: If an enzyme (for which no mitochondrial isoenzymes exist) occurs both in liver and in heart mitochondria, the enzyme has exactly the same localization in both cases.

11.3.1 Mitochondria

Mitochondria are organelles typically ranging in size from 0.5 to 1 μm in length, found in the cytoplasm of eukaryotic cells. Mitochondria contain inner and outer membranes, separated by a intermembrane space (C-side) and the space enclosed by the inner membrane is called matrix (M-side) (Fig. 11.1). The intermembrane space is usually 60–80 Å in width and contains some enzymes. The matrix, however, is very viscous and rich in protein, enzymes, and fatty acids. Both the inner and outer membranes are constructed with tail-to-tail bilayers of phospholipids into which mainly hydrophobic proteins are embedded. One portion of the lipid molecule is hydrophilic (water-attracting) and the other portion is hydrophobic (lipid-attracting). The self-assembled lipid bilayer is in a dynamic and liquid-crystalline state. The outer membrane contains proteins and lipids and numerous transport proteins, which shuttle materials in and out of the mitochondrion. The outer membrane is 60–70 Å thick and permeable to small molecules, including salts, sugars, and coenzymes. The inner membrane contains all the enzymes and fewer lipids than the outer membrane. The inner membrane is permeable to small neutral molecules such as water, oxygen, and carbon dioxide, while its permeability to charged molecules such as proton and ions is limited. The number of mitochondria in a cell depends on the cell's function. Cells with heavy energy demands, such as muscle cells, have more mitochondria than other cells.

The inner membrane houses the electron transport chain and adenosine triphosphate synthesis. The inner membrane has numerous folds called cristae, which have a folded structure that increases the surface area where ATP synthesis occurs (Fig. 11.1b).

Mitochondria contain deoxyribonucleic acid (DNA) and ribosomes, protein-producing organelles in the cytoplasm. The DNA directs the ribosomes to produce proteins as enzymes (biological catalysts) in adenosine triphosphate production. Mitochondria are involved in the transport and regulation of Ca2+, protein import, cell aging and death, and obesity. Mitochondria from different organ systems, such as the liver, heart, and brain, display morphological and functional differences. Mitochondria are the major source of reactive oxygen species throughout the respiratory chain. These oxygen radicals may affect the function of the enzyme complexes involved in energy conservation, electron transfer, oxidative phosphorylation, and aging (Fig. 11.2).

Experimental evidence shows that mitochondria exhibit anisotropy. Three-dimensional images show that inner membrane involutions (cristae) have narrow and long tubular connections to the intermembrane. These openings may lead to lateral gradients of ions, molecules, and macromolecules between the compartments of mitochondria. This type of structure may influence the magnitude of local pH gradients produced by chemiosmosis and the internal diffusion of adenine nucleotides. The mitochondria have elongated tubes aligned approximately in parallel and are embedded in a multilamellar stack of endoplasmic reticulum, which could be related to the specific function of the mitochondria (Ovadi and Saks, 2004).

Mitochondrial Permeability Transition: Structure of Pore

Under physiological conditions, mitochondrial membrane permeability is tightly regulated, and mitochondria reserve an electrochemical gradient across the inner mitochondrial membrane (IMM), created through ETC and exclusion of H+ from the matrix to the intermembrane space. The intact mitochondrial membrane potential, ΔΨm, is essential required for the mitochondrial physiological activity, energy synthesis, Ca2+ and pH homeostasis in cells, and also sequencing caspase activators. In apoptosis and necrosis, initially the membrane permeability increases at the IMM with ΔΨm collapse. Under mild stimulus, transient and reversible pore is formed, which allows entry of water and solutes with molecular mass up to 980 Da, metabolites and inorganic ions into the matrix. Cyclophilin D (Cyp-D, a peptidyl-prolyl cis-trans isomerase) in the matrix binds to adenine nucleotide translocator (ANT) at the IMM and the pore is formed. Cyclosporin A (CysA) binds to Cyp-D and inhibits Cyp-D binding to ANT and suppresses the pore formation. More intense insults make the IMM pore irreversible, and a nonselective mega channel, called permeability transition pore (mPTP), opens fully, which increases the membrane permeability to solutes with molecular mass up to 1500 Da, and influx of solutes causes expansion of the matrix and rupture of the outer mitochondrial membrane (OMM). Cytochrome c and other caspase activator proteins released from the matrix into the cytoplasm activate caspase and finally apoptosis, whereas necrosis is induced when ATP levels decrease abruptly to less than 50% [35].

The mPTP is a pore with c.3-nm diameter and formed by assembly of proteins, but the exact composition and regulatory mechanism are still matter of debate [36–38]. The major proposed proteins include ANT at the IMM, voltage-dependent anion channel (VDAC, porin) at the OMM, Cyp-D at the matrix. TSPO, the Bcl-2 protein family, the hexokinase bound to VDAC at the OMM and the creatinine kinase in the intermembrane space are also bound to the mPTP and associated with the regulation. Recently, the mitochondrial phosphate carrier, ubiquinol-cytochrome c-reductase core protein II, has been proposed as the real regulator of the mPTP through binding to Cyp-D [39].

The mPTP opening is the point of “no return” in most apoptosis. In addition to Cyp-D or phosphate carrier, Bcl-2 protein family regulates the pore formation at the OMM, either in preventing (Bcl-2, Bcl-xL, Mcl-1, and Bcl-w), or promoting way (Bax, Bak, and Bad). Bcl-2 protein functions primarily during the initiation of apoptosis, and under physiological condition, these antiapoptotic and proapoptotic species form the heterodimer and antagonizes each other, whereas in apoptosis Bcl-2 proteins are activated or displaced with either species and form neuroprotective or apoptogenic homodimers. A pore called mitochondrial apoptosis-inducing channel is formed by homodimerization of Bax and Bak at the OMM [40].

8.5.3 Electron Transport Chain

The electron transport chain is the last step in the conversion of glucose into ATP, as illustrated in Figure 8.26. It involves a series of enzyme catalyzed chemical reactions that transfer electrons from (NADH+H+)andFADH2 (donor molecules) to acceptor molecules. Ultimately the electron transport chain produces 32 molecules of ATP from one molecule of glucose through hydrogen oxidation, and also regenerates NAD and FAD for reuse in glycolysis. The overall reaction is given by

(NADH+H+)+12O2+3ADP+3P⟶NAD++4H2O+3ATP

and

(8.113)FADH2+12O2+2ADP+2P⟶FAD+3H2O+2ATP

The electron transport chain activity takes place in the inner membrane and the space between the inner and outer membrane, called the intermembrane space. In addition to one molecule of ATP created during each Krebs cycle, three pairs of hydrogen are released and bound to 3NAD+ to create 3(NADH+H+), and one pair of hydrogen is bound to FAD to form FADH2 within the mitochondrial matrix. As described before, two cycles through the Krebs cycle are needed to fully oxidize one molecule of glucose, and thus 6(NA DH+H+) and 2FADH2 molecules are created.

The energy stored in these molecules of (NADH+H+)andFADH2 is used to create ATP by the release of hydrogen ions through the inner membrane and electrons within the inner membrane. The energy released by the transfer of each pair of electrons from (NADH+H+ )andFADH2 is used to pump a pair of hydrogen ions into the intermembrane space. The transfer of a pair of electrons is through a chain of acceptors from one to another, with each transfer providing the energy to move another pair of hydrogen ions through the membrane. At the end of the acceptor chain, the two electrons reduce an oxygen atom to form an oxygen ion, which is then combined with a pair of hydrogen ions to form H2O. The movement of the hydrogen ions creates a large concentration of positively charged ions in the intermembrane space and a large concentration of negatively charged ions in the matrix, which sets up a large electrical potential. This potential is used by the enzyme ATP synthase to transfer hydrogen ions into the matrix and to create ATP. The ATP produced in this process is transported out of the mitochondrial matrix through the inner membrane using carrier facilitated diffusion and diffusion through the outer membrane. In the following description, we assume all of the hydrogen and electrons are available from these reactions. In reality, some are lost and not used to create ATP. Other descriptions of the electron transport chain have additional sites and are omitted here for simplicity.

We first consider the use of (NADH+H+) in the electron transport chain. During the first step, a pair of electrons from NADH+H+ are transferred to the electron carrier coenzyme Q by NADH dehydrogenase (site 1 and Q in Figure 8.26), and using the energy released, a pair of hydrogen ions are pumped into the intermembrane space.

Next, the coenzyme Q carries the pair of electrons to the cytochrome bc1 complex (site 2 in Figure 8.26). When the pair of electrons are transfered from the cytochrome bc1 complex to cytochrome c (site C in Figure 8.26), the energy released is used to pump another pair of hydrogen ions into the intermembrane space through the cytochrome bc1 complex.

In the third step, cytochrome c transfers electrons to the cytochrome c oxidase complex (site 3 in Figure 8.26), and another pair of hydrogen ions are pumped through the cytochrome c oxidase complex into the intermembrane space. A total of 6 hydrogen ions have now been pumped into the intermembrane space, which will allow the subseqent creation of 3 molecules of ATP.

Also occuring in this step, the cytochrome oxidase complex transfers the pair of electrons within the inner membrane from the cytochrome c to oxygen in the matrix. Oxygen then combines with a pair of hydrogen ions to form water.

As described previously, the transfer of hydrogen ions into the intermembrane space creates a large concentration of positive charges and a large concentration of negative charges in the matrix, creating a large electrical potential across the inner membrane. The energy from this potential is used in this step by the enzyme ATP synthase (site 4 in Figure 8.26) to move hydrogen ions in the intermembrane space into the matrix and to synthesize ATP from ADP and P.

The ATP in the matrix is then transported into the intermembrane space and ADP is transported into the matrix using a carrier-mediated transport process (site 5 in Figure 8.26). From the intermembrane space, ATP diffuses through the outer membrane into the cytosol, and ADP diffuses from the cytosol into the intermembrane space.

In parallel with (NADH+H+), FAD H2 goes through a similar process but starts at coenzyme Q, where it directly provides a pair of electrons. Thus, FADH2 provides two fewer hydrogen ions than (NADH+H+).

The focus of this section has been the synthesis of ATP. Glycolysis and the Krebs cycle are also important in the synthesis of small molecules such as amino acids and nucleotides, and large molecules such as proteins, DNA, and RNA. There are other metabolic pathways to store and release energy that were not covered here. The interested reader can learn more about these pathways using the references at the end of this chapter and the website http://www.genome.jp.

3.3.2.1 Structure and Morphology

In the 19th century, morphologists revealed the morphology of mitochondria as grains (chondria) and filaments (mito) under light microscope. Later, more systematic morphology was established by selective staining approaches. Today, by the use of electron microscopy and development of specimen preparation ultrastructure of mitochondria understanding about mitochondrial characteristics and morphology is perfected. Mitochondria are semiautonomous organelles which do not need the nucleus of the cell for its own division. Mitochondria morphology consist of double membrane which differs in characteristics and functions. The double membrane arrangement compartmentalize intra structure of organelle consists of an outer membrane, inner membrane, intermembrane space, and the matrix (Fig. 1). Outer membrane has a smooth and permeable structure that possess porins, for example, transmembrane proteins, that permit free diffusion of many molecules up to 10 kDa into intermembrane space. The intermembrane space located between outer and inner membrane contains proteins have a critical role in mitochondrial energetics and apoptosis (process of programmed cell death). Unlike the outer membrane, the inner membrane is highly impermeable to the ions and molecules. Usually transportation through the inner membrane requires special transporters. Together with specialized transporters the inner membrane also contains enzymes and proteins of the ETC and the matrix of the mitochondria contains proteins, enzymes that are responsible for metabolic reactions.

Morphology, localization, size, and shape of mitochondria may vary depending on the cell they belong to. The conventional shape of mitochondria usually appears as spherical or rod that float in the cytoplasm; but the exact shape is not fixed and changes upon the physiological conditions of the cell. Thus, the mitochondria may look like club, racket, vesicular, round, or ring. The mitochondria of fibroblast usually characterized as filaments; in hepatocytes it is more likely to appear like ovoid or uniformly spheres [1]. Moreover, in the same cell different mitochondria populations can be seen, for example, in skeletal muscle cells mitochondria is normally ovoid, but the structure can vary such; ones located close to the sarcolemma is rounder and smaller than that are embedded among the myofibrils [2,3]. The size and number of mitochondria can vary among different cell types between 0.5 and 7 micrometers. The size of mitochondria may change in health and disease. For example, the size of mitochondria of venous smooth muscles are smaller than those which have renal failure [4]. In that vein, there are incidences of observation of giant mitochondria in the aged or metabolically injured cells [5,6].

The number of the mitochondria in a cell and their location in a tissue depend on its function. The mammalian red blood cells have no mitochondria; 15%–20% of the mammalian liver cells have mitochondria. In the mitochondria containing liver cells, the number is usually more than 2000 [7–9]. In skeletal muscles of healthy young individuals, mitochondria may constitute 4%–15% of the cell volume [10]. In the nervous system, neurons (the cells that generate and transmit signals) usually have tens of mitochondria to provide energy for this costly process [11]. Astrocytes supply additional energy to the neurons. There is usually more than one astrocyte providing additional energy to each neuron. The astrocytes may have 1 or 2 mitochondria to support the energy demand of the neurons [12]. Among all the tissues, muscle cells have the largest number of mitochondria to be able to respond to the high-energy demand while performing their tasks (i.e., muscle contraction).

Electron transport chain in eukaryotes

In aerobic unicellular eukaryotes which possess traditional mitochondria (e.g., Acanthamoeba and Naegleria), the ETC is comprised of four complexes (I-IV), each of which are composed of multiple proteins (see Fig. 4). Each complex either receives electrons from a coenzyme carrier or directly from one of the other complexes. These proteins sit on the inner mitochondrial membrane and can pump protons extracted from the oxidized substrate into the intermembrane space. This establishes the electrochemical gradient (ΔpH) and largely contributes towards the mitochondrial membrane potential (ΔΨM) across the inner mitochondrial membrane, which is important for the final ATP synthesis step. The ETC begins with complex I (NADH coenzyme Q reductase, also known as NADH:quinone oxidoreductase). This enzyme removes 2e− from NADH, the main electron carrier derived from the Krebs cycle, and subsequently transfers them to coenzyme Q (ubiquinone), producing the reduced product ubiquinol (QH2). This reaction is facilitated by FMN, which also accepts two protons from the dissociation of water in the cytoplasm, becoming its reduced form FMNH2. Each electron from the FMNH2 is transferred to an iron-sulfur (Fe-S) cluster before being passed on to ubiquinone (Q). This releases two protons which are translocated into the intermembrane space, and recycles FMNH2 back to its oxidized form, FMN. The transfer of electrons from the Fe-S cluster to Q is a two-step reaction, whereby transfer of the first electron results in the formation of a semiquinone intermediate (the free radical form of Q), and transfer of the second electron results in reduction of this semiquinone to QH2. This process, known as the Q cycle, results in four H+ being translocated across the membrane. The energy needed to actively transport these protons into the intermembrane space is derived from the electron current which is established through the continuous oxidation and reduction of electrons across the complex at a ratio of 4H+ per 2e− derived from NADH.

The resulting QH2 is mobile within the membrane and is also capable of bypassing complex I by receiving electrons from succinate via complex II (succinate dehydrogenase or succinate-CoQ reductase) or other electron donors (e.g., fatty acids and glycerol-3-phosphate). All such electrons are transferred to Q via FADH2. The pathways of electron transport though complex II is similar to that of complex I, but without the translocation of protons across the membrane.

The electrons are next transferred from QH2 in complex III (cytochrome bc1 or CoQH2-cytochrome c reductase) to two molecules of cytochrome c, which is water soluble and located within the intermembrane space. Complex III is composed of several proteins containing heme or metal centers, including b-type hemes (bL and bH), a c-type heme (c1) and Fe-S protein (“Rieske”). Complex III is conserved across most respiring organisms. The transfer of electrons to cytochrome c by complex III is coupled with the translocation of four protons, of which two are used to reduce QH2 to quinol and a further two are released from two molecules of ubiquinol (see Eq. 7).

(7)QH2+2CytcFeIII+2H+in→Q+2CytcFeII+4H+out

Complex IV (cytochrome c oxidase) transfers four electrons (derived from four molecules of cytochrome c) to O2, reducing it to two molecules of H2O. This complex is composed of cytochromes a and a3, where the latter acts as the terminal oxidase. During this process, eight protons are taken from the mitochondrial matrix, four of which are used to produce H2O and the remaining four are pumped into the intermembrane space. This complex is comprised of several copper and heme groups, including Fe3+. The latter makes complex IV a target for CN− poisoning, which blocks electron transport thereby depleting ATP synthesis, resulting in cell death. The oxidase test is a standard biochemical test for the identification of microbes which possess cytochrome c oxidase. Here, the reagent, tetra-methyl-p-phenylenediamine dihydrochloride (e.g., Kovac's oxidase reagent) acts as an artificial electron acceptor for the oxidase enzyme inducing a color change from clear to purple, indicating an oxidase positive result. Pseudomonas, Campylobacter, Vibrio and Neisseria species are examples of oxidase positive organisms.

The final step in respiration involves coupling the ETC, and the resulting electrochemical gradient, to oxidative phosphorylation via the F0F1 ATP synthase (or complex V). This culminates in the synthesis of ATP, which is a reversable reaction depending on the application. The F0 part of the enzyme spans the membrane providing a channel through which H+ ions can flow back into the mitochondrial matrix. The protons move sequentially through the α, β and δ subunits of the F0 unit. Once this revolution is complete, the protons are released back into the mitochondrial matrix. This process releases the free energy derived from the ETC, which is used by the F1 component of complex V, located at the matrix end of the complex, to drive ATP synthesis.

The overall structure of ATP synthases is conserved across the domains of life, which suggests an early evolutionary origin in biological energy conservation. This structure allows the complex to behave as a motor (see complex V structure in Fig. 4). The flow of protons through subunit a of F0 causes the c proteins to rotate. This torque is transmitted to F1 via the coupled rotation of the γε subunits. These subunits also transfer energy to F1 which is converted to potential energy via confirmational changes of the β subunits. This allows the binding of ADP + Pi, the substrates for ATP synthesis. When this is complete, the β subunits return to their original confirmation. The ATPase usually consumes 3H+ for every ATP produced. As mentioned, the reversibility of the F1/F0 motor is facilitated by the hydrolysis of ATP, which induces the torque to allow the γ subunit to rotate in the opposite direction. Here, protons are also translocated in the opposite direction, i.e., inside the cytoplasm or mitochondrial matrix, thus generating the proton motive force, rather than dissipating it. This explains why some anaerobic microbes which cannot undergo oxidative phosphorylation also synthesize ATPase and use this for important cellular processes such as motility and transport.