Cellular respiration represents the biochemical process in which the gradual degradation of organic compounds (electron donors), such as glucose, succinate, pyruvate and lactate, is coupled to the generation of a proton motive force (PMF), which is ultimately used to drive the production of ATP. Respiration comprises a series of oxidation reactions that are catalyzed by membrane localized enzymes in which electrons are passed from one molecule to another concomitant with the membrane transfer of protons. In the last enzymatic step, electrons are passed on to the terminal electron acceptor, thereby reducing it. Numerous molecules possess sufficient oxidizing power to serve as electron acceptor, although oxygen is Nature’s most potent one. Thus, in aerobic respiration oxygen represents the terminal electron acceptor, while in anaerobic respiration other molecules fulfill this role. In eukaryotes, the respiratory machinery is located in the mitochondrial inner membrane. Figure 1 (adapted from 1) shows the five consecutive respiratory enzyme complexes, collectively called respiratory chain, of the mitochondrial inner membrane. Complex I to IV are connected by small molecules - ubiquinol (UQ) a lipid derivative, or cytochrome c - that shuttle electrons between the respiratory complexes. The first two enzymes of the respiratory chain are typically dehydrogenases that abstract electrons from NADH (complex I) or succinate (complex II), respectively. For complex I, this reaction is accompanied by the membrane transfer of protons in contrast to complex II. Complex III also functions as proton pump, while complex IV represents the last enzyme of the respiratory chain and transfers electrons onto oxygen, yielding water. This step also results in the membrane translocation of protons. The membrane transfer of protons by complex I, III and IV results in the formation of a PMF, which is utilized by complex V to produce ATP through chemiosmosis (1). In bacteria, the components of the respiratory chain are localized to the cytoplasmic membrane and are organized similarly as in mitochondria. Bacterial respiratory chains are extremely diverse and variable albeit less efficient than their mitochondrial counterpart (2). For example, the respiratory chain of Escherichia coli comprises 15 primary dehydrogenases, able to oxidize numerous organic electron donor substrates, and ten terminal oxidases for the reduction of six different electron acceptors (3,4).
This respiratory versatility enables E. coli to switch between aerobic and anaerobic respiration by inducing the expression of the required respiratory enzymes depending on the physiological conditions. Though related, the E. coli aerobic respiratory system differs mechanistically from the mitochondrial respiratory chain as it lacks cytochrome c as well as enzymes homologous to complex III and IV (3). Instead, E. coli contains two terminal oxidases that directly reduce oxygen and allow it to grow at different oxygen concentrations. These are the cytochrome bo-oxidase and bd-oxidase, which are conserved in bacteria and archaea. The bo-oxidase displays a low affinity for oxygen and is present under normal atmospheric conditions, while the bd-oxidase has a high affinity for O2 and is expressed at microaerobic conditions (3,4). Owing to its high affinity for oxygen, this enzyme is crucial during host infection by pathogenic bacteria and therefore bd-oxidases are of interest as potential antimicrobial drug target. The E. coli bd-oxidase is made up of two membrane-embedded subunits, CydA and CydB, and contains three heme cofactors (5). Functionally, the enzyme couples the oxidation of ubiquinol with the reduction of oxygen to water, thereby facilitating the membrane transfer of two protons and contributing to the generation of a PMF (5). Recently, a detailed structure of E. coli bd-oxidase was reported providing profound mechanistic insight (6). Here, I will discuss this structure as well as its current functional understanding.
Aerobic respiratory chain of Escherichia coli
E. coli possesses an extremely versatile respiratory system that allows it to grow under both aerobic and anaerobic conditions because, depending on the physiological conditions, the expression of different respiratory enzymes is induced. These include about 15 primary dehydrogenases that oxidize numerous electron donors as well as ten terminal oxidases for reduction of six different electron acceptors. Organizationally, the E. coli respiratory system is less complex when compared to the mitochondrial respiratory chain as it is in principle made up of a dehydrogenase and a terminal oxidase that are connected through electron-shuttling quinones (3,4). Thus, the dehydrogenases act as quinone reductase and the terminal oxidases function as quinol oxidase. The major function of the E. coli aerobic respiratory chain is to transfer protons out of the cytoplasm, thereby establishing a PMF. It is therefore functionally similar to the mitochondrial respiratory system, although the E. coli respiratory chain is energetically less efficient and employs a different mechanism for protein transfer. This because E. coli lacks enzymes homologous to mitochondrial cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) (3,4). Figure 2 shows a schematic overview of the E. coli aerobic respiratory system, revealing that there are only two coupling sites, namely: NADH dehydrogenase and quinol oxidase. NADH dehydrogenase is the first enzyme of the respiratory chain and is the only one that is able to pump protons. The enzyme is homologous to mitochondrial NADH dehydrogenase (complex I) and is made up of 13 subunits that form a membrane-embedded part as well as a peripheral arm that is exposed to the cytoplasm. The latter part contains one FMN as well as nine iron-sulfur (Fe-S) clusters as redox cofactors. NADH dehydrogenase is involved in aerobic and anaerobic respiration (7). Additionally, the E. coli respiratory system contains succinate dehydrogenase. This is a tetrameric enzyme equipped with FAD and three Fe-S clusters as redox cofactors. Succinate dehydrogenase is localized to the cytoplasmic membrane and catalyzes the oxidation of succinate to fumarate in aerobic growing cells (8). During this reaction, proton release and uptake occur at the same side of the cytoplasmic membrane and overall there is thus no membrane translocation of protons.
Hence, this enzyme does not contribute to PMF formation. Succinate oxidation is an essential step of citric acid metabolism and succinate dehydrogenase, therefore, couples the TCA cycle to the respiratory system. The dehydrogenases and terminal oxidases of the E. coli respiratory chain are linked only by electron-shuttling quinones (ubiquinone or menaquinone) as this bacterium lacks cytochrome c (3,4). Aerobically growing cells contain two terminal oxidases, cytochrome bo-oxidase and bd-oxidase, that directly reduce oxygen to water (3,4). Of these, the bo-oxidase displays a low affinity for oxygen, while the bd-oxidase has a high affinity for O2 and therefore these enzymes enable E. coli to grow at atmospheric and microaerobic conditions, respectively. The bo-oxidase (CyoABCD) comprises three subunits and is equipped with copper and two hemes as redox cofactor (9). Moreover, the bo-oxidase is related to the mitochondrial cytochrome c oxidase (complex IV) and it is therefore not surprising that it functions as proton pump, thereby directly contributing to the generation of a PMF. The bd-oxidase (CydAB) is made up of two subunits and contains three hemes as redox cofactor (5). Although this enzyme does not function as a proton pump, it contributes to the formation of a PMF protons that are produced during quinol oxidation are released into the periplasm, while cytoplasmic protons are used for the reduction of oxygen (5). In sum, dehydrogenases oxidize a variety of organic electron donors at the inner face of the cytoplasmic membrane and transfer the electrons to the quinone pool in the membrane. Reduced quinones provide electrons to either cytochrome bo-oxidase and bd-oxidase, that, in turn, reduce oxygen to water. Protons are transferred across the membrane by two different proton-pumping respiratory enzymes (NADH dehydrogenase and bo-oxidase), whereas additional protons are translocated to the periplasm by bd-oxidase. This generates a PMF that is ultimately used by the ATPase complex to drive the production of ATP from ADP and Pi.
Biochemical and structural features of cytochrome bd-I oxidase
Cytochrome bd-oxidases are exclusively found in bacterial respiratory chains as terminal oxygen reductase. These enzymes are unrelated to heme-copper oxidases, including mitochondrial cytochrome c oxidase. In fact, bd-oxidases represent a separate family of quinol-dependent oxygen reductases, transferring electrons from reduced quinones (ubiquinol or menaquinol) to oxygen (5). These enzymes are typically made up of two membrane-embedded subunits with three hemes (b558, b559, and d) as redox cofactor. Although they contribute to PMF formation, bd-oxidases don’t function as a proton pump. Transmembrane charge separation by these enzymes is achieved through releasing protons that are produced during quinol oxidation in the periplasm, while cytoplasmic protons are used for the reduction of oxygen. Unlike heme-copper oxidases, cytochrome bd-oxidases are much less sensitive to inhibitors such as azide and cyanide (5). Interestingly, many bacterial proteomes contain more than one bd-oxidase variant. E. coli, for example, expresses two bd-oxidases (bd-I and bd-II), which differ in the length of their quinol binding-domain (Q-loop). The bd-II variant features a short (S) binding domain and is encoded by the AppCD gene cluster, whereas bd-I contains a long (L) one and is encoded by the CydABX genes. The physiological role of bd-II oxidase is not fully understood but the bd-I variant appears to be the predominant one and in addition to PMF generation it is involved in a variety of other physiological functions. For example, the E. coli bd-I oxidase supports oxidative protein folding and is involved in heme biosynthesis, while other bd oxidases of the L subgroup are crucial for the survival of many important pathogens during infection inside host cells, which typically involves oxygen deprived conditions (5). E. coli bd-I, the best studied bd-oxidase, comprises two membrane integral subunits CydA and CydB with a total of 17 or 18 predicted transmembrane domains (TMDs) and mass of 57 and 43 kDa, respectively. CydA contains all redox cofactors (heme b558, heme b559, and heme d) and therefore the catalytic activity - reduction of oxygen - is restricted to this subunit. Recently, detailed structures of bd-I from E. coli were obtained by cryo-EM (6,10). Figure 3 shows the structure of this enzyme at 2.7 Å (6) in surface (left and center panel) and ribbon representation (right panel), revealing that the enzyme comprises a heterooligomeric complex made up of a CydAB core and two auxiliary subunits, namely: CydX and CydH. The two core subunits, CydA and CydB, are conformationally the same and comprise 9 TMDs that arranged in two four-helix bundles. Both subunits interact via hydrophobic residues of TMDs 2, 3 and 9. In contrast, CydX and CydH are single spanning membrane proteins. The TMD of CydX comprises 37 residues and this subunit is located in a groove formed by TMD 1 and 6 of CydA. CydH represents a newly identified subunit of the E. coli bd-I oxidase with a mass of 3 kDa and is present in a cleft between TMD 1 and 9 of CydA. CydH and CydX comprise non-catalytic subunits that are probably required for assembly or stability of the oxidase complex. CydA features a soluble domain of about 136 residues located in the periplasm between TMD 6 and 2 that is known as the Q-loop. This domain contains conserved residues (Lys252 and Glu257) that are critical for quinone binding. Moreover, all redox cofactors are present in CydA, indicating that the catalytic activity – reduction of oxygen – is restricted to this subunit.
An overview of the heme arrangement in CydA is provided in the left panel of figure 4, showing that the three heme cofactors are organized triangularly near the periplasmic surface. Heme b558 represents the initial electron acceptor and is located in the membrane next to the Q-loop. It is connected to the protein backbone through interactions with Met393, His186, Lys252, Lys183 and Asp239. Of these, Met393 and His186 are the two axial ligands that directly interact with the heme iron. Heme b558 receives electrons from quinol which are subsequently shuttled to heme b595 and heme d of the active site. Electron transfer between heme b558 and the hemes of the oxygen reduction site is probably facilitated by Trp441. This residue bridges the distance between the initial electron acceptor and the active center. Heme b595 is bound to the enzyme via interactions with Glu445, Arg448 and Arg9. Both arginine residues form salt bridges with Glu445, the axial ligand. Heme d, the oxygen binding and reduction site, is localized to the center of CydA near the interface of CydB. It is bound to the enzyme through interactions with its axial ligand, His19. Oxygen (shown as red spheres) is located on the opposite side of the heme in a hydrophobic pocket made up by the conserved Phe104, Ile144 and Glu99. Collectively, these residues form the oxygen binding site. Moreover, the enzyme contains a hydrophilic proton-conducting channel that starts at the cytoplasmic CydAB interface and runs to the oxygen reduction site with Ser102, Glu107 and Ser140 (right panel of figure 4) facilitating proton transfer to heme d for oxygen reduction. The oxygen-conducting channel starts at Trp63 that is located between TMD1 and TMD9 and represents a hydrophobic pathway that runs to the oxygen-binding site of heme d.
Cytochrome bd-oxidases catalyze the four-electron reduction of di oxygen to water without formation of reactive oxygen species (ROS) and generate a transmembrane proton gradient (membrane potential). To this end, the primary dehydrogenases of the respiratory chain oxidize the electron donor, thereby reducing ubiquinol. Reduced ubiquinol can diffuse laterally through the lipid bilayer towards the bd-oxidase. Following oxidation of ubiquinol, two protons are released into the periplasmic space and two electrons are transferred to the hemes of the oxygen reduction center. At the same time, four protons are derived from the cytoplasm for the reduction of oxygen. Electron transfer from heme b558 to the active site hemes is coupled to vectorial proton transfer from the cytoplasm to the periplasm through the proton-conducting channel of the enzyme (5). Specifically, generation of membrane potential is associated with electron transfer from heme b558 to the active site hemes, whereas transfer from heme b595 to heme d is not coupled to PMF formation.
Different enzymological techniques have established relatively stable intermediates of the cytochrome bd-oxidase catalytic cycle (figure 5 adapted from 6). Under aerobic conditions the enzyme is mainly in the one electron-reduced state bound to O2 (A1). In the absence of oxygen, the reduced form of the enzyme with one electron (R1) can be observed. Oxidation of the first quinol converts the A1-state to the fully reduced state (A3). Cleavage of the dioxygen bond and release of the first water molecule yields the oxoferryl state of the enzyme (F). This represents the critical step in the catalytic cycle where production of ROS must be avoided. It was previously proposed that the dioxygen bond was broken concomitantly with a 4-electron reduction, which prevents the formation of peroxide intermediates. However, the structural data presented above favor a sequential electron transfer from heme b558 via heme b595 to heme d (6). Oxidation of a second quinol results in the one-electron reduced hydroxo-bound R1 state, while release of a second water, reduction of heme d and binding of another oxygen molecule restores the A1 state.
Following the development of oxygenic photosynthesis by cyanobacteria at least 2.5 billion years ago (11), oxygen levels rose significantly to establish the current oxidizing atmosphere. This, in turn, triggered the evolvement of aerobic respiration using O2 as terminal electron acceptor yielding H2O and H+. The oxidizing power of oxygen is harvested in aerobic respiration by an array of membrane-embedded redox enzymes, the respiratory chain, that employ electrons derived from the initial oxidation of the carbon source to reduce oxygen, which is coupled to the generation of a PMF and this is ultimately used to drive the production of ATP. In mitochondria, cellular respiration is performed by the respiratory chain located in the mitochondrial inner membrane (figure 1), while in bacteria the respiratory chain is localized to the cytoplasmic membrane. Although organizationally related to the mitochondrial respiratory chain, their bacterial counterparts are in general less complex and energetically less efficient, while offering greater metabolic flexibility. E. coli, for example, possesses an extremely versatile respiratory system (figure 2) that allows it to grow under both aerobic and anaerobic conditions. Depending on the physiological conditions, the expression of different respiratory enzymes is induced. These include about 15 primary dehydrogenases that oxidize numerous electron donors as well as ten terminal oxidases for reduction of six different electron acceptors. Under aerobic conditions, two terminal oxidases, cytochrome bo-oxidase and bd-oxidase, that directly reduce oxygen to water are present in the E. coli cytoplasmic membrane (3,4). Of these, the bo-oxidase displays a low affinity for oxygen, while the bd-oxidase has a high affinity for O2. The bo-oxidase (CyoABCD) comprises three subunits and is equipped with copper and two hemes as redox cofactor (9). This enzyme is related to the mitochondrial cytochrome c oxidase (complex IV) and likewise it functions as proton pump, thereby directly contributing to the generation of a PMF. The bd-oxidase (CydAB) is made up of two core subunits and contains three hemes as redox cofactor (5). Although it does not function as a proton pump, the bd-oxidase contributes to the formation of a PMF by releasing protons that are produced during quinol oxidation are released into the periplasm, while cytoplasmic protons are used for the reduction of oxygen. Cytochrome bd-oxidases are exclusively found in bacterial respiratory chains, including important human pathogens. In fact, these enzymes are crucial for the survival of pathogenic bacteria during infection inside host cells and therefore bd-oxidases are of interest as potential antimicrobial drug target (5). It can therefore be expected that the detailed structure of the bd I-oxidase from E. coli discussed above (6) will not only improve the mechanistic understanding but also facilitate the design of novel antimicrobials that target this enzyme.
1. Kühlbrandt W. (2015). Structure and function of mitochondrial membrane protein complexes. BMC Biol. :13:89.
2. Skulachev VP, Bogachev AV, Kasparinsky FO. (2013). Structure of Respiratory Chains of Prokaryotes and Mitochondria of Protozoa, Plants, and Fungi. In: Principles of Bioenergetics. Springer, Berlin, Heidelberg,
3. Yasuhiro A, Gennis R.B. (1987). The aerobic respiratory chain of Escherichia coli. Trends Biochem Sci. 12: 262-266.
4. Unden G, Dünnwald P. (2008). The Aerobic and Anaerobic Respiratory Chain of Escherichia coli and Salmonella enterica: Enzymes and Energetics. EcoSal Plus. ecosalplus.3.2.2.
5. Borisov VB, Gennis RB, Hemp J, Verkhovsky MI. 2011. The cytochrome bd respiratory oxygen reductases. Biochim Biophys Acta. 1807: 1398-1413
6. Safarian S, Hahn A, Mills DJ. et al. (2019). Active site rearrangement and structural divergence in prokaryotic respiratory oxidases. Science. 366: 100-104.
7. Friedrich T, Dekovic DK, Burschel S. 2016. Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (respiratory complex I). Biochim Biophys Acta. 1857:214-223.
8. Cecchini G, Schröder I, Gunsalus RP, Maklashina E. 2002. Succinate dehydrogenase and fumarate reductase from Escherichia coli. Biochim Biophys Acta. 1553: 140-157.
9. Abramson J, Riistama S, Larsson G. et al. 2000. The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site. Nat Struct Biol. 7: 910-917.
10. Theßeling A, Rasmussen T, Burschel S. et al. 2019. Homologous bd oxidases share the same architecture but differ in mechanism. Nat Commun. 10(1):5138.
11. Allen JF, Martin W. 2007. Evolutionary biology: out of thin air. Nature. 445: 610-612.