Cytochrome bd oxidase is an integral membrane protein that catalyzes the reduction of oxygen to water using quinol as the reducing substrate.[1] The full reaction is O₂ + 4H+ + 4e- → 2H₂O. The reaction is electrogenic but it is not coupled to a proton pump. Instead, bd oxidase utilizes internal water molecules to provide the four protons needed for the reduction reaction and an external ubiquinone molecule for the four electrons needed.[2] bd oxidase plays a key role in protecting the organism from high oxidative stress. In a gram-negative bacteriaheterotrophs like Geobacillus thermodenitrificans, bd oxidase prevents free radicals in the intracellular space. Other organisms, like humans, have mechanisms that do the same thing but are more intricate due to the organism’s higher levels of complexity.
There are two main types of respiratory cytochrome oxidases: the heme/copper oxidases and the heme-only cytochrome bd quinol oxidase, which is what bd oxidase falls under.[3] Heme-only cytochrome bd quinol oxidases are associated with microaerobic dioxygen respiration, and they have a high affinity for oxygen.
The G. thermodenitrificans is a facultative aerobic thermophilic bacterium that utilizes the bd oxidase mechanism. The oxygen enters the enzyme through the selective oxygen entry site that funnels the extracellular oxygen to Heme D in the active site. The electrons for the reaction are provided by a ubiquinone molecule bound to the Q loop. The protons for the reaction are provided by one of two potential proton pathways, either the CydA pathway or CydB pathway. Both of the proton pathways utilize the intracellular water molecules for the proton source, and shuttle them to Heme B595.
The focus of this page is to explain the structure and function of the G. thermodenitrificans’ bd oxidase. The overall structure contains 19 transmembrane helices that are arranged in a nearly oval shape.[1] The protein contains two structurally similar subunits, CydA, seen in blue, and CydB, seen in red, each containing nine helices, and one smaller subunit, CydX, in teal, with one transmembrane helix. These subunits interact using hydrophobic residues and symmetry at the interfaces. The CydX subunit, whose function is not currently known, is positioned in the same way as CydS, a separate subunit that is found in the bd oxidase homologue from E. coli bd oxidase, but is not found in G. thermodenitrificans. Due to its similar structure and position to CydS, CydX has been hypothesized to potentially stabilize Heme B558 during potential structural rearrangements of the Q loop upon binding and oxidation of ubiquinone (Figure 1), the function of CydS in E. coli[1] The Q loop is a hydrophilic region above Cyd A. The lack of hydrogen bonding in this hydrophobic protein allows the protein to be flexible and go through a large conformational change for reduction of dioxygen. Heme B595 is mostly involved in the proton pathway, and Heme D is involved with the oxygen pathway.
Other structures of bd oxidase exist that contain a variety of potential routes for the different reactants of the reduction of oxygen. For example, the bd oxidase of E. coli contains a different orientation of the Hemes and many different mechanisms of proton shuttling. G. thermodenitrificans was chosen because of the interest in the unique proton pathways, as described in the “Potential Proton Pathways” section.
Active Site
Figure 1. The active site of bd oxidase for G. thermodenitrificans. Heme B558 (pink; left), Heme B595 (pink; right), and Heme D (green). Important residues shown in blue. Measurements are shown in Å.
The active site for bd oxidase in G. thermodenitrificans is located in subunit Cyd A. The site consists of three iron hemes: Heme B558, Heme B595, and Heme D that are held together in a rigid triangular arrangement due to Van der Waals interactions.[1] The length between each heme's central iron is relatively constant which serves to shuttle protons and electrons from one heme to another efficiently (Figure 1). Heme B558 is hypothesized to act as an electron acceptor, orientated toward the extracellular side by His 186, Met 325, and Lys 252 (Figure 1).[1] With Heme B595 closest in proximity to the intracellular side, Heme B559 is likely the proton acceptor with two potential proton pathways. Both Heme B558 and Heme B595 then shuttle their respective ions directly to Heme D as this is the shortest pathway.
Potential Oxygen Entry Site
Heme D is the hypothesized spot for the oxygen to enter the protein. Heme D (shown in green) is directly connected to the protein surface on CydA and contains a solvent accessible substrate channel. This channel and accessibility allow for oxygen to easily bind to Heme D and eventually be reduced to two H₂O molecules. This process requires a proton and electron source, both described in the later sections.
Electron Source
An electron source is needed in order for the redox reaction of O₂ to occur. Cytochrome bd oxidase uses the quinol molecule ubiquinone as an electron donor (Figure 2).
Figure 3. Two potential sources of protons: CydA and CydB pathway.
Because there is no proton pump present, the proton transfer mechanism is facilitated by 2 potential proton pathways via intracellular water molecules.
One potential proton pathway is formed from the four-helix bundle (a1-4) of CydA. It is called the CydA pathway. The residues along this pathway help facilitate the movement of the protons. The location and negative charge characteristic of Glu108, together with previous mutagenesis experiments, supports the proposal that this glutamate residue is a redox state-dependent mediator of proton transfer to a charge compensation site. In other words, it acts like a proton shuttle.[1] The Glu101 residue, which is the last residue in this pathway, could be the protonatable group eventually used upon Heme B595 reduction. More research needs to be done to determine whether the CydA pathway is solely providing protons for charge compensation, or whether Glu108 can be a branching point that is able to pass protons via the Heme B595 propionates to the oxygen-binding site.[1]
Another potential entry site is close to the a1-4 four-helix bundle of CydB and is referred to as the CydB pathway. In this pathway, Asp25 is thought to be the equivalent of the Glu108 in the CydA pathway.[1] The other residues help facilitate the movement of the proton very similarly to the CydA pathway. The CydA pathway is the most accepted source of protons as less is known about the CydB pathway.
As mentioned above, the purpose of the bd oxidase is to reduce O₂ to 2H₂O using quinol as the reducing substrate, yielding the overall reaction of O₂ + 4H+ + 4e- → 2H₂O. The oxygen comes from the extracellular side of the protein, and enters through the oxygen entry site to Heme D. This pathway is depicted in orange in Figure 4.
The electrons required for the reduction mechanism come from a ubiquinol molecule (Figure 2) that simultaneously binds to the Q loop and gets oxidized giving 4e- to Heme B558. Once at Heme B558, the 4e- will be shuttled directly to Heme D to be used in the reaction. The electron pathway is depicted in blue in Figure 4.
The protons that are required in the pathway are not provided by a pump, but rather via intracellular water. The potential proton pathways utilize amino acids with properties that help shuttle the protons from the intracellular side of the protein to Heme B595 in the active site. The CydA pathway passes through the CydA subunit, shown in purple in Figure 4. The CydB pathway proceeds through the CydB subunit, shown in green in Figure 4.
When all of these elements of the reduction reaction aggregate in the active site at their respective hemes, the protons and electrons are shuttled to Heme D, where the actual reduction occurs. The 2H₂O molecules are then expelled from Heme D, shown in red in Figure 4. The shuttling of these electrons and protons also helps assist with creating the electric chemical potential in the cellular membrane.
Structure Similarity to bd oxidase found in E. coli
Figure 5. Alignment of bd oxidase for the organisms G. thermodenitrificans (PDB: 5doq) shown in blue and E. coli (PDB: 6rko) shown in purple.Figure 6. Heme arrangements for the organisms G. thermodenitrificans and E. coli. Heme D shown in green; Heme B595 and Heme B558 shown in pink
The structure of bd oxidase for G. thermodenitrificans is highly similar to the structure of bd oxidase in E. coli, with the only major difference being the length of the Q-loop.[2] All of the structural similarities and differences between the two proteins can be seen in the alignment of their main structures (Figure 5). Although only having one significant difference in structure, this shift in the Q-loop causes the two proteins to have different active sites (Figure 6). In particular, the hemes of bd oxidase in E. coli are arranged differently than the hemes of bd oxidase in G. thermodenitrificans. The main reason for this change in heme arrangement is because of the oxygen binding site being located differently in E. coli, thus causing a different active site arrangement in the protein.[2]
↑ 1.01.11.21.31.41.51.61.71.8Safarian S, Rajendran C, Muller H, Preu J, Langer JD, Ovchinnikov S, Hirose T, Kusumoto T, Sakamoto J, Michel H. Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases. Science. 2016 Apr 29;352(6285):583-6. doi: 10.1126/science.aaf2477. PMID:27126043 doi:https://dx.doi.org/10.1126/science.aaf2477
↑ 2.02.1Thesseling A, Rasmussen T, Burschel S, Wohlwend D, Kagi J, Muller R, Bottcher B, Friedrich T. Homologous bd oxidases share the same architecture but differ in mechanism. Nat Commun. 2019 Nov 13;10(1):5138. doi: 10.1038/s41467-019-13122-4. PMID:31723136 doi:https://dx.doi.org/10.1038/s41467-019-13122-4
Biological Importance of O₂ reduction
Oxygen toxicity is a fatal problem among all organisms, but can easily occur in prokaryotes due to their low oxygen tolerance. In prokaryotes, the cytochrome bd oxygen reductases function to quickly reduce the concentration of O₂ into H₂O to protect the cell from detrimental effects. Without proper functioning of these enzymes, or if O₂ concentrations are too high, the concentrations of the intermediates formed from the reduction reaction will increase and can be detrimental. As a result, cytochrome bd oxidases facilitate growth in both pathogenic and commensal bacteria causing them to be a vital enzyme for cellular growth and division. Their importance in anaerobic prokaryotes makes bd oxidases useful targets for drug development to combat bacterial infection.[4]
References
↑Giuffre A, Borisov VB, Arese M, Sarti P, Forte E. Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress. Biochim Biophys Acta. 2014 Jul;1837(7):1178-87. doi:, 10.1016/j.bbabio.2014.01.016. Epub 2014 Jan 31. PMID:24486503 doi:https://dx.doi.org/10.1016/j.bbabio.2014.01.016
↑Safarian S, Hahn A, Mills DJ, Radloff M, Eisinger ML, Nikolaev A, Meier-Credo J, Melin F, Miyoshi H, Gennis RB, Sakamoto J, Langer JD, Hellwig P, Kuhlbrandt W, Michel H. Active site rearrangement and structural divergence in prokaryotic respiratory oxidases. Science. 2019 Oct 4;366(6461):100-104. doi: 10.1126/science.aay0967. PMID:31604309 doi:https://dx.doi.org/10.1126/science.aay0967
↑Das A, Silaghi-Dumitrescu R, Ljungdahl LG, Kurtz DM Jr. Cytochrome bd oxidase, oxidative stress, and dioxygen tolerance of the strictly anaerobic bacterium Moorella thermoacetica. J Bacteriol. 2005 Mar;187(6):2020-9. doi: 10.1128/JB.187.6.2020-2029.2005. PMID:15743950 doi:https://dx.doi.org/10.1128/JB.187.6.2020-2029.2005
