Introduction
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. [2]. The four electrons are provided by CARSON ADD HERE QUICK SUMMARY OF ELECTRON SOURCE, and the oxygen enters through a selective entry site.
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.
Cytochrome bd oxidase plays a key role in protecting gram-negative heterotrophs from high oxidative stress (ie. preventing free radicals in intracellular space in prokaryotes). [4]. Other organisms, like humans, have mechanisms that do the same thing but are more intricate due to the organism’s higher levels of complexity.
The Geobacillus thermodenitrificans organism utilizes the bd oxidase mechanism. The oxygen enters the enzyme through the selective that funnels the extracellular oxygen to in the active site. The electrons for the reaction are provided ADD MORE SPECIFIC DESCRIPTION OF ELECTRONS TO ORGANISM. The protons for the reaction are provided by one of two , either the or . Both of the proton pathways utilize the intracellular water molecules for the proton source, and shuttle them to .
Structure
The focus of this page is to explain the structure and function of the Geobacillus thermodenitrificans’ bd oxidase. The contains that are arranged in a nearly oval shape (Fig 1.).[2] The protein contains two structurally similar subunits, , seen in blue, and , seen in red, each containing nine helices, and one smaller subunit, , in teal, with one transmembrane helix. The subunits are interacting 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, which is found in E. coli bd oxidase. Due to its similar structure and position, it has been hypothesized to potentially stabilize during potential structural rearrangements of the Q loop upon binding and oxidation of quinol. [2] The 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. is mostly involved in the proton pathway, and 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. Geobacillus 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
Geobacillus 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 Geobacillus 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 due to Van der Waals interactions [2]. The between each heme's central iron is relatively constant which serves to shuttle protons and electrons from one heme to another efficiently (Fig. 1). It is suggested that Heme B558 acts as an electron acceptor, orientated toward the extracellular side by His186, Met325, Lys182, and Lys252 (Fig. 1) [2]. With Heme B559 closest in proximity to the intracellular side, it is suggested it functions as the proton acceptor with two potential proton pathways. It is then proposed that both heme B558 and B595 shuttle their respective ions directly to Heme D based on this being the shortest pathway. Heme D is thus indicated to be the oxygen binding site due to proximity and orientation to the exterior surface of the protein [2].
Potential Oxygen Entry Site
is the hypothesized spot for the to enter the protein . Heme D (seen 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 water 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. The chemical structure of ubiquinone is shown in Fig. 2.
Figure 2. Chemical structure of ubiquinone.
As shown in the overall , the is on the extracellular surface and provides a binding site for ubiquinone
[2]. As mentioned in the Active Site section, Heme is closest in proximity to the Q loop and thus is the suggested electron acceptor. This suggestion is further supported by the conservation of Trp374 (Fig. 1) often found as intermediate electron receptors in biological electron transfer chains
[2].
Potential Proton Pathways
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 via intracellular water molecules.
One potential proton pathway is formed from the of CydA. It is called the . The location of Glu108 in our structure supports the proposal that this glutamate residue is a redox state-dependent mediator of proton transfer. In other words, its main role is to allow charge to be alleviated at this site. [2] With the CydA pathway leading to Glu101, this residue could be the protonatable group used for charge compensation upon heme b595 reduction. It is still unknown whether protons entering the CydA pathway can be transferred from Glu101 to Glu378, to allow the spread of the negative charge of a second electron used to reduce the two high-spin hemes. Proto- nation of Glu378 could alternatively also be accomplished by a proton accessing from the extracellular side. 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.
Another potential entry site is related to the a1-4 four-helix bundle of CydB. Therefore, this is called the . In this pathway, Asp25 is thought to be the equivalent of the Glu108 in the CydA pathway [2]. The other residues help facilitate the movement of the proton very similarly to the CydA pathway. There is less known about the CydB pathway, and therefore, the CydA pathway is the most accepted source of protons.
Overall Oxygen Reduction Mechanism Summary
Figure 4. overall oxidation-reduction mechanism summary.
As mentioned above, the purpose of the bd oxidase is to reduce O₂ to 2H₂O using quinol as the reducing substrate, and having 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 protons that are required in the pathway are not provided by a pump, but rather via intracellular water. The potential pathways utilize amino acids with charges 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, and is shown in purple in Figure 4. THe CydB pathway proceeds through the CydB subunit, and is shown in green in Figure 4.
As shown above, the electrons required for the reduction mechanism come from a ubiquinol molecule (Fig. 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 reduction of O₂. The electron pathway is depicted in blue in Figure 4.
When all of these elements of the reduction aggregate in the active site, the protons and electrons are shuttled to Heme D, where the actual reduction occurs. The 2H₂O molecules are then expelled, as seen in red in Figure 4. The shuttling of these electrons and protons also helps assist with the electric chemical potential in the cellular membrane.
Biological Importance of Reducing O₂
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 family functions 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 of the vitality of reducing O2 in prokaryotes, knowledge on Bd oxidases can help develop drugs that target these enzymes to combat bacterial infection [5].
Structure Similarity to bd oxidase found in E. coli
Figure 5. Alignment of bd oxidase for the organisms
Geobacillus thermodenitrificans (blue) and
E. coli (purple).
Figure 6. Heme arrangements for the organisms
Geobacillus thermodenitrificans and
E. coli. Heme D (green); Heme B595 and Heme B558 shown in pink
The structure of bd Oxidase for
Geobacillus thermodenitrificans is highly similar to the structure of bd Oxidase for
Ecoli with the only noticeable difference being the length of the Q-loop.
[6] The similarity and differences between the two proteins can be seen in the alignment of their main structures (Fig.5). Although only having one noticeable difference in structure, this difference causes the two proteins to have different active sites (Fig. 6). In particular, the are arranged differently than the . The main reason for this change in heme arrangement is because of the being located differently in
E. coli, thus causing a different active site arrangement in the protein
[2]. For
