spinqualitylabelsall models bd oxidase (PDB: 5doq) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Contents 1 Introduction 2 Structure 2.1 Active Site 2.2 Potential Oxygen Entry Site 2.3 Electron Source 2.4 Potential Proton Pathways 3 Overall Oxygen Reduction Mechanism Summary 4 Structure Similarity to bd oxidase found in E. coli

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 and an external ubiquinone molecule for the four electrons needed.^[2]

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 bacteria, more specifically 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 is a facultative aerobic thermophilic bacterium that 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 by ubiquinone molecule bound to the . 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.^[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 ubiquinone (Fig. 1).^[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

The active site for bd oxidase in Geobacillus thermodenitrificans is located in subunit Cyd A. The site consists of three iron hemes: , , and 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 acts as an electron acceptor, orientated toward the extracellular side by (Fig. 1).^[2] With 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 and shuttle their respective ions directly to based on this being the shortest pathway.

Potential Oxygen Entry Site

is the hypothesized spot for the 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. The chemical structure of ubiquinone is shown in Fig. 2. As shown in the the is on the extracellular surface and provides a binding site for ubiquinone.^[2] As mentioned in the Active Site section, Heme and thus is the suggested electron acceptor. This suggestion is further supported by the conservation of often found as intermediate electron receptors in biological electron transfer chains.^[2]

Potential Proton Pathways

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 . It is called the . The residues along this pathway help facilitate the movement of the protons. The location of in the structure is a key residue in this pathway. Its location within the pathway and negative charge characteristic implies that this glutamate residue is a redox state-dependent mediator of proton transfer. In other words, it acts like a proton shuttle.^[2] The , which is the last residue in this pathway, could be the protonatable group eventually used upon reduction. More research needs to be done to determine whether the CydA pathway is solely providing protons for charge compensation, or whether can be a branching point that is able to pass protons via the propionates to the oxygen-binding site.^[5]

Another potential entry site is related to the of . Therefore, this is called the . In this pathway, is thought to be the equivalent of the 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 , and therefore, the is the most accepted source of protons.

Overall Oxygen 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 . 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 utilize amino acids with charges that help shuttle the protons from the intracellular side of the protein to . in the active site. The passes through the , and is shown in purple in Figure 4. The proceeds through the , 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 and gets oxidized giving 4e^- to . Once at the 4e^- will be shuttled directly to 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 , 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.

Structure Similarity to bd oxidase found in E. coli

The structure of bd oxidase for Geobacillus thermodenitrificans is highly similar to the structure of bd oxidase in E. coli 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]