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 <scene name='83/832931/Full/4'>Cytochrome bd oxidases</scene> are quinol-dependent [https://en.wikipedia.org/wiki/Transmembrane_protein transmembrane] (Fig. 1) terminal [https://en.wikipedia.org/wiki/Oxidase oxidases] found exclusively in [https://en.wikipedia.org/wiki/Prokaryote prokaryotes].<ref name="Safarian">PMID: 27126043</ref>  With a very high oxygen affinity, bd oxidases play a vital role in the [https://en.wikipedia.org/wiki/Oxidative_phosphorylation oxidative phosphorylation] pathway in both gram-positive and gram-negative bacteria. Cytochrome ''bd'' oxidase's responsibility in the oxidative phosphorylation pathway also allows it to act as a key survival factor in the bacterial stress response against antibacterial drugs <ref name="Safarian">PMID: 31604309</ref>, hypoxia, cyanide, [https://en.wikipedia.org/wiki/Nitric_oxide nitric oxide], and H<sub>2</sub>O<sub>2</sub><ref name="Harikishore">PMID: 31939065</ref>. With their essential roles in bacterial survival, ''bd'' oxidases have been pursued as ideal targets for antimicrobial drug development. <ref name="Boot">PMID: 28878275</ref>
 [[Image:Transmembrane_bd_ox.png|550 px|center|thumb|''Figure 1''. Cartoon model of cytochrome bd-oxidase in ''E. coli''. Dashed lines represent borders of [https://en.wikipedia.org/wiki/Cytoplasm cytoplasmic] and [https://en.wikipedia.org/wiki/Periplasm periplasmic] regions.  A quinol bound in the periplasmic <scene name='83/832924/Q_loop/3'>Q-loop</scene> is [https://en.wikipedia.org/wiki/Redox oxidized] and releases protons into the periplasmic space, generating a [https://en.wikipedia.org/wiki/Electrochemical_gradient proton gradient].  Protons and oxygen atoms from the cytoplasmic side enter cytochrome ''bd'' oxidase through specific channels.  Oxygen is [https://en.wikipedia.org/wiki/Redox reduced] to water, which is released into the cytoplasmic space.  Blue = CydA; green = CydB; yellow = CydX; pink = CydS.  [[https://www.rcsb.org/structure/6RX4 PDB: 6RX4]]]]
-The overall mechanism of ''bd'' oxidases involves an exergonic [https://en.wikipedia.org/wiki/Dioxygen_in_biological_reactions reduction of molecular oxygen] into water (Fig. 2). During this reaction, a proton gradient is generated in order to assist in the conservation of energy. <ref name="Belevich">PMID: 17690093</ref> Unlike other terminal oxidases, bd oxidases do not use a proton pump. Instead, bd oxidases use a form of vectorial chemistry that releases protons from the quinol oxidation into the positive, periplasmic side of the membrane. Protons that are required for the water formation are then consumed from the negative, cytoplasmic side of the membrane, thus creating the previously mentioned proton gradient.
+The overall mechanism of ''bd'' oxidases involves an exergonic [https://en.wikipedia.org/wiki/Dioxygen_in_biological_reactions reduction of molecular oxygen] into water (Fig. 2). During this reaction, a proton gradient is generated in order to assist in the conservation of energy. <ref name="Belevich">PMID: 17690093</ref> Unlike other terminal oxidases, bd oxidases do not use a proton pump. Instead, bd oxidases use a form of vectorial chemistry that releases protons from the quinol oxidation into the positive, periplasmic side of the membrane. Protons that are required for the water formation are then consumed from the negative, cytoplasmic side of the membrane, thus creating the proton gradient.
-[[Image:proton graadient.jpg|300 px|left|thumb|Figure 2: Overall schematic representation of the reductive cycle of cytochrome bd oxidase. <ref name= "Giuffre">PMID: 24486503</ref>; In this cycle, molecular oxygen is reduced  into water using the quinol as a reducing substrate. The three hemes essential to the electron transfer are located near the periplasmic space. The membrane potential is generated mainly from proton transfer from the cytoplasm towards the active site on the periplasmic side of the membrane. Heme b<sub>558</sub> is involved in quinol oxidation and heme d serves as the site where O<sub>2</sub> binds and becomes reduced to H<sub>2</sub>O.]]
+[[Image:proton graadient.jpg|550 px|center|thumb|''Figure 2''. Overall schematic representation of the reductive cycle of cytochrome bd oxidase. <ref name= "Giuffre">PMID: 24486503</ref> In this cycle, molecular oxygen is reduced into water using the quinol as a reducing substrate. Cytochrome ''bd'' oxidase releases 2 H<sup>+</sup> for each 2 electrons transferred due to the menaquinol oxidation site located on the outer face of the cytoplasmic membrane. <ref name="Fischer">PMID: 29784883</ref> The ''bd'' oxidase completes a redox loop when coupled with quinone [https://en.wikipedia.org/wiki/Dehydrogenase dehydrogenases] that receive electrons from [https://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NADH], [https://en.wikipedia.org/wiki/Pyruvic_acid pyruvate], [https://en.wikipedia.org/wiki/Lactic_acid D-lactate], or [https://en.wikipedia.org/wiki/Acyl-CoA acyl coenzyme A]. The three hemes essential to the electron transfer are located near the periplasmic space. Heme b<sub>558</sub> is involved in quinol oxidation and heme d serves as the site where O<sub>2</sub> binds and becomes reduced to H<sub>2</sub>O. The membrane potential is generated mainly from proton transfer from the cytoplasm towards the active site on the periplasmic side of the membrane.]] This page will focus on the structure and overall function of the ''bd'' oxidase in [https://en.wikipedia.org/wiki/Escherichia_coli ''E. coli'']. This ''bd'' oxidase is part of the long(L) quinol-binding domain subfamily of terminal oxidases. The L-subfamily of ''bd'' oxidases are responsible for the survival of acute infectious diseases such as ''E. coli'' and [https://en.wikipedia.org/wiki/Salmonella ''Salmonella'']. The cytochrome ''bd'' oxidase's three <scene name='83/832931/Heme/4'>heme</scene> groups, its periplasmically exposed <scene name='83/832924/Q_loop/3'>Q-loop</scene>, and <scene name='83/832942/Four_subunits_labelled_6rx4/3'>four protein subunits</scene> will be the primary focus when explaining how the structure of ''bd'' oxidase allows it to catalyze the reduction of molecular oxygen into water.
-This page will focus on the structure and overall function of the ''bd'' oxidase in [https://en.wikipedia.org/wiki/Escherichia_coli ''E. coli'']. This ''bd'' oxidase is part of the long(L) quinol-binding domain subfamily of terminal oxidases. The L-subfamily of ''bd'' oxidases are responsible for the survival of acute infectious diseases such as ''E. coli'' and [http://www.example.com ''Salmonella'']. The 6RX4's three <scene name='83/832931/Heme/4'>heme</scene> groups, its periplasmically exposed <scene name='83/832924/Q_loop/3'>Q-loop</scene>, and <scene name='83/832942/Four_subunits_labelled_6rx4/2'>four protein subunits</scene> will be the primary focus when explaining how the structure of ''bd'' oxidase allows it to catalyze the reduction of molecular oxygen into water.
 ==Structure==
 === Subunits ===
-Cytochrome ''bd'' oxidase is made up of four individual subunits.<ref name="Alexander">PMID:31723136</ref> The two major subunits, CydA and CydB, are each composed of one peripheral [https://en.wikipedia.org/wiki/Alpha_helix helix] and two bundles of four [https://en.wikipedia.org/wiki/Transmembrane_protein transmembrane] helices. The <scene name='83/832924/Cyda_subunit/6'>CydA subunit</scene> plays the most important role in the oxygen [https://en.wikipedia.org/wiki/Redox reduction reaction] as it contains the Q-loop as well as all three [https://en.wikipedia.org/wiki/Heme heme] groups. The <scene name='83/832924/Cydb_subunit/2'>CydB subunit</scene> harbors the <scene name='83/832924/Ubiquinone/3'>ubiquinone</scene> molecule which provides structural support to the subunit that mimics the three hemes found in CydA.<ref name="Safarian">PMID: 31604309</ref><ref name="Safarian2">PMID: 27126043</ref> The remaining two subunits, CydS and CydX, are both single helix structures that assist in the oxygen reduction reaction. Unique to ''E. coli'', the <scene name='83/832924/Cyds_subunit/4'>CydS subunit</scene> binds to CydA to block oxygen from directly binding to heme b<sub>595</sub>. The <scene name='83/832924/Cydx_subunit/4'>CydX subunit</scene> promotes the assembly and stability of the oxidase complex. CydX is composed of 37 mostly hydrophilic [https://en.wikipedia.org/wiki/Amino_acid amino acid] residues, including <scene name='83/832924/Glu25/2'>Glu25</scene> that is exposed to the cytoplasm and prevents the helix from fully entering the membrane. <ref name="Alexander">PMID:31723136</ref>
+Cytochrome ''bd'' oxidase is made up of four individual subunits.<ref name="Alexander">PMID:31723136</ref> The two major subunits, CydA and CydB, are each composed of one peripheral [https://en.wikipedia.org/wiki/Alpha_helix helix] and two bundles of four [https://en.wikipedia.org/wiki/Transmembrane_protein transmembrane] helices. The <scene name='83/832924/Cyda_subunit/7'>CydA subunit</scene> plays the most important role in the oxygen [https://en.wikipedia.org/wiki/Redox reduction reaction] as it contains the Q-loop as well as all three [https://en.wikipedia.org/wiki/Heme heme] groups. The <scene name='83/832924/Cydb_subunit/4'>CydB subunit</scene> harbors the <scene name='83/832924/Ubiquinone/5'>ubiquinone</scene> molecule which provides structural support to the subunit that mimics the three hemes found in CydA.<ref name="Safarian">PMID: 31604309</ref><ref name="Safarian2">PMID: 27126043</ref> The remaining two subunits, CydS and CydX, are both single helix structures that assist in the oxygen reduction reaction. Unique to ''E. coli'', the <scene name='83/832924/Cyds_subunit/6'>CydS subunit</scene> binds to CydA to block oxygen from directly binding to heme b<sub>595</sub>. The <scene name='83/832924/Cydx_subunit/6'>CydX subunit</scene> promotes the assembly and stability of the oxidase complex. CydX is composed of 37 mostly hydrophilic [https://en.wikipedia.org/wiki/Amino_acid amino acid] residues, including <scene name='83/832924/Glu25/4'>Glu25</scene> that is exposed to the cytoplasm and prevents the helix from fully entering the membrane. <ref name="Alexander">PMID:31723136</ref>
 ===Q-Loop===
-Another significant structural feature of bd oxidase is the <scene name='83/832924/Q_loop/3'>Q-loop</scene> which is located between TM helices 6 and 7 of the CydA subunit.<ref name="Alexander">PMID:31723136</ref> The periplasmic Q-loop in ''E. coli'' stretches over a length of 136 amino acid residues, making it much longer than the Q-loop in [https://en.wikipedia.org/wiki/Geobacillus_thermoglucosidasius ''Geobacillus Thermodenitrificans''].<ref name="Safarian">PMID: 27126043</ref> With five helices acting as a flap covering heme b595, the Q-loop is likely involved in [https://en.wikipedia.org/wiki/Hydroquinone quinol] binding and oxidation. The [https://en.wikipedia.org/wiki/N-terminus N-terminal end] of this Q-loop is very flexible and likely functions as the hinge that allows for quinone binding while the [https://en.wikipedia.org/wiki/C-terminus C-terminal end] is much more rigid which provides stabilization for the enzyme.<ref name="Alexander">PMID:31723136</ref>
+Another significant structural feature of bd oxidase is the <scene name='83/832924/Q_loop/3'>Q-loop</scene> which is located between TM helices 6 and 7 of the CydA subunit.<ref name="Alexander">PMID:31723136</ref> The periplasmic Q-loop in ''E. coli'' stretches over a length of 136 amino acid residues, making it much longer than the Q-loop in [https://en.wikipedia.org/wiki/Geobacillus_thermoglucosidasius ''Geobacillus Thermodenitrificans''].<ref name="Safarian">PMID: 27126043</ref> With five helices acting as a flap to cover heme b<sub>558</sub>, the Q-loop is likely involved in [https://en.wikipedia.org/wiki/Hydroquinone quinol] binding and oxidation. The <scene name='83/832924/Q_loop_n-terminus/1'>N-terminal end</scene> of this Q-loop is very flexible and likely functions as the hinge that allows for quinone binding while the <scene name='83/832924/Q_loop_c-terminus/1'>C-terminal end</scene> is much more rigid which provides stabilization for the enzyme.<ref name="Alexander">PMID:31723136</ref>
 == Molecular Function ==
 === H and O channels ===
-[[Image:O_AND_H_CHANNEL.png|300 px|right|thumb|'''Figure 3'''. H and O-channels of cytochrome bd-oxidase in ''E. coli''. Channels are outlined in gray, water is shown as spheres, and relevant amino acids are labeled above.  [[https://www.rcsb.org/structure/6RX4 PDB:6RX4]]]]
+[[Image:O_AND_H_CHANNEL.png|300 px|right|thumb| ''Figure 3''. H and O-channels of cytochrome bd-oxidase in ''E. coli''. Channels are outlined in gray, water is shown as spheres, and relevant amino acids are labeled above.  [[https://www.rcsb.org/structure/6RX4 PDB:6RX4]]]]
 The hydrogen and oxygen channels (Fig. 3) are essential for H<sup>+</sup> and O<sub>2</sub> molecules to reach the active site of cytochrome ''bd'' oxidase. A [https://en.wikipedia.org/wiki/Chemiosmosis#The_proton-motive_force proton motive force] generated by the oxidase<ref name= "Safarian">PMID:31604309</ref> allows protons from the cytoplasm to flow through a hydrophilic <scene name='83/832931/Overall_h_channel/2'>H-channel</scene> full of water (pink dots), entering at <scene name='83/832931/Start_of_h_channel/2'>Asp119<sup>A</sup></scene> and moving past <scene name='83/832931/Start_of_h_channel/2'>Lys57<sup>A</sup>, Lys109<sup>B</sup>, Asp105<sup>B</sup>, Tyr379<sup>B</sup>, and Asp58<sup>B</sup></scene><ref name="Alexander">PMID:31723136</ref> where they can be transferred to the active site with the help of the conserved residues <scene name='83/832931/End_of_h_channel/4'>Ser108<sup>A</sup>, Glu107<sup>A</sup>, and Ser140<sup>A</sup></scene><ref name= "Safarian">PMID:31604309</ref>.  A smaller <scene name='83/832931/O_channel_overall/3'>O-channel</scene> also exists that transitions from hydrophobic to hydrophilic as it gets closer to the active site. This channel allows oxygen to reach the active site, starting near <scene name='83/832931/Ochannel/2'>Trp63</scene> in CydB and passing by <scene name='83/832931/Ochannel/2'>Ile144<sup>A</sup>, Leu101<sup>A</sup>, and Glu99<sup>A</sup></scene><ref name= "Safarian">PMID:31604309</ref>, which assists with the binding of oxygen to the active site.  The O-channel channel is approximately 1.5 [https://en.wikipedia.org/wiki/Angstrom Å] in diameter<ref name="Alexander">PMID:31723136</ref>, which may help with [https://en.wikipedia.org/wiki/Chemical_specificity selectivity].
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 Three <scene name='83/832931/Heme/6'>hemes</scene> are present in the <scene name='83/832924/Cyda_subunit/6'>CydA subunit</scene>. These three hemes form a triangle to maximize subunit stability<ref name="Safarian">PMID:31604309</ref><ref name="Alexander">PMID:31723136</ref><ref name="Safarian2">PMID:27126043</ref>, which is an evolutionary conserved feature across bd oxidases<ref name="Safarian">PMID:31604309</ref>.  Heme b<sub>558</sub> acts as the primary [https://en.wikipedia.org/wiki/Electron_acceptor electron acceptor] by [https://en.wikipedia.org/wiki/Catalysis catalyzing] the [https://en.wikipedia.org/wiki/Hydroquinone#Redox oxidation of quinol]<ref name="Alexander">PMID:31723136</ref>. Conserved <scene name='83/832931/Met393/1'>His186 and Met393</scene> help to stabilize heme b558<ref name="Alexander">PMID:31723136</ref>. Heme b<sub>558</sub> [https://en.wikipedia.org/wiki/Electron_transfer transfers] the electrons to heme b595, which transfers them to the active site heme d<ref name= "Safarian">PMID:31604309</ref>.  Multiple residues help stabilzie this electron trasnfer including a conserved <scene name='83/832931/Trp441/6'>Trp441</scene> that assists heme b<sub>595</sub> in transferring electrons to heme d<ref name="Safarian2">PMID:27126043</ref>.  A conserved <scene name='83/832931/Hemeb595/2'>Glu445</scene> is also essential for charge stabilization of heme b<sub>595</sub><ref name="Alexander">PMID:31723136</ref>, while <scene name='83/832931/Hemeh19/3'>His19</scene> stabilizes heme d<ref name="Safarian2">PMID:27126043</ref>. As heme d collects the electrons from heme b<sub>595</sub>, <scene name='83/832931/Heme_d/3'>Glu99</scene> in the O-channel facilities the binding of oxygen to heme d, and <scene name='83/832931/Heme_d/3'>Ser108, Glu107, and Ser140</scene> in the H-channel facilitate proton transfer to heme d<ref name="Safarian">PMID:31604309</ref>. Similar to the three hemes, the <scene name='83/832931/Uq8/3'>ubiquinone-8</scene> (UQ-8) molecule found in the <scene name='83/832924/Cydb_subunit/2'>CydB subunit</scene> mimics the triangular formation to stabilize the subunit<ref name="Safarian">PMID:31604309</ref>.
 ===Mechanism===
-Quinol transfers two electrons to heme b<sub>558</sub> and releases two protons into the periplasmic space as the initial [https://en.wikipedia.org/wiki/Electron_donor electron donor].  <scene name='83/832931/Heme/6'>Heme b558</scene> transfers the electrons to <scene name='83/832931/Heme/6'>heme b595</scene>, which transfers the electrons to <scene name='83/832931/Heme/6'>heme d</scene>.  Concurrently, the <scene name='83/832931/Overall_h_channel/2'>H-channel</scene> collects protons from the cytoplasmic side using the proton gradient generating by quinol and the <scene name='83/832931/O_channel_overall/3'>O-channel</scene> collects oxygen atoms.  The protons and oxygen flow to the active site heme d (Fig. 3).  With electrons, oxygen, and protons available, heme d can successfully reduce dioxygen to water (Fig. 2, 4).  [[Image:mech4.png|500 px|center|thumb|''Figure 4''. General mechanism of cytochrome bd-oxidase in ''E. coli''. Electrons are passed from quinol to heme b<sub>558</sub> to heme b<sub>595</sub> to heme d. Protons and oxygen atoms flow into the H-channel and O-channel to heme d. Heme d catalzyes the reduction of oxygen to water.]]
+A reduced quinol with two electrons received from NADH, pyruvate, D-lactate, or acyl coenzyme A transfers these electrons to heme b<sub>558</sub> and releases two protons into the periplasmic space as the initial [https://en.wikipedia.org/wiki/Electron_donor electron donor].  <scene name='83/832931/Heme/6'>Heme b558</scene> transfers the electrons to <scene name='83/832931/Heme/6'>heme b595</scene>, which transfers the electrons to <scene name='83/832931/Heme/6'>heme d</scene>.  Concurrently, the <scene name='83/832931/Overall_h_channel/2'>H-channel</scene> collects protons and the <scene name='83/832931/O_channel_overall/3'>O-channel</scene> collects oxygen atoms from the cytoplasmic side.  The protons and oxygen flow to the active site heme d (Fig. 3).  With electrons, oxygen, and protons available, heme d can successfully reduce dioxygen to water (Fig. 2, 4).  Using oxygen as the final electron acceptor generates an exergonic reaction that can be coupled with the movement of protons against their gradient when quinol releases two protons into the periplasmic space and when the H-channel uptakes protons from the cytoplasmic side and transfers them to heme d<ref name="Safarian">PMID: 31604309</ref><ref name="Alexander">PMID:31723136</ref>.  [[Image:mech4.png|500 px|center|thumb|''Figure 4''. General mechanism of cytochrome bd-oxidase in ''E. coli''. Electrons are passed from quinol to heme b<sub>558</sub> to heme b<sub>595</sub> to heme d. Protons and oxygen atoms flow into the H-channel and O-channel to heme d. Heme d catalyzes the reduction of oxygen to water.]]
 == Relevance ==
 The cytochrome ''bd'' oxidase is essential for [https://en.wikipedia.org/wiki/Pathogenic_bacteria pathogenic bacteria] to thrive in the human body because it enhances bacterial growth and [https://en.wikipedia.org/wiki/Bacterial_growth colonization].  Any alteration of the ''bd'' oxidase Cyd subunits will most likely produce a nonfunctional [https://en.wikipedia.org/wiki/Mutant mutant] cytochrome ''bd'' oxidase<ref name="Moosa">PMID: 28760899</ref>, which inhibits bacterial growth.  If ''E. coli'' are missing or possess ineffective CydA and B subunits, bacterial growth ceases.<ref name="Hughes">PMID: 28182951</ref>.  With [https://en.wikipedia.org/wiki/Colitis colitis], ''E. coli'' mutants that were missing CydAB colonized poorly in comparison to the [https://en.wikipedia.org/wiki/Wild_type wild type] levels of colonization<ref name="Hughes">PMID: 28182951</ref>.  The cytochrome ''bd'' oxidase is the main component in [https://en.wikipedia.org/wiki/Biological_functions_of_nitric_oxide#Effects_in_bacteria nitric oxide] (NO) tolerance in bacteria, which is released by [https://en.wikipedia.org/wiki/Neutrophil neutrophils] and [https://en.wikipedia.org/wiki/Macrophage macrophages] when the [https://en.wikipedia.org/wiki/Host_(biology) host] is infected<ref name="Shepherd">PMID: 27767067</ref>. ''E. coli'' growth seen in [https://en.wikipedia.org/wiki/Urinary_tract_infection urinary tract infections] is mainly due to the NO resistant ''bd'' oxidase. Without the CydA  and CydB subunits, bacteria could not colonize in high NO conditions<ref name="Shepherd">PMID: 27767067</ref>.  Cytochrome ''bd'' oxidases are essential for life in other pathogenic bacteria such as [https://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''M. tuberculosis''].  Deletion of the CydA and CydB subunits dramatically decreased the growth of ''M. tb'' compared to the wild type when exposed to [https://en.wikipedia.org/wiki/Imidazopyridine imidazo[1,2-]][https://en.wikipedia.org/wiki/Imidazopyridine pyridine], a known [https://en.wikipedia.org/wiki/Enzyme_inhibitor inhibitor] of respiratory enzymes<ref name="Arora">PMID:25155596</ref>.  [https://en.wikipedia.org/wiki/Downregulation_and_upregulation Upregulation] of the cytochrome ''bd'' oxidase Cyd genes resulted in a mutant strain of ''M. tb'' that was [https://en.wikipedia.org/wiki/Antimicrobial_resistance resistant] to imidazo[1,2-α]pyridine<ref name="Arora">PMID:25155596</ref>.