Monday, June 3, 2019

Electron Transport Chain in Respiratory Complex I

Electron Transport Chain in Respiratory interwoven IIntroductionEvery organism depends on sinew to survive, in order to principal(prenominal)tain an organized read, homeostasis, finished metabolism and different biochemical reactions. Energy is generated in a number of different ways depending on the organism. Mammals create energy through the breakdown of organic tittles, such as carbohydrates, proteins and lipids, that yields other compounds that drives cellular processes. One such compound is adenosine triphosphate (Adenosine triphosphate) an essential energy-carrying mote that is synthesised by respiration through a series of enzyme protein daedales set up in the mitochondria. Complex I (NADHubiquinone oxidoreductase) is one of those essential protein multiform embedded in mammalian mitochondria. NADH produced by the Krebs tricarboxylic acid cycle and - oxidisation of fatty acids, is oxidate to initiate the mechanistic pathway of Complex I, ultimately reducing ubiquin one and tack proton-motive depict crossways the inner membrane of the mitochondria. It is this proton gradient that leave support the generation of ATP from ATP synthase and other core processes.Signifi bungholet research has been conducted on Complex I, particularly from Bovine heart mitochondria, however to date many aspects of this enzyme is facilitate poorly understood due to its complex structural arrangement and pathways undertaken. To decipher its mechanism, will eventually lead to a greater scaning in the role of Complex I in many diseases and dysfunctions.MitochondriaMitochondria ar small sub-cellular organelles involved in a series of processes primarily with its role in the respiratory system. Occupying almost 10% to 30% of cell volumes of sizes ranging among 0.75 and 3m, the unique shape of a mitochondrion allows the process to take place, with its key structural feature being a double membrane.1 These two membranes atomic number 18 separated by the intermembr ane space and general butt in the central ground substance. Whereas the verbotener membrane is inundated by porins to facilitate the movement of solutes of about 12 kDa or less the inner membrane is impermeable to solutes but presents the ideal environment for the establishment of an electrochemical proton gradient, by the front end of numerous protein complexes.Additional compartments of the organelle include the cristae and the mitochondrial matrix, which comprises a plethora of enzymes involved in ATP metabolism.Additionally, a range of studies have also indicated the ability of mitochondria to unionize dynamic networks of interconnected tubules that regulates the cell structure to adapt to its specific function when required. As a result, during disruption of such networks, cellular dysfunction can occur, leading to a number of neural related syndromes such as Parkinsons and Alzheimers.2,3 Aside from the base role of energy metabolism, the mitochondria also power other co re cellular functions such as apoptosis, calcium handling and the constitution of iron southward globs.The pastime sections discuss the main enzymes involved in the negatron transport range of a function that lead to the generation of ATP, particularly respiratory complex I, which will be the main focus of this thesis.Respiratory ComplexesComplex IIAlso cognize as succinate ubiquinone oxidoreductase, complex II is a 120 kDa enzyme lie downing of four nuclear-encoded subunits which are arranged in two bailiwicks.4 It is this distinctive arrangement which allows this enzyme to oxidise succinate to fumarate which is coupled to the production of ubiquinol through the reducing of ubiquinone in the mitochondrial inner membrane. While it is involved with cofactors, this enzyme complex does not directly contribute to the proton motive force in order to establish a chemical gradient.4,5Succinate+ Q Fumarate + QH2 par 1Two of the enzymes subunits SdhA and SdhB form a deliquescent, succinate dehydrogenase subcomplex and forms the succinate/fumarate binding put whereas SdhB contains three iron-sulphur clusters which are embedded to the mitochondrial membrane by the remaining SdhC and SdhD subunits.4 These latter subunits contain a heam group and ubiqionone binding places. When a flavin dinucleotide, which is ligated to SdhA, it oxidises succinate, the negatrons produced in this process are passed down through the iron-sulphur clusters. The negatrons subsequently allow the reduction of ubiquinone to ubiquinol.6,7Complex terceComplex common chord or ubiquinolcytochrome c oxidoreductase is an 240 kDa enzyme which is make up of 11 subunits. Its structure comprises of two ubiquinone binding sites Qo, present towards the mitochondrial membrane, catalyses the oxidation of ubiquinol to ubiquinone and Qi, present towards the matrix, catalyses the reduction of ubiquinone to ubiquinol.8,9Complexes I and II produces ubiquinol from the reduction of ubiquinone, which binds to the Qo site on complex III. During this process, an electron is passed along the iron-sulfur cluster reducing it and moving it towards cytochrome c1 and cytochrome c resulting in a compliancyal change. The change ca usances a second electron to be headred through another pathway formed of cytochromes bL and bH towards to Qi binding site, in where it allows the formation of a semiquinone anion through the reduction of an already bound ubiquinone. analogue to this, a second quinol is oxidised at Qo allowing the electron to be transferred through the first pathway of Rieske iron-sulphur cluster and cytochrome c1 and the second electron follows the second pathway mentioned to a higher place to Qi, reducing the semiquinone anion to ubiquinol.10 The oxidation at Qo releases four protons into the inter-membrane space of the mitochondria and the reduction at Qi results in the uptake of two protons from the matrix which are transferred into the inter-membrane space during ubiquin ol oxidation. This have it off cycle allows the reduction of two cytochrome c molecules.9QH2 + 2 cyt c3+ + 2H+in Q + 2 cyt c2+ + 4H+outEquation 2Oxidation and reduction cycles in Complex III results in the movement of four protons into the inter-membrane space maintains the proton motive force used by ATP synthase to synthesise ATP.8Complex IVComplex IV, also know as cytochrome c oxidase, is an enzyme, which comprises of 13 subunits, of which three are encoded by the mitochondrial genome. The enzyme catalyses the oxidation of cytochrome c which leads to the reduction of group O to water allowing the translocation of four protons across the mitochondrial inner membrane.11,12The oxidation of cytochrome c produces electrons that are transferred to an active site where molecular oxygen is cut back. This reduction producing water releases free energy required for the pumping of four protons from the matrix of the mitochondria into its inner-membrane space. This movement of protons is facilitated through two know proton channels the K-channel passes two protons for the reduction of oxygen and the D-channel allows the movement of newly translocated protons.13O2 + 4 cyt c2+ + 8H+in 2 H2O + 4 cyt c3+ + 4H+outEquation 3The translocated protons and the reduction of oxygen to water allows ATP synthase to generate ATP as this contributes to the proton motive force similar to Complex III.Complex V in the main known as ATP Synthase, this enzyme complex operates by utilising the proton chemical gradient established in the intermembrane space by the preceding complexes, to drive the synthesis of ATP from ADP and inorganic Phosphate. With an average size of 580 kDa, the enzyme is composed of 16 subunits organised in two aquaphobic and deliquescent domains the hydrophobic domain forms a proton semiconducting focalize through the inner membrane bandage the hydrophilic domain, containing three copies of and subunits, spreads into the matrix. The two domains are linked b y an asymmetric central stalk and a circumferential stalk, which acts as a stator to prevent the F1 domain rotating freely during contact action. The interfaces between the two subunits forms the binding sites for ADP and inorganic Phosphate. 14,15ADP + P+ nH+in ATP + nH+outEquation 4Complex IComplex I, is the first and largest enzyme involved the electron transfer chain of the mitochondrion. Alternatively known as NADHubiquinone oxidoreductase, its primary role is to oxidise NADH and ultimately reduce ubiquinone.16NADH + H+ + Q + 4H+in NAD+ + QH2 + 4H+outEquation 5Just like the other protein complexes, the potential energy released from the redox reaction indoors the complex, translocates four protons across the inner membrane for every molecule of oxidized NADH and removes two additional protons from the matrix for the reduction of quinone. The processes contribute to the overall electrochemical gradient which is to be used by ATP synthase to synthesise ATP.17StructureTo date , complex I has been found in a variety of neologisms, including many prokaryotes. The complex I from bovine heart mitochondria is primarily used in studies due to its close sequential identity with the clement complex I enzyme. The mammalian complex I is one of the most complex and largest enzymes known, with a combined mass of 980 kDA and composed of at least 45 different polypeptide subunits with 14 strictly conserved core subunits that are necessary for function and also common across the among all known complex I.16 The importation of the additional subunits in complex I among different species still remain a secret. It is known some be involved in protection against activated oxygen species generation and some are required needed for proper assembly and stability of the enzyme.16,18As observed by single-particle electron microscopy (EM) for some(prenominal) bacterial and mitochondrial enzymes, the determined structure of the enzyme closely resembles to an L shape, with seven hydrophobic core subunits that constitutes the membrane tail domain and seven hydrophilic core subunits that constitutes peripheral (hydrophilic) ramp up domain protruding into the mitochondrial matrix which is known as the catalytic domain as it includes all redox centres and binding site while the membrane domain consists mostly of hydrophobic subunits. 16While the full structure of the eukaryotic complex is not still well characterised, in 2006, Sazanov group successfully report structure of the hydrophilic domain of complex I from Thermus thermophiles bacteria.20The Peripheral Arm of complex IThe peripheral arm of the complex is composed of seven soul subunits, that together, houses the NADH-oxidizing dehydrogenase module, which provides electron input into a noncovalently-bound flavin mononucleotide (FMN) molecule. The molecule sequentially transfers the electron to a chain of nine iron-sulphur (Fe-S) clusters, eight of which are found in the bovine enzyme. Additionally , the hydrophilic arm also comprises of a Q-module, which conducts electrons to the quinone-binding site for quinol production. 16,20All of theseWithin the respiratory chain complexes, thither are three different types of Fe-S clusters, two of which, are found in complex I Two binuclear 2Fe-2S and six tetranuclear 4Fe-4S clusters.As the name suggests, the binuclear clusters are composed of two iron atoms that function as bridged by two acid-labile sulphur atoms. Each iron atom is also coordinated by an additional two sulphur atoms found on the skirt cysteine residues from the protein complex. In the tetranuclear Fe-S clusters, four iron atoms and four sulphur atoms are arranged in a cube with each iron atom also ligated to sulphur cysteine-residue on the surrounding protein, similar to binuclear Fe-S.22Due to their conformational arrangements and redox capabilities provided by the iron atom, these clusters act as electron transfer agents or also known as ferrodoxins. The detection of these clusters can be achieved by EPR (electron paramagnetic resonance) which is successfully achieved in many studies. However, out of the two binuclear and six tetranuclear iron-sulfur clusters found in complex I, only two binuclear and four tetranuclear clusters are EPR active.22Figure 1. structures of the iron-sulphur clusters found in complex I.As previously mentioned, seven of the eight clusters, form a 95 -long extensive chain directly from the flavin site to the quinone binding site on the interface of the membrane domain. Even though the distances between these chains may seem far apart, as much as 14 , distances are close enough to allow electron transfer to occur.23,24However, the presence of the eight cluster is still not well understood. Cluster 2Fe24 found on the opposite side of the Flavin site, is believed not to be involved in electron transfer pathway. While it was just a theory with no rise, it has been proposed that this additional cluster functions as an ele ctron store that accepts an electron from the flavosemiquinone species preventing the generation of reactive oxygen species during enzyme turnover.24Membrane Domain of complex IThe membrane domain comprises the proton-translocating module which catalyses proton transport. With the exception of subunit ND1 and the quinone binding site, found on the interface of the peripheral arm, the membrane domain functions totally independently from the two arms of complex I.Within the membrane domain, there are four structural subunits that have been determine to be possibly involved with proton translocation these include subunits ND2, ND4 and ND5. There is also an additional transporter which believed to be either ND1, ND6 or ND4L. Each believed to be transporting one proton per catalytic cycle. Each individual subunits are composed of charged residues and helices that creates half-channels that allow the passage of proton to occur. The membrane structure is also held together by a long -heli x chain that spans across its entire length. Its feature is to maintain and support the integrity of the membrane domain.26Overall Mechanism of complex IThe mammalian complex I includes 45 known proteins, out of which 14 core subunits comprises of both hydrophilic and hydrophobic domains as explained above.16The mechanism through the electron transfer chain starts with a Flavin mononucleotide (FMN) molecule which is non-covalently bound to the 51kDa subunit through hydrogen bonds at the top of the hydrophilic domain. FMN molecule oxidises NADH leading to the reduction of iron-sulphur clusters (Fe-S) which transfers electrons from Flavin to the quinone-binding site 51. This electron transfer distorts the conformation of the protein through changes in its redox state leading to alterations in pKa values of its side chains these alterations allows four hydrogen ions being pumped out of the mitochondrial matrix.24It is believed NADH gets oxidised to NAD+ through a hydride transfer avoid ing the formation of the unstable NAD. Radical.24 This oxidation process occurs when the nicotinamide ring of the NADH lies above the flavin isoalloxazine system, allowing the electron donor hydride (C4 of the 27 nicotinamide ring) and acceptor (N5 of the flavin) to come in spite of appearance 3.5 of each other and transfer electrons.28As explained above, NADH oxidation leads to transfer of electrons through seven iron-sulphur clusters chain between Flavin and quinone reduction binding site in the membrane.20 It is the final Fe-S cluster that donates the electrons to the bound ubiquinone substrate which is believed to be accessed through an entry point in the membrane to the binding site.21These iron-sulphur clusters are best spy using a technique called electron paramagnetic resonance (EPR). Previous studies have observed five reduced Fe-S clusters through EPR from Bovine compliex I reduced by NADH, and their spectra are represented N1b, N2, N3, N4 and N5.25 This technique will be further explained throughout this thesis.A much recent field by Roessler et al. (2010) used EPR to understand the tunnelling electron transfer pathway through these clusters. Previous studies have already established EPR signals N1b, N2 and N3 are detected from 2Fe cluster in the 75 kDa subunit (position 2), and from 4Fe clusters in the PSST (position7) and 51 kDa subunits (position 1) respectively along the clusters chain due to interactions with ubisemiquinones and flavosemiquinone. As the other EPR signals have yet failed to be assigned to a particular cluster, Roessler et al. (2010) went on to use double electron-electron resonance (DEER) spectroscopy to detect N4 and N5. Their results demonstrate that N4 is assigned to the first 4Fe cluster in the TYKY subunit (position 5), and N5 to the all-cysteine ligated 4Fe cluster in the 75 kDa subunit (position 3).25The study propose an alternating energy potential profile for electron transfer along the chain between the actives sit es, in B.taurus, which enhances the rate of a single electron travelling through the empty chain subsequently leading to more efficient energy conversion in complex I.25Followed by the iron-sulfur cluster is the site of quinone reduction. A study performed by Sazanov and Hinchliffe has identified a supposed binding site for the quinone head group from T. thermophilus complex I hydrophilic domain between the 49 kDa and PSST subunits.20 This alleged site is close to the cluster where the ubiquinone substrate accepts electrons from the chain and it has also been acknowledged the 49 kDa and PSST subunits play an historic role in quinone binding and catalysis.29Nevertheless, it is believed that additional hydrophobic subunits may also be involved in quinone binding and these are still being investigated.Even though the mechanism of NADH oxidation and ubiquinone reduction is relatively well understood, how this oxidoreduction leads to quinone reduction and subsequent protons pumping acro ss the mitochondrial membrane from complex I still remain a mystery. A number of theories for complex I mechanism have been proposed based on the proton-pumping systems of the other mitochondrial respiratory complexes. These theories have been outlined belowA direct coupling mechanism as demonstrated by complex IV through cytochrome c oxidase where the proton transfer is determined by a gating reaction occurring at the same time as the electron transfer reaction that started it.30An indirect coupling mechanism as seen in complex V (ATP synthase) explained previously. A study performed by Efremov et al., suggests that within complex I, one proton is translocated by a directly coupled mechanism at the Fe-S clusters and the rest are moved when quinone reduction drives conformational changes to the four-helix bundle of Nqo4 and of Nqo6 in complex I, subsequently affecting the C-terminal helix of Nqo12. The C-terminal has been identified by the authors running agree to the membrane. T he effect on this helix consequently leads to the other three helices to tilt which results in proton translocation.31A Q-cycle-like mechanism as represented by complex III where quinol is used as a carrier to transport protons across the mitochondrial membrane. A study completed by Dutton and co-workers suggested the complete reverse of this mechanism for complex I featuring the presence of two ubiquinone binding sites one facing the inter-membrane space, Qo, and the other facing the mitochondrial matrix, Qi. The quinone substrate would bind at Qi, and be reduced by one electron from a quinol already bound at Qo and another electron from the Fe-S cluster subsequently leading to two protons being taken up from the matrix while the formed semiquinone specie is still bound at Qo. Following the uptake of the protons, semiquinone is oxidised to ubiquinone.32 Nevertheless, further studies conducted have found no evidence of ubiquinol oxidation signifying complex I do not work through th is mechanism.30,33While the first isolation of complex I from bovine heart mitochondria by Joe Hatefi et al occurred 40 years ago, information on its overall mechanism of action is still very limited particularly the mechanism of redox-proton coupling occurring in the membrane domain. To further understand this, new studies are being conducted to trap ancestor intermediates formed at the interface of the peripheral and membrane arm to establish the pathway that initiates proton translocation.Semiquinone originsSemiquinones are catalytic intermediates formed within complex I during the reduction of quinones at the quinone binding site and can exist in neutral or anionic form. Due to the presence of the unpaired electron, semiquinone intermediates can be studied using EPR spectroscopy.There are numerous pathways in which the formation of semiquinones can occur from quinone. The scheme below, proposed by Roessler and Hirst, illustrates the three main possible routes taken to obtain q uinol.Pathways A and B involves with the generation of a neutral semiquinone radical specie based on the transferring of a proton and electron. On the other hand, pathway C which follows through pathway B involve with the generation of an anionic radical specie generated from an electron transfer. All pathways lead to formation of quinol by series of electron transfer and protons. The pathway shown in grey which occurs from the protonation of the neutral semiquinone radical specie will result in a 1-electron-2-centre bond which are energetically unstable.27Aside from one study, majority of the studies till date, have proved the existence of semiquinones by observing EPR signals using submitochondrial particles (SMPs). As the name suggests, these are inverted membrane vesicles housing the entire electron transport chain containing all enzyme complexes.34 However, since quinone cofactors are used by majority of the other complexes, distinguishing the semiquinone signals with each comp lex, has been far from successful.More recently, there has been a wave of research focalization on the identification of semiquinone radicals only if from complex I, however these have proved even more challenging as the organic intermediates produced very low intensity signals.Within complex I, there are two species of semiquinone that have been identified SQNf and SQNs.35,36 Based on their EPR properties, SQNf or fast relaxing semiquinones has been reported only during the presence of an established proton gradient across the membrane. On the other hand, SQNs or slow relaxing semiquinones, are not effected by proton gradient. The presence of two semiquinones has also lead to the possibility of complex I to contain two separate quinone binding sites Due to SQNf having a spin-spin interaction with Fe-S cluster N2, it is theorised that SQNf binding site is located close to the cluster at around 12 estimated distance, in contrast, SQNs binding site is suggested to be located around 30 from N2 cluster.22,25,37Within the complex, the SQNf is believed to be involved in proton pumping and its site aids the system by acting as bound co-factor site that facilitates the transfer of one electron from one site to another allowing the formation of a binding pocket for the SQNs in equilibrium with the ubiquinone pool of the membrane.22,25,32,35,38The presence of two separate quinone binding sites still remains a mystery and cannot be totally ruled out even though it has been suggested that SQNf and SQNs signals are detected from the same semiquinone species located from different sites or present in catalysis states.39A recent potential way of observing semiquinone intermediates via EPR is through the use of liposomes. Liposomes containing just Complex I or proteoliposomes, will facilitate the capture of semiqinone within its native environment and hopefully provide an insight in the mechanism of Complex I and the binding of Q10.LiposomesLiposomes are spherical nanoves icles used in a variety of applications. Composed of a phospholipid bilayer, these small vesicles have an aqueous solution core surrounded by a hydrophobic membrane. Hydrophobic chemicals associate with the bilayer while the hydrophilic solutes dissolved in the core cannot readily pass through the bilayer essentially mimicking the cellular phospholipid bilayer. Due to these features, liposomes can be incubused both with hydrophobic or hydrophilic molecules and are excellent drug carriers or in this case house protein complexes. Liposomes are also not naturally occurring and must be artificially generated using lipid extracts by aggregating them.40As liposomes are formed from naturally occurring lipids of low intrinsic toxicity, they are biodegradable and non-toxic. The functionality of liposomes is dependent based on three main factors. These include size, bilayer composition and liposome surface properties.40Phospholipids are one the essential components in the formations of lipos omes and can be divided into synthetic and natural phospholipids. They consist of two fatty acids hydrophobic chains linked to a hydrophilic (polar) head group, and they have either glycerol or sphingomyeline as the back bone. Having both hydrophobic and hydrophilic components, make phospholipids having amphipathic molecules.41 The diversity of the hydrophilic head group molecules and hydrophobic chains length allows the formation of different phospholipids which affects the surface charge and bilayer permeability of the liposomes.40The length and degree of saturation of the hydrocarbon acyl chains determines the stability of the liposomal membrane, by affecting the temperature at which the membrane changes from a closely packed gel human body to a fluid phase. The surface charge of the liposomes is determined by the charge of the lipid forming it which can be altered by modifying lipids with hydrophilic moieties to membrane bilayers.40Liposomes can be composed of naturally-derived phospholipids such as cholesterol, one of the commonly used lipids in liposome formation. It enhances the stability of the lipid bilayer and form highly ordered and rigid membrane with fluid like characteristics. Other phospholipids, synthetic and non-synthetic, can also be used for the formation of the liposomes such as pure wetting agent components like DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).42Classifications of liposomesLiposomes are classified according to their morphological sizes and lamellarity, depending on their composition and method of formation.40Multilamellar vesicles (MLVs) consists several concentric phospholipid bilayers or lamellar ranging between light speednm to 20 m in size depending on the method of preparation. These large bilayers allow the integration of lipophilic molecules and proteins.Small unilamellar vesicles (SUVs) single phospholipid bilayer and sized between 20 nm to 100nm. Ideal for encapsulation small compounds and proteins.Large unila mellar vesicles (LUVs) single phospholipid bilayer with size ranging from 100 nm to 1 m. They are known to have larger aqueous core compared with or MLVs, making them suitable to useful to load with numerous compounds.Oligolamellar vesicles (OLVs) vesicles similarly structured to MLVs but consists of anywhere between two and five phospholipid bilayers.Multivesicular liposomes (MVLs) When a large liposome vesicle similar in size to an MLV, enclose a group of liposomes, then the subsequent vesicle is known as multivesicular liposome (MVL).Figure 1.40The current state of research on liposomes have primarily been focusing on the administration of drugs and other compounds to biological systems since it overcome challenges associated with reaching the target, making them very useful in the cosmetic and pharmaceutical industries.40Furthermore, it should be noted, some surfactant based phospholipids can mimic the biological systems helping construct important model systems for the resea rch on enzymes and membranes. Many recent publications concerning liposomes have been focused on using this mimetic chemistry, which deals with models, mimicking cellular membrane to facilitate the research into their structures as well as the mechanisms both in vivo and in vitro.40Aims of ProjectThe current state of research on complex I remain largely focused on the determination of the mechanism since only a fraction has been found. Fully agreement will help solve many diseases and other complication caused by complex I.Whereas the mechanism of the reactions between NADH and iron sulphur clusters have been established, little is known about the mechanism of proton translocation as well as the role and existence of semiquinones that will lead into revealing more information into the function of the enzyme. The work depict in the following records, using the best technique available, EPR, will aim to be using current studies of using liposomes to mimic cellular conditions, simila r to the mitochondrial membrane, for complex I in order to obtain data regarding reduction of Q10 and proton translocation.MaterialsPreparation of Complex I from Bovine MitochondriaPreparation of Complex I proteoliposomes Stock solutions of 25 mgmL-1 of POPC in chloroform was transferred to a glass homogeniser with the required amount of ubiquinone-10 contained in chloroform. The chloroform was removed under Argon. An alternative approach is to remove under vacuum cleaner using rotary evaporator. The resulting phospholipid film was resuspended in 675 L of buffer (10 mM Tris-SO4 (pH 7.5) and 50 mM KCl), and extruded 25 times through a Whatman 0.1 m pore membrane. The liposome mixture was solubilised with the addition of 160 L of octyl-glucoside from an aqueous 10% stock solution, sonicated for 10 min, and further incubated on ice for 10 min. The following steps were carried out at 4 C. 0.2 mg of AOX (50 L of 7.8 mgmL-1) and 0.2 mg of complex I (10 L of 20 mgmL-1) were added to the s olubilised lipids and incubated for a further 10 min, followed by the addition of 100 L of SM2 Biobeads. The mixture wa

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