Class Note for BIOC 460 at UA
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Date Created: 02/06/15
Biochemistry 460 Dr Tischler REDUCTION OXIDATIONRESPIRATORY CHAIN Related Reading Chapter 18 502517 in Stryer 639h edition OBJECTIVES 1 De ne the reduction potential difference AE0 and discuss its relationship to the free energy difference AG of such a reaction 2 Describe the essential features of oxidative phosphorylation and the role of reduction oxidation reactions in the electron transport respiratory chain for this process 3 For the respiratory chain major complexes I to IV a name the components that immediately donate electrons to and accept electrons from each b explain how energy from the respiratory chain complexes is conserved and which ones produce suf cient energy to make ATP 4 Explain how pH and charge gradients are formed across the mitochondrial inner membrane DEFINITIONS Oxidative phosphorylation includes the coupling of the oxidation of NADH or FADH2 by the respiratory chain with the synthesis of ATP via a gradient of protons across the inner mitochondrial membrane Substrate level phosphorylation describes the formation of ATP or GTP linked to exergonic chemical reactions in metabolic pathways that provide the energy for phosphorylation Examples discussed previously include phosphoglycerate kinase and pyruvate kinase in glycolysis succinyl CoA synthetase in the citric acid cycle and creatine phosphokinase Reduction potential E0 de nes how well one substance reduces another donate electrons The oxidants accept electrons from a reductant Reduction oxidation redox couple describes the pair of molecules of which one is reduced and the other is oxidized Examples of redox pairs include lactatepyruvate NADHNAD and FADHzFAD Each pair constitutes half a reaction that is matched up with another pair to complete a reaction Two pairs are needed with the reductant of one pair donating electrons and the oxidant of the other pair accepting the electrons AEo is de ned as the standard reduction potential difference between two half reactions similar to the AG that de nes the standard energy difference The value AEo must be positive for the reaction to be favorable Recall that AG must be negative for a reaction to be favorable RedoxRespiratory Chain 1 ESSENTIAL FEATURES OF OXIDATIVE PHOSPHORYLATION Oxidative phosphorylation consists of two separate processes Fig l The oxidative portion generates energy that is used by the phosphorylation process to make ATP oxidative process Phosphoryla on 39 process 39 39 39 A I 39 r39 I 02 I A PPi I l inner H2 Ie outer membrane H ATP membrane intermembrane matrix Space Figure 1 Essential features of oxidative phosphorylation Redox reactions of the respiratory chain ultimately use electrons to reduce oxygen to water During this process energy that is generated moves protons from the matrix to the intermembrane space and these protons readily cross the outer membrane to equilibrate with the cytoplasmic protons The inward movement of protons from the intermembrane space to the matrix recovers this energy to promote formation of ATP in the matrix from ADP plus inorganic phosphate P The oxidative process is the electron transport chain also called the respiratory chain As will be described in more detail below this chain consists of a series of reductionoxidation redox reactions Recall that a redox reaction consists of two redox pairs with a reductant passing electrons to an oxidant In so doing the reductant becomes oxidized and the oxidant becomes reduced The half reactions are represented by Reductantl Oxidant1 e39 Oxidant2 e39 Reductantz Thus the overall reaction is Reductant1 Oxidantz Reductant2 Oxidantl Because it is not practical to measure electron concentrations directly the electron energy potential of a redox system is determined from the electrical potential of the half reactions relative to a standard half reaction When the reactants and products are in their standard state the potential is de ned as the standard redox potential E0 If the pH is in the biological range pH 7 its potential is de ned as the standard biological redox potential and designated Eo The difference in the reduction potentials between two half reactions is the AEO RedoxRespiratory Chain 2 The free energy difference AG of a typical reaction is calculated from AG nFAE0 Recall that reactions which give off energy have a negative AG so that according to this equation the same will be true for redox reactions that have a positive AEO The redox reactions of the respiratory chain ultimately cause the reduction of oxygen to water Fig l The energy generated from some of these reactions of the respiratory chain is harnessed to move protons PP from the mitochondrial matrix across the inner membrane to the intermembrane space and ultimately the cytoplasm These protons must be pumped out because of the high degree of impermeability of the inner membrane and because the protons must move against their gradient Because the outer membrane is very permeable the protons equilibrate with the cytoplasm The phosphorylation event involves using the energy harnessed from the respiratory chain through the movement of protons back into the matrix to phosphorylate ADP to ATP ADP Pi ATP Lactate dehydrogenase reaction example The NADHNAD couple potential is more negative than the potential of the lactatepyruvate couple Table 1 Therefore NADH is a better reductant than lactate and will preferentially reduce pyruvate rather than lactate preferentially reducing NAD Table 1 Reduction potential of the lactatepyruvate and NADHNAD redox pairs Oxidant Reductant E0 Pyruvate Lactate 0 l 9 V NAD NADH 032 v The optimal potential difference is calculated by subtracting the more negative Eo value from the more positive one In this case the value for the NADVNADH couple 032 v is subtracted from the value for the pyruvatelactate couple 019 v because the former is more negative Thus AEo 019 V 032 V 013 V for the lactate dehydrogenase reaction Arithmetically subtracting the reactions gives Pyruvate NAD Lactate NADH which after rearrangement yields Pyruvate NADH Lactate NAD The conversion of pyruvate to lactate therefore is energetically the preferred direction of the lactate dehydrogenase reaction based on the reduction potential difference Recall however that the less favorable direction can occur by manipulating the concentration of substrate and products so that the AG value becomes negative thus making the AE value positive in that direction In writing the overall reaction the better reductant is generally written on the left since it is more capable of donating electrons by virtue of its more negative reduction potential As noted above NADH is a better reductant than is lactate RedoxRespiratory Chain 3 Overall respiratorv electron transport chain reaction Table 2 Reduction potential of the HZO1202 and NADHNAD redox pairs Oxidant Reductant E0 1202 H20 082 v NAD NADH 032 v The same logic applies to the entire respiratory chain as for the lactate dehydrogenase reaction Table 2 In this instance the respiratory chain contains numerous components Fig 1 However the overall chain involves donation of electrons by NADH to reduce oxygen to water Consequently the potential difference is calculated as AEo 082 V 032 V 114 V The overall reaction becomes NADH H4r 1202 gt NAD H20 RESPIRATORY ELECTRON TRANSPORT CHAIN Redox reactions play an important role in energy metabolism In glycolysis the glyceraldehyde3 phosphate dehydrogenase reaction provides energy to produce a highenergy phosphate intermediate NADH and FADHZ produced as products of various dehydrogenases including pyruvate dehydrogenase and those in the citric acid cycle and the fatty acid Boxidation pathway ultimately are used to produce energy via oxidative phosphorylation Thus the energy contained in glucose and fatty acids is conserved in new forms Similarly the respiratory chain uses redox reactions to harness and conserve energy in new ways that that can be harnessed then to produce ATP EO 003v Succinate II AEO39 007V AEO39 053V 1 EO 032v IV NADH gt Coenzyme Q gt Iquot gtCytochrome C gt H O 2 E 39 010 E 39 029V AEO 042v O V AEO 019v O Eo 082v gt electron ow Figure 2 Overview of the respiratory chain showing the progression of reduction potentials from strong to weak reductants culminating in oxygen as the ultimate electron acceptor The E0 value for each of the major components is given adjacent to that component The AEO values are the potential differences across the four complexes calculated as the difference between the given Eo values RedoxRespiratory Chain 4 As noted above redox reactions consist of two redox pairs In comparing the reduced components of two redox pairs one of these reduced components will more readily donate its electrons than the other component This difference in characteristic ability to donate electrons determines the preferred direction of a redox reaction The reactions of the respiratory chain are arranged so that the best electron donor NADH is at the beginning of the chain and the strongest electron acceptor oxygen is at the end of the chain The components in between proceed in sequence to the right towards better electron acceptors while to the left are the better electron donors Fig 2 Note that the stronger reductants to the left have a more quotnegativequot reduction potential value and that this value becomes progressively more positive as the chain moves towards molecular oxygen For each complex the reduction potential difference is provided Fig 2 NADH in reducing coenzyme Q also known as ubiquinone generates a potential difference AEO of 042 v complex I Reduced CoQ via complex III reduces cytochrome C with a AEO of 019 v Oxygen accepts electrons from complex IV that receives its electrons from cytochrome C with a 053 v potential difference The smallest difference 003 v occurs when electrons donated by succinate from complex II reduces coenzyme Q As noted above the free energy from the respiratory chain can pump protons out of the mitochondrial matrix across the inner mitochondrial membrane Approximately 016 v of energy is required to pump two protons Consequently the energy produced by complexes I III and IV is suf cient for accomplishing this whereas complex II generates insuf cient energy to do so Complexes I and IV each pump four protons H and complex III pumps two protons following the oxidation of each NADH molecule for a total of 10 protons If on the other hand succinate is the initial electron donor then complex I is bypassed and then only a total of six protons can be pumped As will be described further below the inability of complex II to pump protons has implications in terms of the amount of ATP produced from NADH versus FADHZ the attached product of succinate dehydrogenase Components Table 3 Summary of the redox complexes of the electron transport chain Complex Prosthetic groups Function Poisons designation I NADHQ FMN avin oxidizes NADH to NAD Rotenone reductase mononucleotide transfers electrons to FeS coenzyme Q II 7 SuccinateQ FAD FeS oxidizes succinate to fumarate reductase with reduction of FAD to FADHZ electron transfer to CoQ III Cytochrome heme b heme c1 transfers electrons between Antimycin A reductase FeS coenzyme Q and cytochrome C that becomes reduced IV Cytochrome heme aa3 Cu oxidizes cytochrome C Carbon monoxide C oxidase reduces 1202 to H20 Cyanide RedoxRespiratory Chain 5 The principal part of the chain consists of three complexes I III IV Each of these complexes consists of integral proteins of the inner membrane and interacts via mobile carriers of electrons Fig 2 Table 3 A fourth complex II contains succinate dehydrogenase Recall that succinate dehydrogenase part of the citric acid cycle contains bound FAD Many of the proteins in these complexes contain iron as a functional group that aids in the transfer of electrons from one protein to the next There is sufficient energy produced within three of these complexes IIIIIV to pump protons out of the mitochondrion as discussed below Controlled in ux of these protons back into the matrix provides energy to produce one ATP for each of these three complexes Complex I 39 Complex I is named NADH Q reductase Fig 3 NADH is oxidized via NADH dehydrogenase and its electrons are used to reduce coenzyme Q ubiquinone that links complex I to complex III Fig 2 Complex I includes a avin containing functional group avin mononucleotide FMN as well as iron sulfur FeS clusters NADH for complex I comes from a variety of sources including pyruvate dehydrogenase citric acid cycle reactions ketone oxidation and fatty acid Boxidation Additionally NADH derived from glycolysis can be used to produce malate in the cytoplasm that is transported into the mitochondria to be oxidized by NAD thus producing NADH for the respiratory chain Thus under aerobic conditions NAD is regenerated for glycolysis via this mechanism rather than via lactate dehydrogenase The energy conserved from complex I is sufficient for the production of 1 ATP This complex is inhibited by rotenone a commonly used rat poison Knocking out complex I markedly lowers ATP production though electrons can still ow from complex II to complex III thus bypassing complex I see Fig 2 Complex I NADHQ reductase NADH Functional groups FMN FeS NAD Figure 3 Complex I of the respiratory chain that links NADH and coenzyme Q DH is dehydrogenase Complex II Succinate FAD Complex II SuccinateCOQ reductase Functional groups FAD FeS Fumarate FADH2 gt Figure 4 Complex II of the respiratory chain SDH is succinic dehydrogenase an enzyme of the citric acid cycle RedoxRespiratory Chain 6 Complex II is also linked to complex III through coenzyme Q Fig 4 Complex II succinate CoQ reductase consists primarily of the succinic dehydrogenase enzyme that is part of the citric acid cycle The fumaratesuccinate redox pair is linked to the FADFADHZ pair with the FAD being a functional group FADHZ passes electrons to coenzyme Q but there is insufficient energy produced by this complex to pump protons for ATP synthesis Because electrons passing through complex II bypass complex I this explains Why FADHZ yields one fewer ATP molecule than does NADH which feeds electrons through complex I see also fatty acid oxidation Complex I Once coenzyme Q is reduced either by complex I or by complex II it then donates electrons to reduce the cytochromes in complex III Complex III is named cytochrome reductase because it provides electrons to reduce cytochrome c Fig 5 The cytochromes in this complex and in other segments of the respiratory chain are hemecontaining compounds The hemes in complex III are functional groups designated b and c Heme function was discussed previously in conjunction with hemoglobin An antibiotic that inhibits complex III is antimycin A Electrons from complex I or II Complex cytochrome reductase Functional groups heme b heme c1 FeS Figure 5 Complex III of the respiratory chain Like complexes I and II it contains FeS clusters but is unique in that it includes heme functional groups Complex IV Complexes III and IV are linked by cytochrome c Fig 6 Cytochrome c is watersoluble and therefore only loosely associates with the cytoplasmic side of the inner membrane It is reduced by cytochrome reductase complex III and then in turn reduces complex IV that contains cytochrome aa3 and is named cytochrome oxidase it oxidizes cytochrome C Cytochrome aa3 contains copper in addition to the heme iron Complex IV is responsible for oxygen consumption by reducing an atom of oxygen to water Complex IV is inhibited by cyanide and carbon monoxide 12 02 2 H H20 Complex Complex IV Cytochrome C oxidase see Fig 5 Functional groups heme a heme a3 Cu Figure 6 Complexes III and IV of the respiratory chain linked by cytochrome c with complex IV reducing oxygen to water RedoxRespiratory Chain 7 Medical Scenario I Against his oncologist s advice PJ purchases laetrile in Mexico to treat his lung cancer Without proper medical guidance he exceeds the recommended dosage considerably guring that it will hasten its purported destruction of proliferating cells Instead the overdose leads to his death You are a student working with the oncologist You study the effects of laetrile and learn that it is a cyanoglycoside found in high concentrations in the pits of certain fruits Hydrolysis of this and similar compounds releases a cyanidecontaining benzaldehyde derivative which itself spontaneously degrades releasing cyanide With your knowledge of the respiratory chain explain why laetrile s improper use led to the patient s death Medical Scenario II Respiratory chain defects like defects of pyruvate dehydrogenase are associated with lactic academia Lactic academia occurs because the high concentration of NADH favors the formation of lactate from pyruvate Blood lactate may be elevated 30fold or more The blood ratio of 3 hydroxybutyrate to acetoacetate which is proportional to the mitochondrial ratio of NADH to NAD ratio may also be elevated Because elevated NADH inhibits pyruvate dehydrogenase blood pyruvate is also increased though to a lesser extent than observed for lactate Elevated pyruvate increases alanine production by transamination via alanine aminotransferase The respiratory chain is composed of over 100 polypeptides cytochromes ironsulfur clusters and ubiquinone hence there are many potential sites for defects Clinically these molecules are grouped into enzyme complexes as de ned in this lecture Defects in each complex of the respiratory chain have been identi ed PROTON PUMP High H H outer inner y a membrane membrane 3 O v quot 39 Intermembrane matrlx 39 H space Figure 7 Generation of a pH gradient H and charge difference negative in the matrix across the inner membrane constitute the protonmotive force that can be used to drive ATP synthesis and transport processes The free energy released from the redox reactions contained in complexes I III and IV of the respiratory chain is conserved by being used to pump protons 4H per complexes I and IV ZIP for complex III from the mitochondrial matrix into the intermembrane space Fig 7 Looking at this simplistically protons are generated by splitting water H20 If OH leaving a hydroxyl anion behind in the matrix Since the outer membrane is freely permeable to protons the protons equilibrate with the cytoplasm so that the pH in the cytoplasm and the intermembrane space become identical The imperrneability of the inner membrane keeps the protons from diffusing back into the matrix Consequently a pH gradient forms across the inner membrane such that the pH in the matrix is more basic lower proton concentration by about 04 pH unit than in the intermembrane space Part of the energy from pumping out protons is conserved by the formation of this pH gradient In turn the in ux of protons can be harnessed for the synthesis of ATP discussed below Because protons carry a positive charge their unequal distribution across the inner membrane also creates a charge difference charge gradient the energy from which can be used to drive certain transport systems The total energy conserved in the pH and charge gradients is termed the proton motive force that links the respiratory chain and ATP synthesis two separate processes RedoxRespiratory Chain 8
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