Harper's Biochemistry Chapter 12 - Biologic Oxidation.PDF

C H A P T E R Biologic Oxidation Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 12 OBJ E C TI VE S Explain the meaning of redox potential and how it c...

C H A P T E R Biologic Oxidation Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 12 OBJ E C TI VE S Explain the meaning of redox potential and how it can be used to predict the direction of flow of electrons in biologic systems. After studying this chapter Identify the four classes of enzymes (oxidoreductases) involved in oxidation you should be able to: and reduction reactions. Describe the action of oxidases and provide examples of where they play an important role in metabolism. Indicate the two main functions of dehydrogenases and explain the importance of nicotinamide adenine dinucleotide (NAD)- and riboflavin-linked dehydrogenases in metabolic pathways such as glycolysis, the citric acid cycle, and the respiratory chain. Identify the two types of enzymes classified as hydroperoxidases; indicate the reactions they catalyze and explain why they are important. Give the two steps of reactions catalyzed by oxygenases and identify the two subgroups of this class of enzymes. Appreciate the role of cytochrome P450 in drug detoxification and steroid synthesis. Describe the reaction catalyzed by superoxide dismutase and explain how it protects tissues from oxygen toxicity. BIOMEDICAL IMPORTANCE cass, known as the cytochrome P450 system. Administration of oxygen can be ifesaving in the treatment of patients with Chemicay, oxidation is defined as the remova of eectrons respiratory or circuatory faiure. and reduction is defined as the gain of eectrons. Thus, oxida- tion of a moecue (the eectron donor) is aways accompanied by reduction of a second moecue (the eectron acceptor). This FREE ENERGY CHANGES CAN BE principe of oxidation–reduction aso appies to biochemica EXPRESSED IN TERMS OF REDOX systems and is an important concept underying the under- standing of the nature of bioogic oxidation. Note that many POTENTIAL bioogic oxidations can take pace without the participation of In reactions invoving oxidation and reduction, the free moecuar oxygen, for exampe, dehydrogenations. The ife of energy change is proportionate to the tendency of reactants higher animas is absoutey dependent on a suppy of oxygen to donate or accept eectrons. Thus, in addition to express- for respiration, the process by which ces derive energy in the ing free energy change in terms of ΔG0′ (see Chapter 11), it form of ATP (see Chapter 11) from the controed reaction of is possibe, in an anaogous manner, to express it numericay hydrogen with oxygen to form water. In addition, moecuar as an oxidation–reduction or redox potential (E′0). Chemi- oxygen is incorporated into a variety of substrates by enzymes cay, the redox potentia of a system (E0) is usuay com- designated as oxygenases; many drugs, poutants, and chemi- pared with the potentia of the hydrogen eectrode (0.0 V at ca carcinogens (xenobiotics) are metaboized by enzymes of this pH 0.0). However, for bioogic systems, the redox potentia 115 116 SECTION III Bioenergetics TABLE 12–1 Some Redox Potentials of Special Interest AH2 1 /2O2 AH2 O2 in Mammalian Oxidation Systems (Red) Oxidase Oxidase System E′0 Volts H+/H2 −.0.42 A H2O A H2O2 NAD+/NADH −0.32 (Ox) A B Lipoate; ox/red −0.29 Acetoacetate/3-hydroxybutyrate −0.27 FIGURE 12–1 Oxidation of a metabolite catalyzed by an oxidase (A) forming H2O and (B) forming H2O2. Pyruvate/lactate −0.19 Oxaloacetate/malate −0.17 oxidase enzyme compex comprises heme a3 combined with Fumarate/succinate +0.03 another heme, heme a, in a singe protein and so is aso termed cytochrome aa3. It contains two moecues of heme, each hav- 3+ 2+ Cytochrome b; Fe /Fe +0.08 ing one Fe atom that osciates between Fe3+ and Fe2+ during Ubiquinone; ox/red +0.10 oxidation and reduction. Furthermore, two atoms of copper Cytochrome c1; Fe3+/Fe2+ +0.22 are present, one associated with each heme unit. Cytochrome a; Fe3+/Fe2+ +0.29 Other Oxidases Are Flavoproteins Oxygen/water +0.82 Favoprotein enzymes contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed in the body from the vitamin ribo- (E′0) is normay expressed at pH 7.0, at which pH of the flavin (see Chapter 44). FMN and FAD are usuay tighty— eectrode potentia of the hydrogen eectrode is −0.42 V. The but not covaenty—bound to their respective apoenzyme redox potentias of some redox systems of specia interest in proteins. Metalloflavoproteins contain one or more metas as mammaian biochemistry are shown in Table 12–1. The rea- essentia cofactors. Exampes of favoprotein oxidases incude tive positions of redox systems in the tabe aow prediction l-amino acid oxidase, an enzyme found in kidney with gen- of the direction of fow of eectrons from one redox coupe to era specificity for the oxidative deamination of the naturay another. occurring l-amino acids; xanthine oxidase, a moybdenum- Enzymes invoved in oxidation and reduction are caed containing enzyme which pays an important roe in the con- oxidoreductases and are cassified into four groups: oxidases, version of purine bases to uric acid (see Chapter 33), and is of dehydrogenases, hydroperoxidases, and oxygenases. particuar significance in uricoteic animas (see Chapter 28); and aldehyde dehydrogenase, an FAD-inked enzyme pres- ent in mammaian ivers, which contains moybdenum and OXIDASES USE OXYGEN AS nonheme iron and acts on adehydes and N-heterocycic sub- A HYDROGEN ACCEPTOR strates. The mechanisms of oxidation and reduction of these Oxidases catayze the remova of hydrogen from a substrate enzymes are compex. Evidence suggests a two-step reaction using oxygen as a hydrogen acceptor.* They form water or as shown in Figure 12–2. hydrogen peroxide as a reaction product (Figure 12–1). DEHYDROGENASES PERFORM Cytochrome Oxidase Is a Hemoprotein TWO MAIN FUNCTIONS There are a arge number of enzymes in the dehydrogenase Cytochrome oxidase is a hemoprotein widey distributed in cass. Their two main functions are as foows: many tissues, having the typica heme prosthetic group pres- ent in myogobin, hemogobin, and other cytochromes (see 1. Transfer of hydrogen from one substrate to another in a Chapter 6). It is the termina component of the chain of respi- couped oxidation–reduction reaction (Figure 12–3). These ratory carriers found in mitochondria (see Chapter 13) and dehydrogenases often utiize common coenzymes or hydro- it functions to transfer eectrons resuting from the oxidation gen carriers, for exampe, nicotinamide adenine dinuceotide of substrate moecues by dehydrogenases to their fina accep- (NAD+). This type of reaction in which one substrate is oxi- tor, oxygen. The action of the enzyme is bocked by carbon dized/reduced at the expense of another is freey reversibe, monoxide, cyanide, and hydrogen sulfide, and this causes enabing reducing equivaents to be transferred within the ce poisoning by preventing ceuar respiration. The cytochrome and oxidative processes to occur in the absence of oxygen, such as during the anaerobic phase of gycoysis (see Figure 17–2). * The term “oxidase” is sometimes used coectivey to denote a 2. Transfer of eectrons from substrate to oxygen in the respi- enzymes that catayze reactions invoving moecuar oxygen. ratory chain eectron transport system (see Figure 13–3).  CHAPTER 12 Biologic Oxidation 117 R R R H H H3C N N O H3C N N O H3C N N O Oxidized Substrate + + NH NH NH substrate H3C N H 3C N H 3C N O O H O H H Oxidized flavin Semiquinone (H+ + e–) (H+ + e –) Reduced flavin (FAD) intermediate (FADH2) FIGURE 12–2 Oxidoreduction of isoalloxazine ring in flavin nucleotides via a semiquinone intermediate. In oxidation reactions, the flavin (eg, FAD) accepts two electrons and two H+ in two steps, forming the semiquinone intermediate followed by the reduced flavin (eg, FADH2) and the substrate is oxidized. In the reverse (reduction) reaction, the reduced flavin gives up two electrons and two H+ so that it becomes oxi- dized (eg, to FAD) and the substrate is reduced. Many Dehydrogenases Depend on NADP-linked dehydrogenases are found characteristicay in biosynthetic pathways where reductive reactions are required, Nicotinamide Coenzymes as in the extramitochondria pathway of fatty acid synthesis These dehydrogenases use NAD+ or nicotinamide adenine (see Chapter 23) and steroid synthesis (see Chapter 26)—and dinucleotide phosphate (NADP+)—or both—which are aso in the pentose phosphate pathway (see Chapter 20). formed in the body from the vitamin niacin (see Chapter 44). The structure of NAD+ is shown in Figure 12–4. NADP+ has a H O H O phosphate group esterified to the 2′ hydroxy of its adenosine H moiety, but otherwise is identica to NAD+. The oxidized forms NH2 NH2 O– of both nuceotides have a positive charge on the nitrogen + atom of the nicotinamide moiety as indicated in Figure 12–4. O P O N N The coenzymes are reduced by the specific substrate of the O H+ dehydrogenase and reoxidized by a suitabe eectron accep- R tor. They are abe to freey and reversiby dissociate from their respective apoenzymes. O OH OH NH2 Generay, NAD-linked dehydrogenases catayze oxido- reduction reactions of the type: N N OH O O P O N C + NAD+ C + NADH + H+ N O– O H When a substrate is oxidized, it oses two hydrogen atoms and two eectrons. One H+ and both eectrons are accepted by NAD+ OH OH to form NADH and the other H+ is reeased (see Figure 12–4). Many such reactions occur in the oxidative pathways of Oxidized metaboism, particuary in gycoysis (see Chapter 17) and substrate/product the citric acid cyce (see Chapter 16). NADH is generated in OH O these pathways via the oxidation of fue moecues, and NAD+ NAD+ + + NADH + H+ C C is regenerated by the oxidation of NADH as it transfers the eectrons to O2 via the respiratory chain in mitochondria, a H process which eads to the formation of ATP (see Chapter 13). Reduced substrate/product FIGURE 12–4 Oxidation and reduction of nicotinamide AH2 Carrier BH2 coenzymes. Nicotinamide coenzymes consist of a nicotinamide ring (Red) (Ox) (Red) linked to an adenosine via a ribose and a phosphate group, forming a dinucleotide. NAD+/NADH are shown, but NADP+/NADPH are identi- cal except that they have a phosphate group esterified to the 2′ OH A Carrier–H2 B of the adenosine. An oxidation reaction involves the transfer of two (Ox) (Red) (Ox) electrons and one H+ from the substrate to the nicotinamide ring of Dehydrogenase Dehydrogenase NAD+ forming NADH and the oxidized product. The remaining hydro- specific for A specific for B gen of the hydrogen pair removed from the substrate remains free as a hydrogen ion. NADH is oxidized to NAD+ by the reverse reaction. FIGURE 12–3 Oxidation of a metabolite catalyzed by cou- R, the part of the molecule unchanged in the oxidation/reduction pled dehydrogenases. reaction. 118 SECTION III Bioenergetics Other Dehydrogenases Depend on species (ROS). ROS are highy reactive oxygen-containing moecues such as peroxides, which are formed during norma Riboflavin metaboism, but can be damaging if they accumuate. They are The flavin groups such as FMN and FAD are associated with beieved to contribute to the causation of diseases such as can- dehydrogenases as we as with oxidases as described earier. cer and atheroscerosis, as we as the aging process in genera FAD is the eectron acceptor in reactions of the type: (see Chapters 21, 44, 54). H C C + FAD C C + FADH2 Peroxidases Reduce Peroxides Using H H H Various Electron Acceptors Peroxidases are found in mik as we as in eukocytes, pate- FAD accepts two eectrons and two H + in the reaction (see ets, and other tissues invoved in eicosanoid metaboism (see Figure 12–2), forming FADH2. Favin groups are generay Chapter 23). Their prosthetic group is protoheme. In the reac- more tighty bound to their apoenzymes than are the nicotin- tion catayzed by peroxidase, hydrogen peroxide is reduced at amide coenzymes. Most of the riboflavin-linked dehydro- the expense of severa substances that act as eectron acceptors, genases are concerned with eectron transport in (or to) the such as ascorbate (vitamin C), quinones, and cytochrome c. respiratory chain (see Chapter 13). NADH dehydrogenase acts The reaction catayzed by peroxidase is compex, but the over- as a carrier of eectrons between NADH and the components of a reaction is as foows: higher redox potentia (see Figure 13–3). Other dehydrogenases such as succinate dehydrogenase, acyl-CoA dehydrogenase, PEROXIDASE and mitochondrial glycerol-3-phosphate dehydrogenase H2O2 + AH2 2H2O + A transfer reducing equivaents directy from the substrate to the respiratory chain (see Figure 13–5). Another roe of the In erythrocytes and other tissues, the enzyme glutathione per- favin-dependent dehydrogenases is in the dehydrogenation oxidase, containing selenium as a prosthetic group, catayzes (by dihydrolipoyl dehydrogenase) of reduced ipoate, an the destruction of H2O2 and ipid hydroperoxides through the intermediate in the oxidative decarboxyation of pyruvate and conversion of reduced gutathione to its oxidized form, pro- α-ketogutarate (see Figures 13–5 and 17–5). The electron- tecting membrane ipids and hemogobin against oxidation by transferring flavoprotein (ETF) is an intermediary carrier of peroxides (see Chapter 21). eectrons between acy-CoA dehydrogenase and the respiratory chain (see Figure 13–5). Catalase Uses Hydrogen Peroxide as Cytochromes May Also Be Regarded Electron Donor & Electron Acceptor as Dehydrogenases Catalase is a hemoprotein containing four heme groups. It The cytochromes are iron-containing hemoproteins in which can act as a peroxidase, catayzing reactions of the type shown the iron atom osciates between Fe3+ and Fe2+ during oxida- earier, but it is aso abe to catayze the breakdown of H2O2 tion and reduction. Except for cytochrome oxidase (described formed by the action of oxygenases to water and oxygen: earier), they are cassified as dehydrogenases. In the respi- ratory chain, they are invoved as carriers of eectrons from CATALASE favoproteins on the one hand to cytochrome oxidase on the 2H2O2 2H2O + O2 other (see Figure 13–5). Severa identifiabe cytochromes occur in the respiratory chain, they are; cytochromes b, c1, c, This reaction uses one moecue of H2O2 as a substrate eec- and cytochrome oxidase (aa3). Cytochromes are aso found in tron donor and another moecue of H2O2 as an oxidant or other ocations, for exampe, the endopasmic reticuum (cyto- eectron acceptor. It is one of the fastest enzyme reactions chromes P450 and b5), and in pant ces, bacteria, and yeasts. known, destroying miions of potentiay damaging H 2O2 moecues per second. Under most conditions in vivo, the peroxidase activity of cataase seems to be favored. Cata- HYDROPEROXIDASES USE ase is found in bood, bone marrow, mucous membranes, HYDROGEN PEROXIDE OR AN kidney, and iver. Peroxisomes are membrane-bound ORGANIC PEROXIDE organees (see Chapter 49) found in many tissues, incud- ing iver. They are rich in oxidases and in cataase. Thus, AS SUBSTRATE enzymes that produce and breakdown H 2O2 are contained Two types of enzymes found both in animas and pants fa within the same subceuar compartment. However, mito- into the hydroperoxidase category: peroxidases and catalase. chondria and microsoma eectron transport systems as Hydroperoxidases pay an important roe in protect- we as xanthine oxidase must be considered as additiona ing the body against the harmfu effects of reactive oxygen sources of H 2O2.  CHAPTER 12 Biologic Oxidation 119 OXYGENASES CATALYZE Cytochromes P450 Are THE DIRECT TRANSFER & Monooxygenases Important in Steroid INCORPORATION OF OXYGEN Metabolism & for the Detoxification of INTO A SUBSTRATE MOLECULE Many Drugs Oxygenases are concerned with the synthesis or degrada- Cytochromes P450 are an important superfamiy of heme- tion of many different types of metaboites. They catayze the containing monooxygenases, and more than 50 such enzymes incorporation of oxygen into a substrate moecue in two steps: have been found in the human genome. They are ocated (1) oxygen is bound to the enzyme at the active site and mainy in the endopasmic reticuum in the iver and intes- (2) the bound oxygen is reduced or transferred to the sub- tine, but are aso found in the mitochondria in some tissues. strate. Oxygenases may be divided into two subgroups, dioxy- The cytochromes participate in an eectron transport chain in genases and monooxygenases. which both NADH and NADPH may donate reducing equiva- ents. Eectrons are passed to cytochrome P450 in two types of reaction invoving FAD or FMN. Cass I systems consist of an Dioxygenases Incorporate Both Atoms FAD-containing reductase enzyme, an iron sufur (Fe2S2) pro- of Molecular Oxygen Into the Substrate tein, and the P450 heme protein, whie cass II systems con- The basic reaction catayzed by dioxygenases is as foows: tain cytochrome P450 reductase, which passes eectrons from FADH2 to FMN (Figure 12–5). Cass I and II systems are we A + O2 → AO2 characterized, but in recent years, other cytochromes P450, Exampes incude the iver enzymes, homogentisate dioxy- which do not fit into either category, have been identified. In genase (oxidase) and 3-hydroxyanthranilate dioxygenase the fina step, oxygen accepts the eectrons from cytochrome (oxidase), which contain iron; and l-tryptophan dioxygenase P450 and is reduced, with one atom being incorporated into (tryptophan pyrroase) (see Chapter 29), which utiizes heme. H2O and the other into the substrate, usuay resuting in its hydroxyation. This series of enzymatic reactions, known as the hydroxylase cycle, is iustrated in Figure 12–6. In the Monooxygenases (Mixed-Function endopasmic reticuum of the iver, cytochromes P450 are Oxidases, Hydroxylases) Incorporate found together with another heme-containing protein, cyto- Only One Atom of Molecular Oxygen chrome b5 (see Figure 12–5) and together they have a major Into the Substrate roe in drug metaboism and detoxification. Cytochrome b5 aso has an important roe as a fatty acid desaturase. Together, The other oxygen atom is reduced to water, an additiona eec- cytochromes P450 and b5 are responsibe for about 75% of the tron donor or cosubstrate (Z) being necessary for this purpose: modification and degradation of drugs which occurs in the A — H + O2 + ZH2 → A — OH + H2O + Z body. The rate of detoxification of many medicina drugs by Class I P450 REDUCTASE Fe2S2 P450 NAD(P)H FAD FADH2 Fe3+ Fe2+ O2+RH H2O+ROH Hydroxylation Class II P450 P450 REDUCTASE P450 NAD(P)H Hydroxylation FAD FMN FMNH2 O2+RH H2O+ROH Cytochrome b5 O2+Oleoyl CoA b5 REDUCTASE NADH b5 FAD FADH2 Stearoyl CoA + H2O Stearoyl CoA desaturase P450 REDUCTASE P450 Hydroxylation FAD FMN FMNH2 O2+RH H2O+ROH FIGURE 12–5 Cytochromes P450 and b5 in the endoplasmic reticulum. Most cytochromes P450 are class I or class II. In addition to cytochrome P450, class I systems contain a small FAD-containing reductase and an iron sulfur protein, and class II contains cytochrome P450 reductase, which incorporates FAD and FMN. Cytochromes P450 catalyze many steroid hydroxylation reactions and drug detoxification steps. Cytochrome b5 acts in conjunction with the FAD-containing cytochrome b5 reductase in the fatty acyl-CoA desaturase (eg, stearoyl-CoA desaturase) reaction and also works together with cytochromes P450 in drug detoxification. It is able to accept electrons from cytochrome P450 reductase via cytochrome b5 reductase and donate them to cytochrome P450. 120 SECTION III Bioenergetics Substrate A-H P450-A-H Fe3+ e– P450 P450-A-H NADPH-Cyt P450 reductase Fe3+ Fe2+ NADP+ FADH2 2Fe2S23+ O2 e– – NADPH + H+ FAD 2Fe2S22+ CO 2H+ P450-A-H Fe2+ O2 H2O P450-A-H Fe2+ O2 – A-OH FIGURE 12–6 Cytochrome P450 hydroxylase cycle. The system shown is typical of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein Fe2S2. Carbon monoxide (CO) inhibits the indicated step. cytochromes P450 determines the duration of their action. Superoxide can reduce oxidized cytochrome c Benzpyrene, aminopyrine, aniine, morphine, and benzphet- O2−. + Cytc(Fe3+) → O2 + Cytc(Fe2+) amine are hydroxyated, increasing their soubiity and aiding their excretion. Many drugs such as phenobarbita have the or be removed by SOD, which catayzes the conversion of abiity to induce the synthesis of cytochromes P450. superoxide to moecuar oxygen and hydrogen peroxide. Mitochondria cytochrome P450 systems are found in In this reaction, superoxide acts as both oxidant and steroidogenic tissues such as adrena cortex, testis, ovary, and reductant. Thus, SOD protects aerobic organisms against the pacenta and are concerned with the biosynthesis of steroid potentia deeterious effects of superoxide. The enzyme occurs hormones from choestero (hydroxyation at C22 and C20 in in a major aerobic tissues in the mitochondria and the cyto- side chain ceavage and at the 11β and 18 positions). In addi- so. Athough exposure of animas to an atmosphere of 100% tion, rena systems catayzing 1α- and 24-hydroxyations of oxygen causes an adaptive increase in SOD, particuary in the 25-hydroxychoecacifero in vitamin D metaboism—and ungs, proonged exposure eads to ung damage and death. choestero 7α-hydroxyase and stero 27-hydroxyase invoved Antioxidants, for exampe, α-tocophero (vitamin E), act as in bie acid biosynthesis from choestero in the iver (see scavengers of free radicas and reduce the toxicity of oxygen Chapters 26, 41)—are P450 enzymes. (see Chapter 44). SUMMARY SUPEROXIDE DISMUTASE In bioogic systems, as in chemica systems, oxidation (oss of PROTECTS AEROBIC ORGANISMS eectrons) is aways accompanied by reduction of an eectron acceptor. AGAINST OXYGEN TOXICITY Oxidoreductases have a variety of functions in metaboism; Transfer of a singe eectron to O2 generates the potentiay oxidases and dehydrogenases pay major roes in respiration; damaging superoxide anion-free radical (O2−.), which gives hydroperoxidases protect the body against damage by free rise to free-radica chain reactions (see Chapter 21), ampi- radicas; and oxygenases mediate the hydroxyation of drugs fying its destructive effects. The ease with which superoxide and steroids. can be formed from oxygen in tissues and the occurrence of Tissues are protected from oxygen toxicity caused by the superoxide dismutase (SOD), the enzyme responsibe for superoxide free radica by the specific enzyme superoxide its remova in a aerobic organisms (athough not in obigate dismutase. anaerobes), indicate that the potentia toxicity of oxygen is due to its conversion to superoxide. Superoxide is formed when reduced favins—present, for REFERENCES exampe, in xanthine oxidase—are reoxidized univaenty by Neson DL, Cox MM: Lehninger Principles of Biochemistry, 7th ed. moecuar oxygen: Macmian Education, 2017. Nichos DG, Ferguson SJ: Bioenergetics, 4th ed. Academic Press, Enz − Favin − H2 + O2 → Enz − Favin − H + O2−. + H+ 2013.