Se Vit E
S (METIONINA –SH)
Se Vit E
S RO-‐OH·∙
H2O2
O= (METIONINA –SH)
Metabolismo oxida?vo celular Biotransformación hepá?ca de xenobió?cos (zona centrolobulillar)
Oxidación de lípidos de membrana
Alteración de estructura y función de proteínas
2 R-‐SH R-‐S-‐S-‐R
CH
CH
C
C O
538CHAPTER 18 Oxidative Phosphorylation
1. Electrons from two molecules of reduced cytochrome c flow down an electron-transfer pathway within cytochrome c oxidase, one stopping at Cu B and the other at heme a 3 . With both centers in the reduced state, they together can now bind an oxygen molecule.
2. As molecular oxygen binds, it abstracts an electron from each of the nearby ions in the active center to form a peroxide (O2
22) bridge between them (Figure 18.15).
3. Two more molecules of cytochrome c bind and release electrons that travel to the active center. The addition of an electron as well as H 1 to each oxygen atom reduces the two ion–oxygen groups to Cu B 2 1 ¬OH and Fe 3 1 ¬OH.
4. Reaction with two more H 1 ions allows the release of two molecules of H 2 O and resets the enzyme to its initial, fully oxidized form.
4 Cyt cred 1 4 H1matrix 1 O2 S 4 Cyt cox 1 2 H2O
The four protons in this reaction come exclusively from the matrix. Thus, the consumption of these four protons contributes directly to the proton gradi-ent. Recall that each proton contributes 21.8 kJ mol 2 1 (5.2 kcal mol 2 1 ) to the free energy associated with the proton gradient; so these four protons contribute 87.2 kJ mol 2 1 (20.8 kcal mol 2 1 ), an amount substantially less than the free energy available from the reduction of oxygen to water. What is the fate of this missing energy? Remarkably, cytochrome c oxidase uses this energy to pump four additional protons from the matrix to the cytoplasmic side of the membrane in the course of each reaction cycle for a total of eight protons removed from the matrix (Figure 18.16). The details of how these protons are transported through the protein are still under study. However, two effects contribute to the mechanism. First, charge neutrality tends to be main-tained in the interior of proteins. Thus, the addition of an electron to a site inside a protein tends to favor the binding of H 1 to a nearby site. Second, conformational changes take place, particularly around the heme a 3 –Cu B center, in the course of the reaction cycle. Presumably, in one conformation, protons may enter the protein exclusively from the matrix side, whereas, in another, they may exit exclusively to the cytoplasmic side. Thus, the overall process catalyzed by cytochrome c oxidase is
4 Cyt cred 1 8 H1matrix 1 O2 S 4 Cyt cox 1 2 H2O 1 4 H1
cytoplasm
Figure 18.17 summarizes the flow of electrons from NADH and FADH 2 through the respiratory chain. This series of exergonic reactions is coupled to the pumping of protons from the matrix. As we will see shortly, the energy inherent in the proton gradient will be used to synthesize ATP.
Toxic derivatives of molecular oxygen such as superoxide radicals are scavenged by protective enzymes
As discussed earlier, molecular oxygen is an ideal terminal electron accep-tor, because its high affinity for electrons provides a large thermodynamic driving force. However, danger lurks in the reduction of O 2 . The transfer of four electrons leads to safe products (two molecules of H 2 O), but partial reduction generates hazardous compounds. In particular, the transfer of a single electron to O 2 forms superoxide ion, whereas the transfer of two electrons yields peroxide.
O2 ¡e2
O2?2 ¡
e2 O2
22
Superoxideion
Peroxide
Peroxide
Cu2+Fe3+
FIGURE 18.15 Peroxide bridge. The oxygen bound to heme a3 is reduced to peroxide by the presence of CuB.
Fe Cu
4 44 H+
4 H+
Pumpedprotons
4 H+
Chemicalprotons
O2 2 H2O
Cyt creduced Cyt coxidized
FIGURE 18.16 Proton transport by cytochrome c oxidase. Four protons are taken up from the matrix side to reduce one molecule of O2 to two molecules of H2O. These protons are called “chemical protons” because they participate in a clearly defined reaction with O2. Four additional “pumped” protons are transported out of the matrix and released on the cytoplasmic side in the course of the reaction. The pumped protons double the efficiency of free-energy storage in the form of a proton gradient for this final step in the electron-transport chain.
O2 + 4 e-‐ + 4 H+ 2 H2O
53918.3 The Respiratory Chain
Both compounds are potentially destructive. The strategy for the safe reduction of O 2 is clear: the catalyst does not release partly reduced intermedi-ates. Cytochrome c oxidase meets this crucial criterion by holding O 2 tightly between Fe and Cu ions.
Although cytochrome c oxidase and other proteins that reduce O 2 are remarkably successful in not releasing intermediates, small amounts of
superoxide anion and hydrogen peroxide are unavoidably formed. Superoxide, hydrogen peroxide, and species that can be generated from them such as the hydroxyl radical (OH ? ) are collectively referred to as reactive oxygen species or ROS. Oxidative damage caused by ROS has been implicated in the aging process as well as in a growing list of diseases (Table 18.3).
What are the cellular defense strategies against oxidative damage by ROS? Chief among them is the enzyme superoxide dismutase. This enzyme scavenges superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen.
O22? 1 2H1
Superoxide dismutase
! O2 1 H2O2
Eukaryotes contain two forms of this enzyme, a manganese-containing version located in mitochondria and a copper-and-zinc-dependent cyto-plasmic form. These enzymes perform the dismutation reaction by a similar
FADH2
Q pool
NADH
Matrix
Intermembranespace
Citricacidcycle
O2 H2O
IIII
IV
Acetyl CoA
H+
Q
QH2
II
H+
CytC
H+
Innermitochondrialmembrane
FIGURE 18.17 The electron-transport chain. High-energy electrons in the form of NADH and FADH2 are generated by the citric acid cycle. These electrons flow through the respiratory chain, which powers proton pumping and results in the reduction of O2.
TABLE 18.3 Pathological conditions that may entail free-radical injury
AtherogenesisEmphysema; bronchitisParkinson diseaseDuchenne muscular dystrophyCervical cancerAlcoholic liver diseaseDiabetesAcute renal failureDown syndromeRetrolental fibroplasia (conversion of the retina into a fibrous mass in premature infants)Cerebrovascular disordersIschemia; reperfusion injury
Information from Michael Lieberman and Allan D. Marks, Basic Medical Biochemistry: A Clinical Approach, 4th ed. (Lippincott, Williams & Wilkins, 2012), p. 437.
DismutationA reaction in which a single reactant is converted into two different products.
Oxidación de nutrientes
R-‐OH·∙ Radical hidroxil
METIONINA-‐(SH)
CISTEÍNA-‐(SH)
GLUTATIÓN
GLICINA
GLUTÁMICO
734CHAPTER 24 The Biosynthesis of Amino Acids
24.4 Amino Acids Are Precursors of Many Biomolecules
In addition to being the building blocks of proteins and peptides, amino acids serve as precursors of many kinds of small molecules that have impor-tant and diverse biological roles. Let us briefly survey some of the biomol-ecules that are derived from amino acids (Figure 24.22).
N
N NH
N
NH2
N
NH
O
NH2
+H3N
NH3+
HHO
HO H
N
NH
Adenine Cytosine Sphingosine Histamine
+H3N COO–
HO
I
I
HO I
I
HOH H
NCH3
HO
HO
N
O
NH2
RNH
NH3+
HO
Thyroxine(Tetraiodothyronine)
Epinephrine Serotonin Nicotinamide unit of NAD+
+
FIGURE 24.22 Selected biomolecules derived from amino acids. The atoms contributed by amino acids are shown in blue.
Purines and pyrimidines are derived largely from amino acids. The bio-synthesis of these precursors of DNA, RNA, and numerous coenzymes will be discussed in detail in Chapter 25 . The reactive terminus of sphingosine, an intermediate in the synthesis of sphingolipids, comes from serine. Histamine, a potent vasodilator, is derived from histidine by decarboxyl-ation. Tyrosine is a precursor of thyroxine (tetraiodothyronine, a hormone that modulates metabolism), epinephrine (adrenaline), and melanin (a com-plex polymeric molecule responsible for skin pigmentation). The neu-rotransmitter serotonin (5-hydroxytryptamine) and the nicotinamide ring of NAD 1 are synthesized from tryptophan. Let us now consider in more detail three particularly important biochemicals derived from amino acids.
Glutathione, a gamma-glutamyl peptide, serves as a sulfhydryl buffer and an antioxidant
Glutathione, a tripeptide containing a sulfhydryl group, is a highly distinc-tive amino acid derivative with several important roles (Figure 24.23). For
example, glutathione, present at high levels (~5 mM) in animal cells, protects red blood cells from oxidative damage by serving as a sulfhy-dryl buffer ( Section 20.5 ). It cycles between a reduced thiol form (GSH) and an oxidized form (GSSG) in which two tripeptides are linked by a disulfide bond.
2 GSH 1 ROOOH ! GSSG 1 H2O 1 ROH
GSSG is reduced to GSH by glutathione reductase, a flavoprotein that uses NADPH as the electron source. The ratio of GSH to GSSG in most cells is greater than 500. Glutathione plays a key role in detoxi-fication by reacting with hydrogen peroxide and organic peroxides, the harmful by- products of aerobic life.
–OOC
O
NH
O
HN COO–
H NH3+
H
SH
!-Glutamate Cysteine Glycine
FIGURE 24.23 Glutathione. This tripeptide consists of a cysteine residue flanked by a glycine residue and a glutamate residue that is linked to cysteine by an isopeptide bond between glutamate’s side-chain carboxylate group and cysteine’s amino group.
(GSH)
Glutathione peroxidase, the enzyme catalyzing this reaction, is remarkable in having a modified amino acid containing a selenium (Se) atom (Figure 24.24). Specifically, its active site contains the selenium analog of cysteine, in which selenium has replaced sulfur. The selenolate ( E-Se 2 ) form of this residue reduces the peroxide substrate to an alcohol and is in turn oxidized to selenenic acid (E-SeOH). Glutathione then comes into action by forming a selenosulfide adduct (E-Se-S-G). A sec-ond molecule of glutathione then regenerates the active form of the enzyme by attacking the selenosulfide to form oxidized glutathione (Figure 24.25).
Nitric oxide, a short-lived signal molecule, is formed from arginine
Nitric oxide (NO) is an important messenger in many vertebrate signal-transduction processes, identified first as a relaxing factor in the cardiovas-cular system. It is now known to have a variety of roles not only in the cardiovascular system, but also in the immune and nervous systems. NO has also been shown to stimulate mitochondrial biogenesis. This free-radi-cal gas is produced endogenously from arginine in a complex reaction that is catalyzed by nitric oxide synthase. NADPH and O 2 are required for the synthesis of nitric oxide (Figure 24.26). Nitric oxide acts by binding to and activating soluble guanylate cyclase, an important enzyme in signal trans-duction ( Section 32.3 ). This enzyme is homologous to adenylate cyclase but includes a heme-containing domain that binds NO.
15N labeling: A pioneer’s account“Myself as a Guinea Pig
. . . in 1944, I undertook, together with David Rittenberg, an investigation on the turnover of blood proteins of man. To this end I synthesized 66 g of glycine labeled with 35 percent 15N at a cost of $1000 for the 15N. On 12 February 1945, I started the ingestion of the labeled glycine. Since we did not know the effect of relatively large doses of the stable isotope of nitrogen and since we believed that the maximum incorporation into the proteins could be achieved by the administration of glycine in some continual manner, I ingested 1 g samples of glycine at hourly intervals for the next 66 hours . . . . At stated intervals, blood was withdrawn and after proper preparation the 15N concentrations of different blood proteins were determined.”
—David SheminBioessays 10(1989):30
Selenocysteine
FIGURE 24.24 Structure of glutathione peroxidase. This enzyme, which has a role in peroxide detoxification, contains a selenocysteine residue in its active site. [Drawn from 1GP1.pdb.]
GSH
GSSG+ H+
ROH
ROOH+ H+E-Se!
Selenolate
E-Se-S-GSelenosulfide
E-SeOHSelenenic acid
H2O GSH
FIGURE 24.25 Catalytic cycle of glutathione peroxidase. [Information from O. Epp, R. Ladenstein, and A. Wendel. Eur. J. Biochem. 133(1983):51–69.]
+H3N COO– +H3N COO– +H3N COO–
H
NHH2N
NH2
H
NHH2N
N+
HOH
H
NHH2N
O+
Arginine N-!-Hydroxy-arginine
Citrulline
+ NO
Nitricoxide
H+
+O2+
NADPH
H2O+
NADP+
O2+
NADPH
H2O+
NADP+
FIGURE 24.26 Formation of nitric oxide. NO is generated by the oxidation of arginine.
73524.4 Amino Acid Precursors
of Biomolecules
2 GSH + ROOH·∙ +H+ GSSG + ROH + H2O
E-‐Se-‐ GLUTATIÓN PEROXDASA
GSSH + NADPH 2 GSH + NADP GSH Reductasa
43915.4 Recurring Motifs
vitamins can be complex; thus, it is biologically more efficient to ingest vita-mins than to synthesize the enzymes required to construct them from simple molecules. This efficiency comes at the cost of dependence on other organ-isms for chemicals essential for life. Indeed, vitamin deficiency can generate diseases in all organisms requiring these molecules (Tables 15.3 and 15.4).
Not all vitamins function as coenzymes. Vitamins designated by the letters A, C, D, E, and K (Figure 15.18 and Table 15.4) have a diverse array of func-tions. Vitamin A (retinol) is the precursor of retinal, the light-sensitive group in rhodopsin and other visual pigments (Section 32.3), and retinoic acid, an important signaling molecule. A deficiency of this vitamin leads to night blind-ness. In addition, young animals require vitamin A for growth. Vitamin C, or ascorbate, acts as an antioxidant. A deficiency in vitamin C results in the for-mation of unstable collagen molecules and is the cause of scurvy, a disease characterized by skin lesions and blood-vessel fragility (Section 27.6). A metab-olite of vitamin D is a hormone that regulates the metabolism of calcium and phosphorus. A deficiency in vitamin D impairs bone formation in growing animals. Vitamin E ( a -tocopherol) deficiency causes a variety of neuromuscular pathologies. This vitamin inactivates reactive oxygen species such as hydroxyl
TABLE 15.4 Noncoenzyme vitamins
Vitamin Function Deficiency
A Roles in vision, growth, Night blindness, cornea reproduction damage, damage to respiratory and gastrointestinal tractC (ascorbic acid) Antioxidant Scurvy (swollen and bleeding gums, subdermal hemorrhaging)D Regulation of calcium Rickets (children): skeletal deformities, and phosphate metabolism impaired growth Osteomalacia (adults): soft, bending bonesE Antioxidant Lesions in muscles and nerves (rare)K Blood coagulation Subdermal hemorrhaging
FIGURE 15.18 Structures of some vitamins that do not function as coenzymes. These vitamins are often called the fat-soluble vitamins because of their hydrophobic nature.
O
O
CH3
H3C
CH3
CH3CH3
CH3
CH3
CH2OH
H3C
H6
CH3
CH3
Vitamin K
Vitamin E (!-Tocopherol)
CH3
CH3
OH
HO
CH2
OH
CH3
1,25-Dihydroxyvitamin D3 (Calcitriol)
CH3
CH3
CH3 CH3
O
HO
H3C3
Vitamin A (Retinol)
Vitamina E (alfa tocoferol)