Aerobic Metabolism The citric acid cycle Krebs discovery

有氧代謝(Aerobic Metabolism) • The citric acid cycle: Krebs’ discovery of the cycle. • pyruvate oxidation的pathway是經由citric acid cycle是Han Krebs 在 1937 年首先提出 的。 • 有氧呼吸所產生的NADH與FADH 2會經由電 子傳遞鏈(簡稱ETC)將電子逐步的傳遞給氧 分子，同時由伴隨大量的ATP生成。

圖 9. 9 脂醯胺(lipoamide)

• Pyruvate dehydrogenase complex是 一個具三種酵素的結構體，包含 pyruvate decarboxylase (E 1)、 dihydrolipoyl transacetylase (E 2) 與dihydrolipoyl dehydrogenase (E 3)。每個酵素在複合體中皆有許多 個重複。

• Pyruvate dehydrogenase complex含有5種 輔酶thiamine(TPP)、lipoic caid、 ribiflavin(FAD)、pantothenic acid(Co. ASH)與niacin(NAD+)。 • 轉換丙酮酸成acetyl-Co. A的第一個步驟是 由pyruvate decarboxylase催化pyruvate 去二氧化碳(decarboxylation)，此酵素需 TPP當輔酶，中間產物hydroxylethyl-TTP (HETTP)。

• 接下來dihydrolipoyl transacetylase將HETTP轉 Acetyl-Co. A，在這個轉換過程中，lipoic acid扮 演重要角色。Lipoic acid結合在此酵素 (dihydrolipoyl transacetylase)的lysine上， 它與HETTP反應形成乙醯化的lipoic acid(acetylated lipoic acid)，然後這acetyl group 再轉給Co. ASH，接著還原態的Lipoic acid 被dihydrolipoyl dehydrogenase上的FAD氧化， 然後NAD+再還原FADH 2。

• 接著α-ketoglutarate dehydrogenase complex催化α-ketoglutarate轉成Succinyl. Co. A。此酵素催化氧化去二氧化碳，其機轉 很類似pyruvate dehydrogenase complex ( 也需要TPP、Co. ASH、lipoic acid、NAD+ 與FAD)。 • 然後succinate thiokinase催化succinyl-Co. A 形成succinate，此反應會伴隨一個GTP的 生成。

• 接著fumarase催化fumarate轉成malate， 隨後malate dehydrogenase催化malate形 成oxaloacetate。

乙醛酸循環(The glyoxylate cycle) • 植物、真菌、類、原生動物與一些細菌因 為具有glyoxylate cycle可以利用二個碳的 化合物，如ethanol、acetate與acetyl-Co. A 製造Glucose，在植物glyoxylate cycle是發 生glyoxysome，而其真核微生物則發生在 cytoplasm。 • 註: 動物無法利用acetyl-Co. A製造glucose(只 能利用lactate、glycerol、alanine與一些α酮酸等)。

圖 9. 13 乙醛酸循環(glyoxylate cycle)

• glyoxylate cycle是由 5個酵素組成，由於它 們比一般動物多了二個酵素，isocitrate lyase與malate synthase。這個很類似citric acid cycle，但至isocitrate時繞過去二氧化 碳(decarboxylation)的途徑，而分解成 glyoxylate與succinate，glyoxylate再與 acetyl-Co. A作用合成malate。Glyoxylate的 作用是產生glucose。 • 檸檬酸循環的調控

• 由丙酮酸轉成acetyl-Co. A進入檸檬酸循環的途徑 中，主要的調控酵素為: • 1 pyruvate dehydrogenase: - acetyl-Co. A • 2 pyruvate carboxylase: + acetyl-Co. A、ATP與 NADH • 3 citrate synthase: - succinyl-Co. A、citrate、 NADH與ATP • 4 isocitrate dehydrogenase: - NADH、ATP。 + NAD+、ADP。 • 5 α–ketoglutarate dehydrogenase: - succinyl. Co. A、NADH

• 在細胞質內的角色如下: • 1由ATP citrate lyase催化形成 acetyl-Co. A與oxaloacetate。acetyl. Co. A可合成脂肪與膽固醇， oxaloacetate可以合成胺基酸。 • 2產生NADPH供合成fatty acid使用。 • 3抑制PFK-1活性，即抑制Glycolysis

Oxidative phosphorylation • Electro-transfer reaction in mitochondria • ATP synthesis 氧化磷酸化反應: 1粒線體中的電子傳遞反應 2 ATP的合成 Regulation of oxidative phosphorylation 氧化磷酸化反應的調節

• 主要有4個complex組成: • 1 Complex I: NADH dehydrogenase complex • 功能: 催化電子由NADH傳至 UQ(ubiquinone)，目前結構功能未知。 • 2 Complex II: Succinate dehydrogenase complex: 主要是由Succinate dehydrogenase與2個硫鐵蛋白組成。功能 是催化電子由FADH 2傳至UQ。

Absorption spectra of cytochrome c in its oxidized (red) and reduced (blue) forms. Also labeled are the characteristic a, b, and g bands of the reduced form.

Prosthetic groups of cytochromes. Each consists of four fivemembered, nitrogen-containing rings in a cyclic structure called a porphyrin. The four nitrogen atoms are coordinated with a central Fe ion, either Fe 2+ or Fe 3+.

• 3 Complex III是由 2個B-type cytochrome、 cytochrome C 1與一個硫鐵分子組成。功能: 將UQH的電子傳遞至cytochrome C • 4 Complex IV: 由cytochrome oxidase組成 • 功能: 催化 4個電子與O 2形成H 2 O。 • 每個complex皆包含數個蛋白質與prothetic group。另外還有兩個參與的分子 Coenzyme Q與Cytochrome C。

Summary of the flow of electrons and protons through the four complexes of the respiratory chain The vectorial equation for the process is: NADH + 11 HN+ + 0. 5 O 2 → NAD+ + 10 H+P + H 2 O

Methods for determining the sequence of electron carriers 還原半電位表

In the presence of an electron donor and O 2, each inhibitor causes a characteristic pattern of oxidized/reduced carriers; those before the block become reduced (blue), and those after the block become oxidized (red). 電子傳遞鏈 中電子傳遞順序與其阻斷劑。

Electron carriers function in multienzyme complexes The electron carriers of the respiratory chain are organized into membrane-embedded supramolecular complexes that can be physically separated. 呼吸鏈 的電子攜帶者是位於膜中的巨大蛋白複合 物，且可分離出。

Separation of functional complexes of the respiratory chain 分離呼吸鏈具功能的複合體

The overall reaction catalyzed by Complex IV 4 Cyt c (reduced) + 8 H+ + O 2 → 4 cyt c (oxidized) + 4 NH+ + 2 H 2 O P Complex IV含銅離子，亦具兩種作用: 1電子 傳遞。2質子的輸送。

Summary of the flow of electrons and protons through the four complexes of the respiratory chain The vectorial equation for the process is: NADH + 11 HN+ + 0. 5 O 2 → NAD+ + 10 H+P + H 2 O

• 1961年，Peter Mitchell提出一個稱為 Mitchell’s model又稱chemiosmotic coupling theory理論 • 1當電子通過ETC時，proton(H+)會由粒線 體的matrix輸送至intermembrane space形 成電化學的proton gradient，是一種 protomotive force，即質子趨動力。

Proton-motive force

• Because the transfer of two electrons from NADH to O 2 is accompanied by the outward pumping of 10 H+, roughly 200 k. J of 220 k. J released by oxidation of a mole of NADH is conserved in the proton gradient. • 估算 1 mole NADH約可打出 10 mole個質 子，花費約200 -220 kj 的能量。

Chemiosomitic model

圖 10. 14 ATP合 成

• Uncoupler: • 可攜帶H+離子進出內膜，導致proton gradient的破壞的物質。使得電子傳遞 與oxidative phosphorylation無法伴隨 發生(coupling)。如2, 4, Dinitrophenol (DNP)。

Two chemical uncouplers of oxidative phosphorylation

圖 10. 13 去偶合劑(uncoupler)

• 1 Glycerol phosphate shuttle: 這個過程 會損失一個ATP，它是利用DHAP被還原成 Glycerol-3 -phosphate再穿過粒線體外膜， 在內膜受glycerol-3 -phosphate dehydrogenase催化再氧化成DHAP釋出FADH 2。 • 2 Malate-Asparate shuttle: • 理論上在交換過程中並無ATP的損失，但需 要轉胺反應的作用。

Mitochondrial production and disposal of superoxide(超氧歧的生成與清除) • The passage of electrons from QH 2 to cytochrome b. L through Complex III, and passage of electrons from Complex I to QH 2, involve the. radical as intermediate. The Q can, with a _. low probability, pass an electron to O 2 in the reaction O 2 + e - →. O 2粒線體會產生superoxide: 在電子傳遞的過程中，電 子會由複合體漏出與氧結合形成超氧歧 (superoxide ·O 2 - ) Q

• The superoxide free radical generated, ·O 2 - is very reactive and can damage enzymes, membrane lipids, and nucleic acids. • Superoxide含自由基對酵素、膜與核酸具 破壞性。

Reactive oxygen species (ROS) • ROS包含Superoxide radical (．O 2 - )、 hydrogen peroxide (H 2 O 2)、hydroxyl radical (．OH)與singlet oxygen ( 1Ｏ 2 )。因為ROS相當reactive，易引起一些鏈 鎖反應。如lipid peroxidation reaction: hydroxyl radical會攻擊一些脂肪酸，引 起鏈鎖反應。食品加 時常會促進這種 radical chain reaction，常需加入抗氧 化劑，阻止radical chain reaction的反應。

• 一些酵素具有阻止oxidative stress的功 能，如superoxide dimutase、catalase與 glutathione peroxidase。 • SOD (superoxide dimutase) • 催化: 2 O 2 + 2 H+ H 2 O 2 + O 2 • 在入類中，Cu-Zn SOD 位於細胞質內 • Mn SOD則在粒線體內，兩者是同功脢

• Glutathione reductase再將GSSG還原成 GSH • GSSG + NADPH + H+ 2 GSH + NADP+ • NADPH主要是由pentose phosphate pathway提供

Mitochondrial production and disposal of superoxide

ATP synthase • Mitochondrial F 1 has nine subunits of five different types, with the composition a 3 b 3 gde. • Each of the three b subunits has one catalytic site for ATP synthesis. • The crystallographic determination of the F 1 structure by John E. Walker and colleagues revealed structural details very helpful in explaining the catalytic mechanism of the enzyme. ATP synthase 的結晶結構由John E. Walker 等解出。

Diagram of the Fo. F 1 complex, deduced from biochemical and crystallographic studies

Binding-change model for ATP synthase

Chemiosmotic coupling allows nonintegral stoichiometries of O 2 consumption and ATP synthesis • If 10 protons are pumped out per NADH and 4 must flow in to produce 1 ATP, the proton-based P/O ratio is 2. 5 for NADH as the electron donor and 1. 5 for succinate. • 1 NADH可產生 2. 5 ATP • 1 FADH 2可產生 1. 5 ATP

Adenine nucleotide and phosphate translocases (located in the inner mitochondrial membrane)

Shuttle systems indirectly convey cytosolic NADH into mitochondria for oxidation 1. malate-asparate shuttle— producing 2. 5 ATP. 肝與心臟細胞 2. Glycerol 3 -phosphate shuttle—producing 1. 5 ATP. 肌肉與腦細胞

Malate-asparate shuttle

Glycerol 3 -phosphate shuttle

Uncoupled mitochondria in brown fat produce heat • Most newborn mammals, including humans, have a type of adipose tissue called brown fat in which fuel oxidation serves not to produce ATP but to generate heat to keep the newborn warm. 大部分哺乳動物的幼兒，其脂 肪組織具有棕色脂肪。可以行脂肪氧化，非產 生ATP而產熱。

• The mitochondria of brown fat have a unique protein in their inner membrane. • Thermogenin, also called the uncoupleing protein, provides a path for protons to return to the matrix without passing through the Fo. F 1 complex. • 因為棕色脂肪粒線體膜上具有獨特的蛋 白Thermogenin又稱uncoupleing protein， 會破壞質子梯度。

Mitochondrial P-450 oxygenases catalyze steroid hydroxylations • Mitochondria are the site of biosynthetic reaction that produce steroid hormones, including the sex hormones, glucocorticoids, mineralocorticoids, and vitamin D hormone. • 粒線體是合成類固醇與維生素D的胞器。 • These hormones are synthesized from cholesterol or a related sterol in a series of hydroxylations catalyzed by the enzymes of the cytochrome P-450 family. 這些荷爾 蒙是由膽固醇經一系的羥化反應而成，而 催化這些反應的酵素為cytochrome P-450 家族的成員。

• In the hydroxylation reactions, one atom of molecular oxygen is incorporated into the substrate and the second is reduced to H 2 O: R-H + O 2 + NADPH → R-OH + H 2 O + NADP+ • 羥化反應的方程式如上。

Mitochondrial P-450 oxygenases catalyze steroid hydroxylations • Cytochrome P-450 enzymes are situated in the inner mitochondrial membrane with their catalytic site exposed to the matrix. • All of P-450 enzyme have a critical heme group. Its absorption at 450 nm gives this family its name。 • Cytochrome P-450酵素位於粒線體內膜，其催 化的位置曝露於基質中。具有血質(heme), 因對 450奈米具有強的吸收故名之。

• The path of electron flow in the mitochondrial P-450 system is involved a flavoprotein and an iron-sulfur protein that carry electrons from NADPH to the P-450 heme. P-450系統的電子流動路徑包含黃素蛋白、 硫鐵蛋白與NADPH。

Mitochondria of adrenal gland, specialized for steroid synthesis

Another large family of P-450 enzymes • Found in the ER of hepatocytes. 另一大族群則 位內質網，催化反應類似於粒線體的P-450 enzymes。主要用於解毒代謝。 • Catalyze reactions similar to the mitochondrial P-450 reactions. • Their substrates include a wide variety of hydrophobic compounds, many of which are xenobiotics－compounds not found in nature but synthesized industrially.

• Hydroxylation of the hydrophobic compounds makes them more water soluble, and they can then be cleared by the kidneys and excreted in urine. • 羥化不溶於水的疏水性的化合物，可增加 其溶解度，有利於排出體外。

Another large family of P-450 enzymes • Found in the ER of hepatocytes. 另一大族群則 位內質網，催化反應類似於粒線體的P-450 enzymes。主要用於解毒代謝。 • Catalyze reactions similar to the mitochondrial P-450 reactions. • Their substrates include a wide variety of hydrophobic compounds, many of which are xenobiotics－compounds not found in nature but synthesized industrially.

• Hydroxylation of the hydrophobic compounds makes them more water soluble, and they can then be cleared by the kidneys and excreted in urine. • 羥化不溶於水的疏水性的化合物，可增加 其溶解度，有利於排出體外。

The role of mitochondria in apoptosis • Besides their central role in ATP synthesis, mitochondria also participate in processes associated with cellular damage and death. 除了 合ATP的角色，粒線體也參與細胞損壞與死亡的 過程。 • Apoptosis: Programmed cell death, in which a cell brings about its own death and lysis, signaled from outside or programmed in its genes, systematically degrading its own macromolecules. 細胞凋亡，是計劃性的細死亡。 當細胞接受到死亡訊息，會增加粒線體外膜的通 透性。有利 於cytochrome c 漏出，漏出的 cytochrome c會活化caspase 9，導致細胞凋亡的 啟動。

• When a cell receives a signal for apoptosis, one consequence is an increase in the permeability of the outer mitochondrial membrane ( the opening of the permeability transition pore complex (PTPC)), allowing escape of the cytochrome c normally confined in the intermembrane space. • The released cytochrome c activates one of the proteolytic enzymes (caspase 9) responsible for protein degradation during apoptosis.

Role of cytochrome c in apoptosis

Mitochondrial genes • Mitochondria contain their own genome, a circular double-stranded DNA (mt. DNA) molecule. 粒線體內有一環狀的 雙股螺旋的DNA。共 16569 bases，無 intron且內含 37個基因。隨細胞分裂而分 裂。只有13 genes被轉譯。絕大部分 (95%以上)的粒線體蛋白是由染色體中 的基因所轉譯再送至粒線體中。

Mitochondrial genes and mutations

Mutations in mitochondrial DNA accumulate throughout the life of the organism • The respiratory chain is the major producer of ROSs in cells, so mitochondrial contents, including the mitochondrial genome, suffer the greatest exposure to, and damage by ROS. • The mitochondrial DNA replication system is less effective than the nuclear system at correcting mistakes made during replication and at repairing DNA damage. • One theory of aging is that this gradual accumulation of defects with increasing age is the primary cause of many of the symptoms of aging, which include progressive weakening of skeletal and heart muscle.

Heteroplasmy in mitochondrial genomes

Different cells in the same tissue are affected to different degrees by the mitochondrial mutation • The cells from human muscle tissue are stained to make wild-type cell blue and cells with mutant cytochrome oxidase brown.

染色體基因突變的遺傳疾病 • 賴博氏遺傳性視覺神經症 ( Leber Hereditary Optic Neuropathy, LHON ) • Myoclonic epilepsy and ragged-red fiber disease (MERRF)(肌陣攣性癲癇發作伴破 碎紅纖維病變)