Lecture 41

Question 1

pyruvate dehydrogenase complex (PDH)

Answer 1

• converts pyruvate to acetyl CoA in the mitochondria; one of the carbons is removed and CoA carries the 2-C portion to donate it in metabolic pathways
• three separate enzymes that work at the same time (E1,E2,E3)
• five different coenzymes (all required for reaction to be efficient)

Coenzymes:

• thiamine pyrophosphate, TPP (from thiamine, vitamin B1)
• lipoamide (can be synthesized by human cells)
• coenzyme-A (CoA) (from pantothenic acid, vitamin B5)
• FAD (from riboflavin, vitamin B2)
• NAD+ (from nicotinamide, vitamin B3/niacin)

pg 1090

Question 2

PDH and human health: vitamin deficiencies

Answer 2

• deficiency of niacin or thiamine causes serious CNS problems
• Wernicke-Korsakoff syndrome (thiamine deficiency) -> often seen in chronic alcohol use disorder
• neurological symptoms occur from deficiency of any vitamins involved in metabolic processes

pg 1091

Question 3

PDH and human health: genetic defects in the PDH complex

Answer 3

Leigh Syndrome: subacute necrotizing encephalomyelopathy

• caused by mutation in either PDH, ETC protein or ATP-synthase
• rare, progressive neurodegenerative disorder
• chronic lactic acidosis

also, deficiency in the activity of the α-subunit of PDH (X-linked dominant)

pg 1091

Question 4

PDH and human health: arsenic poisoning

Answer 4

• arsenite forms a stable thiol with the -SH group in lipoic acid making it unavailable to serve as coenzyme
• affects the brain causing neurological disturbances and death

pg 1091

Question 5

PDH regulation

Answer 5

• complex multi-level regulation
• substrate activation (pyruvate, NAD+, CoA)
• product inhibition (acetryl CoA, NADH)
• covalent modifications: phosphorylation by a kinase deactivates PDH, dephosphorylation by a phosphatase activates PDH
• the PDH kinase and phosphatase can be allosterically regulated

Activators: pyruvate, NAD+, ADP, Ca2+, CoA
Inhibitors: acetyl CoA, NADH, ATP

kinase -> inhibitors: ADP, pyruvate; activators: acetyl CoA, NADH
phosphatase -> Ca2+ (released in muscle cells when contractions occur -> muscle needs energy so allows PDH to work)

pg 1092

Question 6

TCA (Krebs, Citric acid) Cycle: purpose

Answer 6

• TCA = tricarboxylic acid, Krebs = physician who discovered, citric acid = first intermediate produced
• the final pathway where the catabolism of carbohydrates, amino acids, and fatty acids converge and their carbon skeletons are converted to CO₂ (and H2O is produced)
• terminal oxidation of all biomolecules -> energy is carried by NADH and FADH2 for the subsequent production of ATP in the ETC
• located in the mitochondrial matrix, in close proximity to the ETC
• amphibolic pathway -> provides substrates for gluconeogenesis and NEAA synthesis

pg 1094

Question 7

TCA (Krebs) Cycle: Key Steps (irreversible)

Answer 7

1. citrate synthase (oxaloacetate + acetyl CoA -> citrate): produces the first intermediate, regulated by substrate activation and product inhibition
2. isocitrate dehydrogenase (isocitrate -> α-ketoglutarate): rate-limiting step, regulated allosterically (inhibitors are ATP and NADH, activators are ADP and Ca²⁺ -> muscle contraction)
3. α-ketoglutarate dehydrogenase complex (α-ketoglutarate -> succinyl CoA): similar to the PDH complex -> multiple copies of 3 enzymes with same coenzymes as PDH; inhibitors are its products (succinyl CoA) and activators are Ca²⁺ in muscle

pg 1095-1097

Question 8

step 6 of TCA cycle

Answer 8

• succinate dehydrogenase (embedded in inner mitochondrial membrane) always has tightly bound FADH
• FAD is similar to NAD+ (electron carrier) and highly reactive; hydrogen molecules bond are easily lost when exposed to water

pg 1097

Question 9

TCA Cycle summary of regulation

Answer 9

• low energy states activate
• low energy: high AMP/ADP, high NAD+
• high energy states inhibt
• high energy: high ATP, high NADH

pg 1098

Question 10

mitochondria: ETC function

Answer 10

• ETC couple the oxidation of the reduced carries produced by the TCA cycle with the production of ATP by oxidative phosphorylation
• components are embedded in the inner mitochondrial membrane (IMM)
• cristae in IMM increase SA to have a lot of proteins attached and produce ATP very quickly

pg 1099

Question 11

mitochondria: IMM composition

Answer 11

• cardiolipin is a unique lipid in mitochondria
• 2 molecules are esterified through their phosphate groups
• exclusive to the inner mitochondrial membrane
• maintains the structure and function of ETC complexes

pg 1100

Question 12

Barth syndrome

Answer 12

• TAZ gene mutations (~160) result in production of tafazzin proteins with little or no function
• linoleic acid not added to cardiolipin, which causes problems with normal mitchondrial shape and functions such as energy production and protein transport
• tissues with high energy demands are most susceptible
• WBCs have abnormally shaped mitochondria leading to a weakened immune system and recurrent infections
• very rare X-linked disease

pg 1100

Question 13

mitochondria number and oxidative capacity

Answer 13

• oxidative capacity is determined by number of mitochondria in the cell (more mitochondria, greater oxidative capacity)
• erythrocytes have no mitochondria (NO ATP through oxidative phosphorylation -> ONLY glycolysis.. 2 ATP per glucose)
• hepatocytes have very high mitochondrial content (liver is central organ for metabolism)

pg 1101

Question 14

Electron Transport Chain (ETC)

Answer 14

4 protein complexes split into oxidation-reduction components (accepts e- and donates to the next molecule) and mobile components

• oxidation-reduction components: flavin mononucleotide (FMN); Fe-S centers; iron (Fe) in cyt b, c1, c, a, and a3; copper (Cu) in cyt a and a3
• mobile components: coenzyme Q (CoQ) and cytochrome C (cyt c)

pg 1102

Question 15

structure of Fe centers: porphyrins

Answer 15

• porphyrins are cyclic compounds that readily bind metal ions, usually ferrous (Fe²⁺) or ferric (Fe³⁺) iron forming metalloporphyrin (a prosthetic group of some proteins); most common in humans is heme

Heme porphyrin

• structure: one Fe²⁺ coordinated in the center of the tetrapyrrole ring of protoporphyrin IX
• function: a prosthetic group for hemoglobin, myoglobin, the cytochromes in ETC, cytochrome p450 monooxygenase system (CYP), other enzymes (catalase, nitric oxide synthase, and peroxidase)

pg 1103

Question 16

heme in cytochromes vs hemo/myoglobin

Answer 16

• cytochromes: the heme iron interconverts between the oxidized (Fe³⁺) and the reduced (Fe²⁺) states during electron transfer
• hemoglobin and myoglobin: heme iron must remain in the reduced (Fe²⁺, ferrous) state for proper O₂ binding and release

pg 1104

Question 17

heme proteins have color

Answer 17

all porphyrins have absorb visible light because they have conjugated double bonds

pg 1105

Question 18

oxidative phosphorylation and chemiosmosis

Answer 18

• as electrons are transferred, protons are released into intermembrane space
• protons come back in to synthesize ATP
• coupling in normal mitochondria: ATP synthesis is coupled to e- transport through the H+ gradient, change in one has the same effect on the other

pg 1106, 1110

Question 19

energy yield of oxidative phosphorylation

Answer 19

• oxidative phosphorylation yields 11 ATP
• substrate-level phosphorylation yields 1 ATP
• energy yield per 1 acetyl-CoA = 12 ATP
• estimates base on presumptive yield from ETC
• comparatively, energy yield per 1 glucose is 36-38 ATP

pg 1107

Question 20

uncoupling

Answer 20

• H+ flow back through the membrane without generation of ATP
• non-shivering thermogenesis (energy released as heat)
• natural - uncoupling proteins (UCPs) localized in mitochondrial membrane: UCP1 -> in brown adipose tissue; UCP2,3,4,5 -> expressed in tissue specific manner but not well understood
• synthetic uncouplers -> chemicals that increase the permeability of the inner mitochondrial membrane to H+

pg 1111

Question 21

inhibitors of the ETC

Answer 21

• blocking electron transfer by any one of these inhibitors stops electron flow from substrate to oxygen because the reactions of the ETC are tightly coupled like meshed gears
• carbon monoxide (CO): inhibits transfer of electrons from complex IV to oxygen (end electron acceptor)
• cyanide (CN): inhibits transfer of electrons through complex IV to oxygen

pg 1112

Question 22

OXPHOS diseases

Answer 22

mitochondrial diseases that affect proteins involved in oxidative phosphorylation

pg 1113

Question 23

lactic acidosis

Answer 23

mechanisms that increase lactate/lactic acid:

1. decreased oxidation of NADH and FAD(2H) in the ETC results in pyruvate -> lactate and fatty acids -> triglyceride
2. deficiencies or inhibition of TCA cycle enzymes inhibit acetyl-CoA oxidation, leading to increased pyruvate and lactate formation
3. anoxia, ischemia, cyanide, CO poisoning and other interruptions of the ETC prevent electron flow and ATP synthesis, so glycolysis operates anaerobically to produce ATP, and lactate is formed
4. genetic defects in proteins encoded by mtDNA decrease electron transport and ATP synthesis, so glycolysis operates anaerobically to produce ATP, and lactate is formed

pg 1114