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CHEMISTRY 4: RESPIRATORY MODULE

!Chapter 6: Myoglobin & Hemoglobin


!Chapter 32: Porphyrins & Bile Pigments
!Chapter 10: Bioenergetics: The Role of ATP
!Chapter 11: Biologic Oxidation

CHAPTER 32: PORPHYRINS AND BILE PIGMENTS

INTRODUCTION
I. BIOMEDICAL IMPORTANCE
A. Porphyrias
B. Jaundice
II, IMPORTANCE OF METALLOPORPHYRINS AND HEMOPROTEINS
A. Porphyrins
B. Substituent Side Chains in Porphyrins
1. Type III Porphyrin
2. Type I Porphyrin

BIOSYNTHESIS OF HEME

I. SUCCINYL – CoA + GLYCINE


II. SYNTHESIS OF ALA
III. SYNTHESIS OF PORPHOBILINOGEN (PBG
IV. SYNTHESIS OF HYDROXYMETHYLBILANE (HMB)
V. SYNTHESIS OF UROPORPHYRINOGEN
VI. SYNTHESIS OF THE HEME
VII. ALA SYNTHASE
VIII. PROPERTIES OF PORPHYRINS
IX. GENETIC DISORDERS IN PORPHYRINS
A. Porphyrias
B. Photosensitivity

CATABOLISM OF HEME

I. PRODUCTION AND MECHANISM OF BILIRUBIN


A. Uptake of Bilirubin by the Liver
B. Conjugation of Bilirubin with Glucuronic Acid
C. Secretion of Bilirubin
D. Fate of Conjugated Bilirubin
II. HYPERBILIRUBINEMIA CAUSES JAUNDICE
A. Hyperbilirubinemia
B. Jaundice / Icterus
III. DISEASES OF ELEVATED UNCONJUGATED BILIRUBIN
A. Hemolytic Anemia
B. Neonatal Physiologic Jaundice
C. Crigler-Najjar Syndrome: Congenial Non-Hemolytic Syndrome
D. Gilbert Syndrome
E. Toxic Hyperbilirubinemia
IV. DISEASES IN CONJUGATED BILIRUBIN
A. Obstruction of Biliary Tree
B. Dubin-Johnson Syndrome
C. Rotor Syndrome
HEMOGLOBIN AND ITS ROLE IN GAS TRANSPORT
I. BIOMEDICAL IMPORTANCE
II. HEMOGLOBIN
III. RESPIRATORY SYSTEM AFFECTS BLOOD-GAS CONCENTRATION
IV. ALLOSTERIC PROPERTIES OF HEMOGLOBIN IN ITS OXYGENATION
A. Cooperative Binding
B. P50 Expresses the Relative Affinities of different Hemoglobins for Oxygen
C. Oxygenation of Hemoglobin is accompanied by Large Conformational Changes
D. Hemoglobin transports Carbon Dioxide and Protons in to the Lungs
E. Protons Arise from Rupture of Salt Bonds when Oxygen Binds to T-State Hemoglobin
V. PHYSICAL FACTORS THAT AFFECT OXYGEN BINDING
A. High Temperature
B. High Levels of BPG
C. Low pH
VI. SUMMARY OF OXYGEN – CARBON DIOXIDE BINDING
A. In the Lungs
B. In the Peripheral Tissues
VII. MUTANT HUMAN HEMOGLOBINS (not bind with Oxygen)
A. Methemoglobin
B. Hemoglobin-M
C. Hemoglobin-S
VIII. TWO PROCESSES REGULATE H+ DERIVED FROM CO2 TRANSPORT
A. Buffering
B. Isohydric Mechanism
IX. CLINICAL CORRELATIONS
A. Cyanosis
B. Glycosylated Hb

CHAPTER 10: BIONERGETICS (ROLE OF ATP)


INTRODUCTION
I. METABOLISM (Catabolism and Anabolism make-up Metabolism)
A. Catabolism
B. Anabolism
II. LAWS OF THERMODYNAMICS
A. Law of Conservation of Energy
B. Law of Entropy

BIOENERGETICS
I. CONCEPT OF FREE ENERGY
II. KINDS OF CELLULAR REACTIONS
A. Exergonic Reactions
B. Endergonic Reaction
III. REDOX REACTIONS
A. Oxidation
B. Reduction
IV. COMPOUNDS WITH HIGH ENERGY POTENTIAL
A. Substrate Level Phosphrylation
B. Oxidative Phosphrylation
V. CHARACTERISTIC STRUCTURES
A. Enol Phosphate
B. Acid Anhydrides
C. Guanidinium Phosphates
D. Thiol Esters
VI. OXIDATIVE PHOSPHORYLATION
A. Electron Carriers (NAD and FAD: found in Mitochondria)
B. Participating Enzymes
CHAPTER 32: PORPHYRINS AND BILE PIGMENTS

INTRODUCTION
I. BIOMEDICAL IMPORTANCE
-Porphyrins and Iron are needed in the synthesis of Heme
-basic to understanding varied functions of Hemoproteins

**Porphyrins and Iron -needed for synthesis of Heme


**Bile Pigments & Iron -products of degradation of Heme

A. Porphyrias -group of diseases caused by abnormalities in pathway of biosynthesis of Porphyrins


-not very prevalent

B. Jaundice -due to elevation of Bilirubin in the plasma


-elevation is due to overproduction of Bilirubin or to failure of its excretion
-seen in numerous diseases ranging from hemolytic anemias to viral hepatitis and to
cancer of the Pancreas

II, IMPORTANCE OF METALLOPORPHYRINS AND HEMOPROTEINS

A. Porphyrins -cyclic compounds formed by the linkage of four pyrrole rings through –HC=
Methenyl Bridges
-can form complexes with Metal Ions bound to the Nitrogen Atom of the Pyrrole Rings
-proteins that contain Heme (Hemoproteins) are widely distributed in nature
-ex) Iron Porphyrins: Heme
Magnesium-containing Porphyrin: Chlorophyll

IMPORTANT HEMOPROTEINS FUNCTION


Hemoglobin Transport oxygen in blood
Myoglobin Storage of oxygen in muscles
Cytochrome C Involved in Electron Transport Chain
Cytochrome P450 Hydroxylation of Xenobiotics
Catalase Degradation of Hydrogen Peroxide
Tryptophan Pyrrolase Oxidation of Tryptophan

B. Substituent Side Chains in Porphyrins


-Porphyrins in nature are compounds in which various side chains are substituted for the eight
hydrogen atoms numbered in Porphin Nucleus
-has Acetate (A) and Propionate (P) substituents

1. Type III Porphyrin -asymmetric distribution


-reverse order of A – P in ring IV
-more abundant
-includes Heme and its immediate precursors
-ex) Uroporphyrin: 1st isolated from Urine
Coproporphyrin: 1st isolated from Feces

2. Type I Porphyrin -symmetric distribution


BIOSYNTHESIS OF HEME
-occurs in most mammalian cells with the exception of mature erythrocytes (do not contain
mitochondria)
-approximately 85% of Heme Synthesis occurs in Erythroid Precursor Cells in the Bone Marrow and
the majority of the remainder in Hepatocytes
-Glycine is needed in Synthesis

Ferrochelatase
Protoporphyrin III (IX) Heme

Fe2++

**Parent of Heme: Protoporphyrin III


**Enzyme Needed: Ferrochelatase
**Rate Limiting: ALA Synthase (key regulatory enzyme in Hepatic Biosynthesis)

**Ferrous (Fe2++) is used, not Ferric (Fe3++)

Succinyl CoA + Glycine


(Pyridoxal Phosphate)

A-Amino-B-Ketoadipate
(ALA Synthase)

S-Aminolevulinate (ALA) + S Aminolevulinate (ALA)


(2 Molecules)
(ALA Dehydratase)

Porphobilinogen (1st Precursor Pyrrole)


(Uroporphyrinogen I Synthase)

Hydroxymethylbilane

(Uroporphyrinogen III Synthase) (Spontaneous Reaction)

Uroporphyrin III Uroporphyrinogen III Uroporphyrinogen Uroporphyrin I


(Uroporphyrinogen Decarboxylase)

Coproporphyrin III Coproporphyrinogen III Coproporphyrinogen Coproporphyrin I

Protoporphyrinogen III

Protoporphyrin III
(Ferrochelatase)

HEME
I. SUCCINYL – CoA + GLYCINE
-reaction occurs in the Mitochondria
-starting materials to synthesize Heme
-Succinyl CoA is derived from Citric Acid in the Mitochondria

II. SYNTHESIS OF ALA


-Pyridoxal Phosphate is the necessary enzyme to Activate Glycine
-product is A-Amino-B-Ketoadipic Acid
-A-Amino-B-Ketoadipic Acid is decarboxylated to form A-Aminolevulinate (ALA)
-the Rate Controlling enzyme is ALA Synthase (which Catalyzes the reaction sequence)

III. SYNTHESIS OF PORPHOBILINOGEN (PBG)


-2 molecules of ALA are condensed by ALA Dehydratase forming Porphobilinogen (PBG) + 2H20
-ALA Dehydratase is a zinc containing enzyme sensitive to inhibition by lead (lead poisoning)

IV. SYNTHESIS OF HYDROXYMETHYLBILANE (HMB)


-HMB is a Cyclic Tetrapyrrole (ie. Porphyrin)
-occurs by condensation of four molecules in a head-to-tail manner to form a linear tetrapyrrole
-reaction is catalyzed by: Uroporphyrinogen I Synthase (also called PBG Deaminase or HMB Synthase)

V. SYNTHESIS OF UROPORPHYRINOGEN
A. Uroporphyrinogen I -is synthesized when HMB Cyclizes spontaneously
-auto-oxidized by light to its respective Porphyrins

B. Uroporphyrinogen III -is synthesized by the action of Uroporphyrinogen III Synthase

VI. SYNTHESIS OF THE HEME

Uroporphyrinogen Coproporphyrinogen Protoporphyrinogen Protoporphyrin Heme


III III III III

**Uroporphyrinogen Decarboxylase -converts Uroporpphyrinogen III into Coproporphyrinogen III


-decarboxylation of all the acetate groups, which changes them
to Methyl (M) Substituents

**Ferrochelatase (Heme Synthase) -mitochondrial enzyme


-incorporates Ferrous Iron into Protoporphyrin

A. Coproporphyrinogen III -enters the Mitochondria and converted into Protoporphyrinogen III
and then to Protoporphyrin III

B. Protoporphyrin III -Ferrous Iron is incorporated and then it forms Heme

VII. ALA SYNTHASE


-key regulatory enzyme in hepatic biosynthesis of Heme
-occurs in both Hepatic (ALAS1) and Erythroid (ALAS2) forms
-Heme can act as a Negative Regulator of Synthesis of ALAS1
VIII. PROPERTIES OF PORPHYRINS
• Porphyrinogens (no double bonds) are colorless while Porphyrins are Colored
• Sharp Absorption band near 400nm (Soret Band)
• Strong Red Fluorescence when dissolved in strong organic solvents

A. Application: Cancer Phototherapy


-tumors often take up more Porphyrins than do normal tissues
-Hematoporphyrin are administered to a patient with an appropriate tumor
-Tumor is then exposed to an Argon Laser which excited Porphyrins (producing Cytotoxic Effects)

B. Spectrophotometry
-used to test for Porphyrins and Precursors
-Coproporphyrins and Uroporphyrins are of clinical interest because they are excreted in high
amounts in the Prophyrias

IX. GENETIC DISORDERS IN PORPHYRINS

Mutations in DNA

Abnormalities of the
Enzymes of Heme Synthesis

Accumulation of ALA Accumulation of Porphyrinogens


and PBG and/or decrease in Skin and Tissues
in Heme in Cells

Neuropsychiatric Signs Spontaneous Oxidation of


and Symptoms Porphyrinogens to Porphyrins

Photosensitivity

A. Porphyrias -group of disorders due to abnormalities in biosynthesis of Heme


-can be genetic or acquired
-doctors must give drugs which use up Heme

B. Photosensitivity -favoring nocturnal activities


-exhibited by some patients with Porphyrias
CATABOLISM OF HEME
-erythrocytes are destroyed regularly
-when Hemoglobin is destroyed in the body:
1. Globin -degraded to constituent Amino Acids which are reused
2. Iron -iron in heme enters the iron pool and reused
3. Porphyrin -degraded mainly in Reticuloendothelial Cells of the Liver, Spleen, Bone Marrow

**Heme Oxygenase -complex enzyme system which carries out the catabolism of Heme

I. PRODUCTION AND MECHANISM OF BILIRUBIN


-Heme Oxgenase System is involved
-when Heme from Heme Proteins reach the Oxygenase system, Ferrous is oxidized to Ferric (Hemin)
-Hemin is then reduced to Heme by NADPH and oxygen is added to the A-Methynyl Bridge between Pyrroles I
and II of Porphyrin
-Ferrous ion is then oxidized into Ferric form and is released
-Carbon Monoxide is produced and Biliverdin is produced from the splitting of the Tetrapyrrole Ring

**Two Forms of Bilirubin:


1. Unconjugated Bilirubin -free form of Bilirubin en route to the liver from the RE Cells
-goes to the liver; not water soluble
-requires methanol to initiate coupling with Ehrlich’s Diazo Reagent (Vander
Berg Reaction)
-“Indirect Reacting Bilirubin”! Methanol needed to extract it
-the only Bilirubin that can cross the Blood Brain Barrier of CNS causing
Encephalopathy (Kernicterus) bec it is insouble

2. Conjugated Bilirubin -“Direct Acting” with Diazo Reagent


-water soluble
-found in cases of Jaundice due to Biliary Obstruction
-the only Bilirubin that can appear in Urine: Choluric Jaundice

**Choluria -presence of Bilirubin in Urine

**Biliverdin Reductase -soluble enzyme which reduces Methenyl Bridge between Pyrroles to produce the
Bilirubin (yellow pigment)
-1 g of hemoglobin yields 35mg of Bilirubin

**Bilirubin -when formed in peripheral tissues, it is transported to the liver by Albumin


-primary metabolism occurs in the Liver and divided into three processes:
1. Uptake of Bilirubin by Liver Parenchymal Cells
2. Conjugation of Bilirubin with Glucuronate in ER
3. Secretion of Conjugated Bilirubin into Bile

Bilirubin in Albumin
BLOOD
A. UPTAKE

Bilirubin

B. CONJUGATION HEPATOCYTE

Bilirubin Diglucuronide

C. SECRETION
BILE DUCTULE
Bilirubin Diglucuronide
A. Uptake of Bilirubin by the Liver
-in the liver, Bilirubin is removed from Albumin and taken up at sinusoidal surface of
hepatocytes by a carrier-mediated saturable system (Facilitated Transport System)
-once it enters Hepatocytes, it can bind to certain Cystolic Proteins

B. Conjugation of Bilirubin with Glucuronic Acid


-conjugation of Bilirubin is catalyzed by a specific Glucuronosyltransferase which is mainly
located in the Endoplasmic Reticulum
-Bilirubin is convertedinto Bilirubin Diglucuronide:
Biliburin ! B. Monoglucuronide ! B. Diglucuronide

UDP-Glucuronic Acid UDP-Glucuronosyl Transferase Biliburin Diglucoronide


+ +
Bilirubin Monoglucuronide UDP

**Bilirubin -nonpolar and would persist in cells (eg bound to lipids)


-Unconjugated is Insoluble in the Liver
-Conjugated form in the Liver: Bilirubin Diglucuronide

**Conjugation -hepatocytes convert Bilirubin to a polar form (which is readily


excretable in Bile)

**Glucuronosyltransferase -enzyme which uses UDP-Glucuronic Acid as Glucoronosyl


donor and is refered to as Bilirubin-UGT

C. Secretion of Bilirubin
-secretion occurs as an active transport mechanism (Rate Limiting for the entire process of
Hepatic Bilirubin Metabolism)
-protein involved in secretion is MRP-2 (Multidrug Resistance-Like Protein 2) or MOAT

D. Fate of Conjugated Bilirubin


-when conjugated Bilirubin reaches the terminal ileum, Glucoronides are removed by
B-Glucuronidase
-pigment is reduced by the Fecal Flora to a group of colorless compounds: Urobilinogens
-Urobilinogens are oxidized and formes the Colored Urobilins which is responsible for the
color of stools

Glucuronides fecal flora Urobilinogens oxidation Urobilins


(Colorless) (Colored)
II. HYPERBILIRUBINEMIA CAUSES JAUNDICE
A. Hyperbilirubinemia -Bilirubin > 1 mg/dL
-the normal level is less than 1mg /dL
-may be classified as:
!Retention Hyperbilirubinemia -due to overproducion
!Regurgitation Hyperbilirubinemia -due to reflux into bloodstream

**Causes: 1. Increased production of Bilirubin that the normal liver can excrete (Hemolisis)
2. Failure of a damaged liver to excrete Bilirubin produced in normal amounts

B. Jaundice / Icterus -Bilirubin exceeds 2-2.5 mg/dL


-has two types:
!Choluric Jaundice -occurs in regurgitation hyperbilirubinemia
-presence of bile pigments in urine
!Acholuric Jaundice -presence of an excess inconjugated bilirubin

III. DISEASES OF ELEVATED UNCONJUGATED BILIRUBIN


A. Hemolytic Anemia -important causes of unconjugated hyperbilirubinemia

B. Neonatal Physiologic Jaundice -most common cause of unconjugated hyperbilirubinemia


-results from accelerated hemolysis at the time of birth and an
immature hepatic system
-can result in Kernicterus ! Mental Retardation
-Treatments Include:
a. Phenobarbital -because it can induce this Bilirubin
metabolizing system
b. Phototherapy -to promote hepatic excretion of unconjugated
bilirubin

C. Crigler-Najjar Syndrome: Congenial Non-Hemolytic Syndrome


1. Type I -severe congenital jaundice due to mutations
2. Type II -more benign course than type I

D. Gilbert Syndrome -caused by mutations in the Gene Encoding Bilirubin-UGT

E. Toxic Hyperbilirubinemia -due to hepatic parenchymal cell damage, which impairs conjugation

IV. DISEASES IN CONJUGATED BILIRUBIN


A. Obstruction of Biliary Tree -most common
-often due to the gallstone or CA of the head of the Pancreas
-Cholestatic Jaundice

B. Dubin-Johnson Syndrome -consists of Hyperbilirubinemia in childhood or adult life

C. Rotor Syndrome -characterized by Chronic Conjugated Hyperbilirubinemia and Normal Liver

V. SUMMARY OF BIOSYNTHESIS:
• Hemoproteins (Hemoglobin and Cytochromes) contain heme (Heme is an iron-porphyrin compound in which
4 pyrrole rings are joined by Methenyl Bridges
• Biosynthesis of the Heme ring occurs in the Mitochondria and Cytosol via eight Enzymatic Steps
• Porphyrias result from abnormalities in heme biosynthesis (RBC and Liver Cell are the major sites of
Metabolic Expression of Porphyrias
• Catabolism of heme ring is inflated by the enzyme heme oxygenase, producing a linear tetrapyrrole
• Biliverdin is an early product of catabolism and on reduction yields Bilirubin
• In the Liver, Bilirubin is made water soluble by conjugation with 2 molecules of Glucuronic Acid and is
secreted in the Bile
• Jaundice is due to elevation of the level of Bilirubin in the Blood
HEMOGLOBIN AND ITS ROLE IN GAS TRANSPORT
I. BIOMEDICAL IMPORTANCE
-Myoglobin and Hemoglobin maintain a supply of Oxygen essential for Oxidative Metabolism
-illustrate both protein structure – function relationships and the molecular basis of genetic diseases
such as sickle cell disease and the thalassemias

**Myoglobin -stores oxygen as a reserve against oxygen deprivation


**Hemoglobin -transport oxygen to tissues & returns Carbon Dioxide and protons to the lungs
**Cyanide -disrupt physiologic function of Cytochrome Oxidase
**Carbon Monoxide -disrupt the physiologic function of Hemoglobin

II. HEMOGLOBIN
-effective carrier because oxygen is not soluble enough in blood plasma
-control oxygen delivery (since oxygen affinity is responsive to changing physiological conditions)
-Histidine is the Amino Acid Residue in Hemoglbin formation in Oxygen binding
-contains a Heme: cyclic tetrapyrrole with four molecules of Pyrrole and an atom of Fe2+ at the center

A. Deoxyhemoglobin -“T-State” (Taut State)


-cavity admits BPG in between the B-Chains of the Hemoglobin
-low affinity conformation (lost all oxygen)

B. Oxyhemoglobin -“R-State” (Relaxed State)


-cavity is smaller and it no longer accommodates BPG easily so binding of BPG is
much weaker
-high affinity conformation (attachment for all oxygen)

III. RESPIRATORY SYSTEM AFFECTS BLOOD-GAS CONCENTRATION


-Alveoli do not change in size during inhalation and exhalation (only the airways do)
-gas exchange between the airways and the alveoli proceeds by diffusion

A. Importance of Respiratory Anatomy


1. Gas composition of Alveolar Air differs from that of the Atmosphere (Alveoli, being a large
dead space and the gases are not completely replaced by fresh air with each breath)
2. Gases can exchange freely in the blood that flows thru the pulmonary capillaries during
Inspiration and Expiration

B. Unusual Properties of Physiological Oxygen Carrier


1. It should be able to bind Oxygen at an Oxygen-Tension of about 100mmHg (Partial Pressure of
O2 in the Alveoli)
2. It must be able to release Oxygen to the Extrapulmonary Tissues where Oxygen Tension is
Lower

**Hemoglobin is a GOOD Physiological Oxygen


-it is 98% Saturated in the Lungs and only about 33% Saturated in the Working Muscle
-under these conditions, it delivers about 65% of the Oxygen can carry
-it has a Sigmoidal Curve

**P50: Partial Pressure at 50% Saturation


-most common way of expressing Hemoglobin-Oxygen Affinity
-P50 of Hemoglobin is 27 mmHg (at 27mmHg, Hemoglobin is 50% Saturated)
-P50 expresses the relative affinities of different Hemoglobins for Oxygen
-Oxygenation of Hemoglobin is accompanied by large conformational changes
IV. ALLOSTERIC PROPERTIES OF HEMOGLOBIN IN ITS OXYGENATION

A. Cooperative Binding
-permits Hemoglobin to maximize both the quantity of Oxygen loaded at the PO2 of the lungs
and the quantity of Oxygen released at the PO2 of the Peripheral Tissues
-binding of First Oxygen to the deoxyHb molecule shifts the Heme ring and this motion is
transmitted to the Proximal Histidine
-this result to further conformational changes of the other subunits of hemogllbin
-critically important to Aerobic Life

B. P50 Expresses the Relative Affinities of different Hemoglobins for Oxygen


!P50 of Hemoglobin = 27% (better carrier)
!P50 of Myoglobin = Lower

C. Oxygenation of Hemoglobin is accompanied by Large Conformational Changes


-Deoxyhemoglobin (without oxygen) changes to Oxyhemoglobin (with oxygen)

**BPG or 2,3-Biphosphoglycerate
-the higher concentration, the more efficient in the delivery of Oxygen
-the concentration in the Red Blood Cell rise during tissue Hypoxia
-its high levels enhance formation of Deoxyhemoglobin at partial pressure of Oxygen
so that Hemoglobin then delivers more of its Oxygen to tissues

**BPG Mechanism will not compensate for tissue hypoxia when the Partial Pressure of Oxygen
in the lungs fall too low
!At low pressure of Oxygen in Lungs (cancer)
!Cannot be compensated if Pressure is too low

**HbF (Fetal Hemoglobin) -has higher affinity for oxygen


-in presence of BPG, oxygen affinity of HbF is higher than
that of maternal Hb
-optimizes transfer of oxygen from maternal to fetal circulation
D. Hemoglobin transports Carbon Dioxide and Protons in to the Lungs
-after releasing oxygen at the tissues, Hemoglobin picks up CO2 and Protons and transports
them into the Lungs
-Hb carries Carbon Dioxide as Carbamates formed with amino terminal nitrogens of the
polypeptide chains

CO2 + Hb–NH3+ = 2-H + Hb – NH--C–C

**Blood Carbon Dioxide is Present in 3 Forms:


-carbon dioxide from tissues enters the capillaries
-some CO2 become dissolved, some are converted to CarbaminoHb, and some are
converted into Bicarbonates
-Bicarbonates exit the RBC and Cl- enters to balance (Chloride Shift) electroneutrality
!Dissolved Carbon Dioxide = 9%
!Carbaminohemoglobin = 13%
!Bicarbonates (HCO3) = 78%

1. Carbamates -change the charge on amino terminals from (+) to (-), favoring salt bond
formation between the A and B chains
-account for about 15% of the Carbon Dioxide in the Venous Blood
-Carbon Dioxide attached to hemoglobin

2. HCO3 -most common form of Carbon Dioxide


-when it exits the RBC, Cl- enters the cell

3. Dissolved CO2

**Deoxyhemoglobin -binds one proton for every 2-Oxygen Molecules released


-contributes to the buffering capacity of the blood

**Bohr Effect -reciprocal coupling of Proton and Oxygen binding


-dependent upon cooperative interactions between hemes of the
Hemoglobin Tetramer
-Myoglobin has no Bohr Effect

carbonic anhydrase spontaneous


CO2 + H20 H2CO3 2 HCO3- + 2 H+

" CO2 generated in Peripheral Tissues combines with water to form Carbonic Acid
which dissociates into Protons and Bicarbonate ions
" Deoxyhemoglobin binds protons and deliver them to the lungs
" In the Lungs, the uptake of oxygen by Hemoglobin releases protons that combine
with Bicarbonate Ion, forming Carbonic Acid
" Carbonic Acid when dehydrated by Carbonic Anhydrase becomes Carbon Dioxide
and is then exhaled

E. Protons Arise from Rupture of Salt Bonds when Oxygen Binds to T-State Hemoglobin
-conversion to oxygenated R-State break salt bridges involving B-Chain residue at His146
-protons responsible for the Bohr Effect arise from rupture of salt bridges
-the dissociation of Protons from His 146 drives conversion of Bicarbonate to Carbonic Acid
-upon release of Oxygen, the T-Structure and its salt bridges reform
V. PHYSICAL FACTORS THAT AFFECT OXYGEN BINDING

A. High Temperature -weakens Hb’s oxygen affinity


-it shifts the dissociation curve to the right
-more oxygen readily released to Periphery to be utilized by tissue
-in fever, out tissues are greatly metabolizing which is why oxygen is more
available

B. High Levels of BPG -the higher concentration, the more efficient in the delivery of Oxygen
-stabilizes the T-State; Right shift
-in Hypoxia: produce more BPG and increased release of oxygen into tissues

C. Low pH -stablizes the T-state (better unloading of oxygen) by right shift


-a decrease in pH is often associated with increased oxygen demand
-increased metabolic rate increases production of CO2 as in muscular exercise
and Hypoxic Tissue ! Lactic Acid ! Acidosis
-acids produced by Metabolism help release oxygen to support that metabolism

**Adaptation to High Altitude

Prolonged exposure to Increase in Number Concentration of


High altitude of Erythrocytes Hb and BPG

!elevated BPG lowers the affinity of Hb Affinity for Oxygen (decreases P50) which enhances
the release of Oxygen at tissues
VI. SUMMARY OF OXYGEN – CARBON DIOXIDE BINDING

**The Sigmoidal Curve


!at 100mmHg (in lungs), high affinity for oxygen
!at 27mmHg (in tissues), low affinity for oxygen

A. In the Lungs
**Conditions in the Lungs:
-high Pressure for Oxygen (around 100mmHg)
-high pH, low [H+]
-Hemoglobin has High Affinity for Oxygen

**Events Taking Place:


" Blood coming from the peripheral tissues deposit the Bicarbonates and H+ and this in turn
produces Carbon Dioxide which is Exhaled

carbonic andhydrase
HCO3- + H+ H2CO3 CO2 + H20
(carbonic acid)

" The pressure for oxygen in lungs is high, which increases the Hemoglobin affinity for
oxygen. Oxygen binds to the Hemoglobin which releases the Protons (H+) and drives the
exhalation of CO2
" Increase in Pressure of Oxygen promotes proton release which in turn binds with
Bicarbonate to produce Carbon Dioxide

B. In the Peripheral Tissues


**Conditions in the Tissues
-low Pressure for Oxygen (around 27mmHg)
-low pH, high [H+]
-Hemoglobin has Low Affinity for Oxygen

**Events Taking Place:


" In metabolizing tissues, CO2 is produced which is converted into Bicarbonates and Protons
(H+)

carbonic anhydrase spontaneous


CO2 + H20 H2CO3 2 HCO3- + 2 H+

" Blood coming from heart is oxygenated and it unloads the oxygen in the tissues
" Deoxyhemoglobin binds one proton for every two Oxygens released, contributing to the
buffering capacity of blood
" Lower pH in tissues (aided by Carbamation) stabilizes the T-State and enhances the delivery
of Oxygen
" In the tissues, affinity for oxygen is low which caused the release of Oxygen
VII. MUTANT HUMAN HEMOGLOBINS (not bind with Oxygen)

A. Methemoglobin -heme iron is Ferric (Fe3+) instead of Ferrous (Fe2+)


-can neither bind nor transport oxygen
-can arise as side effect of drug like Sulfonamides (oxidation of Fe2+ to Fe3+)

B. Hemoglobin-M -Histidine F8 (His F8) has been replaced by Tyrosine


-the iron forms a tight ionic complex with Phenolate Anion of Tyrosine stabilizing
Fe3+ form: Oxygen affinity is Reduced and Bohr Effect is Absent

C. Hemoglobin-S -the non-polar amino acid valine has replaced the polar surface residue GluG of
the B-Subunit
-Hydrophobic “Sticky Patch” on the surface of B-Subunit of both oxyHbs and
deoxyHbs
-distort the erythrocytes into sickle shape rendering it vulnerable to Lysis in the
Splenic Sinusoides (low PO2 exacerbates the tendency to polymerize)

VIII. TWO PROCESSES REGULATE H+ DERIVED FROM CO2 TRANSPORT


-Hemoglobin (besides carrying oxygen and carbon dioxide) also plays the major role in handling H+
produced in CO2 transport by buffering and by the Isohydric Mechanism

A. Buffering -Hb’s buffering mechanism at ionizable groups with pK values close to the
Intraerythrocute pH
-more of Hb’s buffering ability is provided by the Imidazole side chains of the
Histidine Residues (38 Histidine per Hb Tetramer)

**Hemoglobin -most important non-bicarbonate buffer in the blood


-control of excess H+ generated during normal CO2 Transport
-comparison with other buffers:
!Buffering with Hb -50%
!Other Buffers -10%
!Isohydric Mechanism -40%

B. Isohydric Mechanism -Hb becomes a stronger acid and releases H+ when it becomes oxygenated
(Bohr Effect)
-in the capillaries, where O2 is released from Oxyhemoglobin:

HbO2 + H+ HHb + O2

**CO2 enters the capillaries and is Hydrated:

CO2 + H20 H+ + HCO3

**Combination of the two reactions:

HbO2 + CO2 + H20 HHb + HCO3- + O2


(isohydric carriage of CO2)

IX. CLINICAL CORRELATIONS

A. Cyanosis -patient’s skin or mucous membrane appears gray or (in severe cases) purple
-due to an abnormally high concentration of Deoxyhemoglobin below surface
which is responsible for color
-most commonly caused by diseases of the Cardiac or Pulmonary Systems,
resulting in inadequate Oxygenation of the blood
-in someone with low Hb, it cannot lead to Cyanosis because there is no Hb to
begin with (there will be Anemia)

B. Glycosylated Hb -quantitatively the most significant species of HbA


(HbAic) -formed by covalent binding of a glucose residue to the N-Terminal of B-Chain
at a rate that depends on the concentration of glucose
-forms more rapidly in uncontrolled diabetics
-useful measure of how well diabetes has been controlled over the preceeding
6 – 8 weeks
CHAPTER 10: BIONERGETICS (ROLE OF ATP)

INTRODUCTION
I. METABOLISM (Catabolism and Anabolism make-up Metabolism)

A. Catabolism -comprised of reactions that are usually oxidative


-reason for oxidative reaction is to generate energy from Oxidation of food
-in generating energy, body traps it in the form of High Energy Compounds
-these energized compounds are used for sources of energy to drive reactions
-“Energy Generating”

B. Anabolism -comprised of reactions that are synthetic


-product of these reactions would be formation of substance needed by body
-energy is needed: “Energy Requiring”

**Not all energy are trapped by the body (only 60% are trapped)
**Energy not trapped escapes as Heat (form of energy which cannot be utilized by the Body)

II. LAWS OF THERMODYNAMICS

A. Law of Conservation of Energy


-energy is neither created nor destroyed
-total amount of energy in a system remains constant
-energy may change in form
-energy may be transported from one region to another
-ultimate source of energy: derived from the SUN
-ex) ATP in muscle contraction is transformed from one form to the other
Chemical Energy (ATP) converted to Mechanical Energy

B. Law of Entropy
-if we leave a system on its own, it would always proceed to a state of equilibrium
-state of equilibrium: reactant forming a product; product forming a reactant
-disadvantage of equilibrium: No work is performed (nothing accomplished) Wasting energy!

**Entropy (S) -extent of disorder or randomness


-energy that is unavailable for useful work
-when S is a maximum, system is at equilibrium and no work will take place

**All processes, chemical or biological, leads to progress toward a situation of maximum S


**Living systems (highly ordered) are never at Equilibrium with surroundings

**Living organisms preserve their internal order:


1. Take free Energy (nutrients) from environment
2. Return Equal amount of energy (Heat and Entropy) to the Environment
BIOENERGETICS
I. CONCEPT OF FREE ENERGY
-energy that is useful to the Body (energy associated with work)
-when two Laws of Thermodynamics are combined: Gibb’s Equation

A. Gibb’s Equation

!G = !H - T!S **!G -change in free energy (energy available for work)


**!H -change in Enthalpy
**T -absolute temperature
**!S -change in entropy

B. Change in Free Energy: !G


-predicts the direction in which a reaction will proceed
-difference in energy between products and reactants
-!G = Gproducts – Greactants

A B **If A has greater energy than B, A will make B


**Therefore, reaction proceeds Forward
**Flow is higher to lower
!G = GB - GA

1. Spontaneous Reaction -!G is Negative if GA is Greater than GB


-proceeds to completion
-no need to do anything (just mix the reactants)
-ex) Ninhydrin Reaction, Molisch, Anthrone Test

2. Non-Spontaneous -!G is Positive if GA is Less than GB

C. Standard Free Energy Change (!G0)


**Conditions: -pH = 7
-Temperature = 250 C
-Concentration = 1 M Concentration of reactants and Products

D. !G0 and Keq (when concentration of product and reactant are known, !G0 can be obtained)

A+B C+D

Keq = ( C ) ( D )
(A) (B)

!G0 = - RT ln Keq **R =Gas Constant


=1.987 cal / molo
= - 2.3 RT log Keq
II. KINDS OF CELLULAR REACTIONS

A. Exergonic Reactions -Negative !G0


-Spontaneous Reaction
-accompanied by a liberation of Free Energy (Oxidation)
-Catabolic Reactions: give free energy

B. Endergonic Reaction -Positive !G0


-Non-Spontaneous Reaction
-requires Energy to Proceed (Reduction)
-Anabolic Reaction: needs free energy
-ex) Benedict’s Test needs Heat for reaction to take place

**Coupled Reactions -product of the first reaction must be the substrate of the second reaction
-Endergonic Reaction must be coupled to an Exergonic Reaction to proceed
-amount of energy endergonic needed must be given by exergonic reaction
-amount of energy liberated by exergonic reaction must be enough to drive
the products of endergonic
-ex) Glycolysis

**Glycolysis
+ Pi
1. Glucose Glucose – 6 – Phosphate !G = + 3.3 Kcal / mol (Endergonic)
+ H2O
2. ATP ADP + Pi !G = - 7.3 Kcal / mol (Exergonic)

Coupled Reaction: Glucose + ATP Glucose 6 – Phosphate + ADP

**There is still unused energy which is converted to Heat


III. REDOX REACTIONS
-if there is oxidation, there is also reduction (go hand in hand)
-oxidant oxidizes a substance (which is a reductant): reductant is oxidized
-the way the body traps energy from food
-when we talk of Electrons, we refer to the Electron of Hydrogen (only one Electron)

A. Oxidation -loss of electrons


-when a substance loses / donated electrons: it is Oxidized

**Oxidant -electron acceptor


-the one reduced by reductant

B. Reduction -gain of electrons


-substance which gains electrons: becomes Reduced

**Reductant -electron donor


-the one oxidized by oxidant

**Standard Reduction Potential (E0)


-constant that describes tendency of a compound to be reduced
-conditions: 250 C
pH = 7
1 M Concentration of Electron Donors and Acceptors

**Change in Standard Reduction Potential (!E0)

!E0 = Reductant – Oxidant

**Relationship Between !G0 and !E0


*!E0 =Change in Standard Reduction Potential
0 0
!G = - nF!E *F =Farade (23 Kcal / Vmol)
*n =number of electrons

!if !E is Positive, !G is Negative (Reaction will Proceed)


!if !E is Negative, !G is Positive (Non-Spontaneous Reaction)

**The Food that we eat is Oxidized by and Oxidant: Oxygen


IV. COMPOUNDS WITH HIGH ENERGY POTENTIAL
-high Energy Compounds
-!G0 = -7 to –15 Kcal / Mole is liberated when Energy is released
-contain a very Labile and Reactive Chemical Bond
-amount if Energy liberated should be sufficient enough to participate in Substrate Level Phosphrylation

**ATP -High Energy Compound used by the Body


-synthesized by:
1. Substrate Level Phosphrylation
2. Oxidative Phosphrylation

A. Substrate Level Phosphrylation


-synthesis of AT through High Energy Compounds
-amount of ATP synthesized is only 1 (one) ! Not so efficient
-no oxidation: ATP is synthesized by transferring only of energy
-one high energy bond can be synthesized at each level

B. Oxidative Phosphrylation
-synthesis by Oxidation of the Substrate and the Oxidation liberated the energy
-the Energy is trapped and used in the Synthesis of ATP

V. CHARACTERISTIC STRUCTURES
-used by the body in Substrate Level Phosphorylation

A. Enol Phosphates
o Phosphoenolpyruvic Acid

B. Acid Anhydrides
o 1,3 Biphosphoglyceric Acid (high energy bond: 1)
o ATP, GTP, TTP, CTP

C. Guanidinium Phosphates
o Creatine Phosphate (in muscles for more ATP)

D. Thiol Esters
o Acetyl Coenzyme A (from Phantothenic Acid: Vitamin B)

**Superoxide Anion Oxidase -specific enzyme that protects tissues from oxygen toxicity caused
by free radicals
VI. OXIDATIVE PHOSPHORYLATION
-ATP Synthesis Via the Respiratory Chain (chain involved in cellular respiration)
-ATP Synthesis thru oxidation of substrates
-Oxidation of substrates releases electrons
-Electrons are carried by NADH and FADH2 (carriers of Electrons)

**NAD -becomes NADH when carries electron


**FAD -becomes FADH2 when it carries electrons

A. Electron Carriers (NAD and FAD: found in Mitochondria)

1. NAD -accepts a Hydride Ion (Hydrogen Atom with one extra electron: H+ +)
-carrier of Electrons in Oxidation
-forms NADH + H+ when it accepts two electrons and one proton (the other
Proton is released in the Medium)

2. FAD -removal of Hydrogen Atom


-carrier of Electrons in Oxidation
-forms FADH2 when it accepts two electrons and two protons

3. NADP -major source for Biosynthetic reactions (involves reduction)


-mostly formed and used extra-mitochondrial
-forms NADPH + H+

B. Participating Enzymes (Oxidoreductases)


-for reaction to be fast enough for the body
-reactions in body are not that fast without the Enzymes
-even if free energy allows it to proceed, an enzyme is needed to speed up the reaction
-instead of two electron flowing in a reaction, some electrons may be lost and picked up by
substances (these substance with excess electrons will be very reactive)

1. Dehydrogenases -catalyze oxygenation (enzymes that oxidize: electrons are released)


-linked to NAD and FAD
-cannot use oxygen as Hydrogen acceptor
-transfer of Hydrogen from one substrate to another (removal of Hydrogen)
-often utilize Coenzymes as Electron Carrier
-ex) Lactic Acid ! Lactate

2. Oxidase -Transfer of two electrons from Donor to Oxygen


-forms H2O2 (side product: Hydrogen Peroxide responsible for color)
-in ETC, water is formed instead of H2O2
-uses oxygen as a Hydrogen Acceptor
-ex) Cytochrome Oxidase in ETC

3. Oxygenases -Oxidizing Enzymes


-incorporates oxygen in substances
-Two Types: Dioxygenase and Monooxygenase

a. Dioxygenase -incorporate both atoms of Oxygen in Product


b. Monooxygenase -incorporate one atom of Oxygen as Hydroxyl and as Water

BIOLOGIC OXIDATION

INTRODUCTION
• Source of energy for the body is from the Oxidation of Foods
• All molecules can be degraded into a common compound: Acetyl-CoA
• Acetyl-CoA is a high energy compound (2-Carbon High Energy Component)
• Acetyl-CoA enter TCA and the 2-Carbons are liberated as CO2
• The main purpose is to Yield energy
• Trapping of energy = Trapping of Electrons and are taken up by Electron Acceptors (NAD / FAD)
• Electrons are delivered into an Electron Transport System (ETC) / Respiratory Chain
• Transfer of Electrons from NADH & FADH2 from one member to another until it reaches the last
member: Cytochrome Oxidase
• When electrons are delivered to Oxygen, Water is formed
• When Electrons are moving, Reduction Potential is produced and free energy is released to form the
Phosphodiester Bond to form ADP + Pi to ATP

Carbohydrates Proteins Fat

Glucose Amino Acids Fatty Acids

Pyruvate

Acetyl-CoA

TCA Cycle 2CO2

3 NADH & FADH2

O
e- ! ETC ! !

H 2O
ADP + Pi ATP
PARTICIPATING ENZYMES IN BIOLOGIC OXIDATION
-enzymes involved in Oxidation and Reduction are called Oxidoreductases
-classified into four groups: Oxidases, Dehydrogenases, Hydroperoxidses, Oxygenases

I. OXIDASES
-can use oxygen as a Hydrogen Acceptor
-catalyze removal of Hydrogen from a substrate using Oxygen as an acceptor
-form water of Hydrogen Peroxide as a product

A. Cytochrome Oxidase -hemoprotein distributed in many tissues having the typical heme Prosthetic
group (such as in Hemoglobin, Myoglobin, Cytochrome)
-terminal component of Respiratory Carriers in mitochondria
-transfers electrons from oxidation of substrates by dehydrogenases to the
final acceptor: Oxygen
-poisoned by Carbon Monoxide, Cyanide, Hydrogen Sulfide
-also known as Cytochrome aa3
-two atoms of Copper are present associated with a heme unit

B. Flavoprotein Enzymes -contain Flavin Mononucleotide (FMN) / Flavin Adenine Dinucleotide (FAD)
-ex) L-Amino Acid Oxidase (FMN linked enzyme)
Xanthine Oxidase (conversion of purine bases)
Aldehyde Dehydrogenase (FAD linked enzyme)
II. DEHYDROGENASES
-cannot use oxygen as a Hydrogen Acceptor
-perform two main functions:
1. Transfer of Hydrogen from one substrate to another in a coupled redox reaction
2. Components in the Respiratory Chain of Electron Transport from substrate to Oxygen

A. NAD+ or NADP+ -used by dehydrogenases; both from Niacin


-NAD-linked Dehydrogenases catalyze Oxidoreduction Reactions
-NADP-linked Dehydrogenases found in reductive syntheses

B. FAD -most of Riboflavin-linked Dehydrogenase are concerned w/ Electron Transport

C. Cytochromes -may also be regarded as Dehydrogenase


-iron containing hemoproteins
-in respiratory chain, they are involved as carriers of electrons from Flavoproteins on
one hand to Cytochrome Oxidase on the other

III. HYDROPEROXIDASES
-use hydrogen peroxide or an organic peroxide as substrate and protect body against harmful peroxides
-two types of enzymes found in both animals and plants: Peroxidases and Catalase

A. Peroxidase -reduce peroxides using various Electron Acceptors


B. Catalase -uses Hydrogen Peroxide as Electron Donor and Electron Acceptor

IV. OXYGENASES
-catalyze direct transfer and incorporation of oxygen into a substrate molecule in two steps:
1. Oxygen is bound to enzyme at the active site
2. Bound oxygen is reduced or transferred to the substrate
-can be divided into: Dioxygenase and Monooxygenases

A. Dioxygenase -incorporate both atoms of molecular oxygen into the substrate


-ex) Liver enzymes
B. Monooxygenase -only 1 atom of molecular oxygen to substrate (other oxygen atom reduced to water)
-ex) Cytochrome P450 (for detoxification of many drugs
BIOLOGICAL OXIDATION
-oxidation of compoungs with Oxygen as the Oxidant
-enzymes: NAD and FAD-linked Dehydrogenases (except Cytochrome Oxidase)
-Dehydrogenases cannot give electrons to Oxygen, but Cytochrome Oxidase can
-removal of Hydrogen and formation of Water

!Oxidative Phosphorylation
!The Electron Transport Chain
!Importance

I. OXIDATIVE PHOSPHORYLATION
-oxidation of substrates, coupled by the synthesis of ATP
-if ATP Synthesis is not coupled to Oxidation of Substrates, the energy synthesized by oxidation is
liberated as Heat
-without oxidation, there will be no Phosphorylation (dependent on Oxidation)
-Oxidation is Independent on Phosphorylation, but Phosphorylation is dependent on Oxidation
-rate of Oxidation will increase if there is no Phosphorylation

II. THE RESPIRATORY CHAIN (ETC)


-concerned with the transport of Electrons which is transferred to Oxygen
-involves Redox reactions
-can accept 2 Electrons per cycle
-performed function well if Oxidation is coupled to Phosphorylation
-electrons move from the 1st member to the Oxygen (Reduction potential increases from 1st to O2)
-in the process, Protons are released into intermembrane space which creates a difference in pH
-movement of Electrons generates Reduction Potential create free energy (synthesis of ATP utilizes
about 7Kcal/Mol equivalent to ______Volts !should be > or = to 7Kcal/Mol)

e -s
Substrate H2 H2
(reductant: compound w/ 2e-)

Oxidized Substrate

• When Substrate donates the electrons, substrate is oxidized


• Cytochrome Oxidase forms water
• Electrons and protons transferred vary: NAD only accepts 2e- + 1H+; FAD accepts 2e-- + 2H+
• Ex) If there are two electrons which enter the ETC, two moles of Cytochrome is needed (1:1)

A. The Mitochondria
-powerhouse of the cell: site of ATP synthesis
-has an outer membrane, inner membrane, matrix within
-there is also ATP production in cytoplasm (anaerobic: not so efficient)
-Cristae increases surface area to have much enzymes to synthesize ATP

!Outer Membrane -characterized by enzymes such as Acyl-CoA Synthetase and


Glycerolphosphate Acyltransferase
!Inner Membrane -contains the Enzymes and Coenzymes of the ETC

B. Enzymes and Coenzymes


-occur as complexes in the inner membrane of mitochondria
-has Two Entry Points in membrane: Complex I – NAD + Complex II - FAD
-Redox Potential is the determining factor on where the compound will donate its Electrons
(whether it is more reduced or more oxidized)
-if compound enters Complex II, the compound is more Oxidized (because flow of electrons is
from Complex 1 – Oxygen: as we go nearer oxygen, it becomes more Oxidized)
-electrons are accepted, while protons are released in the Medium

C. Electron Transport Chain Members: all with Dehydrogenase Enzymes


-entry point determine the number of ATP’s produced
-arranged in order of increasing Redox Potential (from least to greatest)

1. Complex I -NAD (2.5 ATPs)


2. Complex II -FAD (1.5 ATPs)
3. Complex III -Cytochrome b & c
4. Complex IV -Cytochrome aa3 (Cytochrome Oxidase)
5. Complex V -ATPase (ATP synthase)

**For each site, one ATP can formed per cycle


**If substrate enters NAD: 2.5 ATP is formed for each cycle of the ETC (P:O Ratio is 2.5)
**If substrate enters FAD: 1.5 ATP is formed for each cycle of the ETC (P:O Ration is 1.5)

D. Electron Transport Chain Mobile Members


1. Coenzyme Q -also called Ubiquinone or CoQ
-oxygenation of more substrates
-links flavoproteins to Cytochrome b (with lowest redox potential)
-acts as a mobile component of ETC that collects reducing equivalents
from flavoproteins and passes them on to the Cytochromes
-has two sites of entry of Electrons: Quinol and Quinone
-receives one electron at a time to undergo changes:
!Quinol Form -Reduced Form
!Semi-Quinone -accepts 1st electron
!Quinone Form -Oxidized

2. Cytochrome C -small Hydrophilic Protein bound loosely to membrane


-accept one electron at a time

**Cytochromes -heme like prosthetic groups


-active site: Iron (Fe2+) accepts / donates electrons only
-iron accepts only one electron at a time (with NO protons)
-iron undergoes a conversion:

Ferric (oxidized state) #! Ferous (reduced state)


Fe 3+ Fe 2+

**Hemoglobin -carrier of oxygen (which binds to Iron in the Heme)


-Heme Iron remains in the Ferrous state when Oxygen binds
-when Iron becomes Ferric (Fe3+): Methemoglobin, it cannot carry O2
3. Iron Sulfur Proteins -FeS non-heme iron
-associated with Flavoproteins and with Cytochrome b
E. The Proton Motive Force
o In some areas, there is a release of Protons from mitochondrial matrix to Intramembranous
space (these protons create a Proton-Motive Forces w/c can drive reactions: enable
dehydration of ADP + Pi ! ATP)
o This causes a change in pH between Intramembranous space and the matrix
o Because of the decrease in pH, tendency of cell is to make it Homgenous and the protons
will now travel and go back into the Matrix by passing through the ATPase Enzyme in
Compartment-V (it contains the ATPase)
o Movement of the Protons produces a Proton Motive Force which drives the Synthesis of
ATP from ADP + Pi (w/o Proton Motive Force: no phosphrylation)

F. Genetic Defects in ETC


-some enzymes are absent in some complexes
-ultimate effect of absence: Low ATP Production (or none depending on protein)

III. INHIBITIONS IN OXIDATIVE PHOSPHORYLATION


A. Inhibitions in ETC
-there are three sites where inhibition can take place
-includes: Barbiturates, Antimycin, Cyanide, Carbon Monoxide

1. Barbiturates -affect at Complex I


2. Antimycin -Complex III
3. Cyanide -Complex IV: binds to Ferric (prevent reduction of Fe3+ of Cytochrome Oxidase)
4. CO -bind to the Oxidase in iron portion
5. H2S -act on Cytochrome Oxidase

B. Inhibitions of ATP Synthase: Oligomycin

C. Uncouplers -substances that separate process of Oxidation from Phosphorylation


-uncouple Oxidative Phosphorylation by binding to membrane & abolish semi-permeability
of the membrane, affecting the movement of the Protons! No proton motive force
-usually Lipophilic (binds to the mitochondrial membrane)
-ex) Dinitrophenol, Thermogenin

**Thermogenin -found in the membrane and instead of the Proton passing from
intramembranous space to the matrix, some protons are attracted to
the Thermogenin
-Protons which enter Thermogenin will not produce a Proton Motive Force
-Proton Motive Force + ATPsynthase = ATP synthesis
-without either the Force or ATPsynthase = NO ATP Synthesis