1
Citric acid cycle
Synthesis of heme. Hemoproteins
© Department of Biochemistry (J.D.) 2013

2
Phase
The conversions of nutrients
ATP yield
I.
Hydrolytic cleavage of nutrients during digestion:
Starch ® maltose ® glucose
Proteins ® peptides ® amino acids
Lipids ® fatty acids
none
II.
Intracellular catabolism of nutrients:
Glc, FA, AA ® ® ® pyruvate ® acetyl-CoA
Production of ATP in glycolysis (2 ATP/Glc)
Production of reduced cofactors (NADH+H+, FADH2)
small
III.
Citrate cycle: acetyl-CoA ® 2 CO2 + red. cofactors + ATP
Respiratory chain: oxidation of reduced cofactors
Aerobic phosphorylation: synthesis of ATP from ADP + Pi
the biggest
Three phases of nutrient catabolism

3
Sources of acetyl-CoA
•oxidative decarboxylation of pyruvate (from glucose and 6 AA)
•β-oxidation of fatty acids
•catabolism of some amino acids (Thr, Trp, Lys, Leu, Ile)
•ketone bodies utilization in extrahepatal
tissues:                                                      acetoacetate ® acetoacetyl-CoA ® 2
acetyl-CoA
•catabolism of ethanol ® acetaldehyde ® acetate ® acetyl-CoA
•

4
Compare different ways of pyruvate decarboxylation
pyruvate
simple decarboxylation
acetaldehyde
acetic acid
oxidative decarboxylation
in vitro
oxidative decarboxylation
in vivo

5
1.thiamin diphosphate (TDP)
2.lipoate
3.coenzyme A
4.FAD
5.NAD+
Oxidative decarboxylation of pyruvate is catalyzed by pyruvate dehydrogenase complex: three enzymes
and five cofactors
mitochondria

6
(1) Decarboxylation of pyruvate
thiazolium ring

7
(2) Transfer of acetyl to lipoate is redox reaction
• hydroxyethyl group is dehydrogenated to thioester during transfer
• one H atom reduces sulfur atom of lipoate to –SH group
• the second H atom goes back to TDP
 II
 0
lipoate attached to enzyme
S-acetyl hydrogen lipoate
(thioester)

8
(3) Transfer of acetyl to coenzyme A
dihydrogen lipoate

9
(4) Transfer of 2H to NAD+ via FAD


10
Balance reaction
Pyruvate dehydrogenase is allosterically inhibited by end products: acetyl-CoA + NADH
CH3-CO-COOH  +  CoA-SH  +  NAD+   ®  CO2  +  CH3-CO-S-CoA  +  NADH+H+

11
Citric acid cycle (CAC)
Krebs cycle, tricarboxylic acid cycle (TCA)
• final common pathway for oxidation of all major nutrients
• located in mitochondria, active in all cells that possess mitochondria
•  acetyl-CoA from metabolism of nutrients is oxidized to two molecules of CO2
(CH3-CO-S-CoA + 3 H2O ® 2 CO2 + 8 H + CoA-SH)
•   CAC products:
     CO2  ®  expired by lungs
     four oxidative steps ® reduced cofactors  ® respiratory chain
    GTP ®  ATP
•  most reactions are reversible, only three reactions are irreversible

12
(1) Oxaloacetate + Acetyl-CoA
Reaction type: condensation
Enzyme: citrate synthase
Cofactor: coenzyme A
Note: exothermic + irreversible
C
C
H
2
C
O
O
H
O
C
O
O
H
+
C
H
3
C
O
S
C
o
A
H
2
O
- CoA-SH
- heat
-
oxalacetate
acetyl-koenzym A
citrate
C
C
H
2
C
O
O
H
C
O
O
H
H
O
C
H
2
C
O
O
H
oxaloacetate
acetyl-CoA
  low-energy compound  Þ
  for backward reaction
  in cytosol ATP needed

13
(2) Citrate ® Isocitrate
Reaction type: isomeration  Enzyme: aconitase  Cofactor: Fe-S
Note: two-step-reaction, intermediate is cis-aconitate
•
C
C
H
2
C
O
O
H
C
O
O
H
C
H
2
H
O
C
O
O
H
C
H
C
H
2
C
O
O
H
C
H
C
O
O
H
H
O
C
O
O
H
          tertiary
hydroxyl group
       secondary
hydroxyl group
citrate
isocitrate

14
(2a) Dehydration of citrate
citrate
C
C
H
2
C
O
O
H
C
O
O
H
C
H
O
C
O
O
H
H
H
H
2
O
C
C
C
O
O
H
H
C
H
2
C
O
O
H
H
O
O
C
cis-aconitate

15
(2b) Hydratation of cis-aconitate
stereospecific reaction
C
H
C
H
2
C
O
O
H
C
H
C
O
O
H
H
O
C
O
O
H
C
C
C
O
O
H
H
C
H
2
C
O
O
H
H
O
O
C
cis-aconitate
H
2
O
isocitrate

16
Aconitase is inhibited by fluoroacetate
dichapetalum
Dichapetalum cymosum
(see also Med. Chem. II, p. 65)
FCH2COOH
reacts with oxaloacetate                          to give fluorocitrate
CAC is stopped
LD50 for human is 1 mg/kg
rat poison

17
(3) Isocitrate ® 2-oxoglutarate
Reaction type: dehydrogenation + decarboxylation
Enzyme: isocitrate dehydrogenase
Cofactor: NAD+   Note: irreversible
isocitrate
2-oxoglutarate

18
(4) 2-Oxoglutarate ® succinyl-CoA
Reaction type: oxidative decarboxylation   Enzyme: 2-oxoglutarate dehydrogenase complex
Cofactors: TDP, lipoate, CoA-SH, FAD, NAD+
Note: irreversible, similar to pyruvate dehydrogenase reaction (five coenzymes)
C
H
2
C
H
2
C
O
O
H
C
C
O
O
H
O
+
N
A
D
H

+
H
+
N
A
D
+
-
C
H
2
C
H
2
C
O
O
H
C
O
S
C
o
A
+
C
O
2
2-oxoglutarate
succinyl-coenzyme A
thioester
macroergic intermediate
H
S
C
o
A

19
(5) Succinyl-CoA + GDP + Pi
Reaction type: substrate phosphorylation
Enzyme: succinyl-CoA synthetase (succinate thiokinase)
Cofactor: coenzyme A
+
C
H
2
C
H
2
C
O
O
H
C
O
S
C
o
A
+
+
G
D
P
P
i
C
H
2
C
H
2
C
O
O
H
C
O
O
H
G
T
P
succinyl-coenzyme A
succinate
origin of oxygen?
+ CoA-SH

20
GTP is formed in three-step reaction
•Chemical energy of macroergic succinyl-CoA
•is gradually transformed into two macroergic
•intermediates and finally to macroergic GTP
•(Passing a hot potato)

21
(5a) Addition of phosphate to succinyl-CoA
mixed anhydride
four oxygen atoms in phosphate
P
O
O
O
O
H
C
O
O
C
H
2
C
H
2
C
O
S
C
o
A
H
S
C
o
A
C
O
O
C
H
2
C
H
2
C
O
O
P
O
O
O
succinyl-CoA
succinylphosphate

22
(5b) Phosforylation of His in the active site of enzyme
substituted
phosphoamide
N
N
H
E
n
z
y
me
C
O
O
C
H
2
C
H
2
C
O
O
P
O
O
O
C
O
O
C
H
2
C
H
2
C
O
O
succinylphosphate
succinate
phospho-His
N
N
E
n
z
y
me
P
O
O
O
H
+

23
(5c) Phosforylation of GDP
N
N
N
N
O
H
2
N
H
O
O
H
O
H
O
P
O
O
O
P
O
O
O
N
N
E
n
z
y
me
P
O
O
O
N
N
N
N
O
H
2
N
H
O
O
H
O
H
O
P
O
O
O
P
O
O
O
P
O
O
O
guanosine diphosphate
guanosine triphosphate
N
N
H
E
n
z
y
me
H
+

24
Distinguish
       -PO32-                                                     HPO42- (Pi)
      phosphate inorganic
P
O
O
O
P
O
O
O
O
H
phosphoryl
phosphate
virtual group
real compound

25
GTP is quickly converted to ATP
GTP  +  ADP
ATP
  +  GDP
nucleoside-diphosphate kinase

26
(6) Succinate ® fumarate
Reaction type: dehydrogenation (-CH2-CH2- bond)
Enzyme: succinate dehydrogenase
Cofactor: FAD
•
•
C
O
O
H
C
H
2
C
H
2
C
O
O
H
+   FAD
C
C
C
O
O
H
H
H
O
O
C
H
-
II
-
II
-
I
-
I
+
F
A
D
H
2
succinate
fumarate

27
Malonate is competitive inhibitor                                  of succinate dehydrogenase
Do not confuse:
malonate × malate
C
O
O
C
H
2
C
H
2
C
O
O
C
O
O
C
H
2
C
O
O
succinate
malonate

28
(7) Fumarate ® L-malate
Reaction type: hydration    Enzyme: fumarase   Cofactor: none
Notes: 1) addition of water on double bond is stereospecific
         2) hydration is not redox reaction
-
II
+
H
2
O
C
O
O
H
C
H
C
H
2
H
O
C
O
O
H
0
fumarate
L-malate
S
 =
-II
S
 =
-II
C
C
C
O
O
H
H
H
O
O
C
H
-
I
-
I

29
Distinguish: hydrolysis × hydration
substrate  +  H2O  ®
product 2
OH
substrate  +  H2O  ®
+
product 1
H
product
OH
H
Hydrolysis = decomposition of substrate by the action of water                             (typical
in esters, amides, peptides, glycosides, anhydrides)
Hydration = addition of water (to unsaturated substrates)

30
Compare: Hydration of fumarate in vivo and in vitro
in vivo: (enzymatic reaction):
only one enantiomer is formed (L-malate)
in vitro: formation of racemate
C
C
C
O
O
H
H
H
H
O
O
C
H
O
H
Enzyme
Substrate
C
C
C
O
O
H
H
H
O
O
C
H
H
H
O
H
H
O
C
C
O
O
H
C
H
2
C
O
O
H
O
H
H
C
C
O
O
H
C
H
2
C
O
O
H
H
H
O
L-malate
D-malate

31
(8) L-malate ® oxalacetate
Reaction type: dehydrogenation
Enzyme: malate dehydrogenase       Cofactor: NAD+
•
C
O
O
H
C
H
C
H
2
H
O
C
O
O
H
+
N
A
D
+
C
O
O
H
C
C
H
2
C
O
O
H
O
+
N
A
D
H
H
+
+
L-malate
oxaloacetate

32
The net equation of citrate cycle
CH3-CO-S-CoA + 3NAD+ + FAD + 2H2O + H+ + HPO42- +
  2 CO2 + CoA-SH  + 3 NADH + 3H+ + FADH2 +
• two C atoms are completely oxidized to 2 CO2
• 8 H atoms are released in the form of reduced cofactors
   (3 × NADH+H+,    1 × FADH2)

33
The energetic yield
•Products of CAC
•1 × GTP
•3 × NADH + H+
•1 × FADH2
•
•Equivalent of ATP (Resp. chain)
•1
•9
•2
•Total:  12 ATP*
* new calculations: 10 ATP

34
Factors affecting CAC
•Energy charge of the cell:
•ATP/ADP ratio and NADH/NAD+ ratio
•Allosteric inhibition
•Inhibition by products
•Supply of oxygen - CAC can proceed only at aerobic conditions (reduced cofactors must be
reoxidized in respiratory chain)

35
Key enzymes for regulation of citrate cycle: irreversible reactions
Enzyme
ATPa
NADHa
Other effect
Pyruvate dehydrogenase
y
y
y acetyl-CoAb
Citrate synthase
y
y citrateb
Isocitrate dehydrogenase
y
y
 Å ADPc
2-OG dehydrogenase
y
y succinyl-CoAb
a allosteric inhibitor – signal of high energy status of cell
b feed-back inhibitor (inhibition by a product)
c allosteric activator

36
Anaplerotic reactions of CAC
•Reactions that supply the intermediates of citrate cycle
•Carboxylation of pyruvate → oxalacetate
•(Reductive carboxylation of pyruvate → malate)
•Transamination of aspartate → oxaloacetate
•Catabolism of Phe + Tyr → fumarate
•Aspartate in the synthesis of urea/purines →  fumarate
•Catabolism of Val, Ile, Met → succinyl-CoA
•Transamination of glutamate → 2-oxoglutarate
•

37
Carboxylation of pyruvate (biotin)
B
i
o
t
i
n
C
O
O
H
H
3
C
C
O
C
O
O
H
B
i
o
t
i
n
H
C
H
2
C
O
C
O
O
H
H
O
O
C
pyruvate
oxaloacetate
pyruvate carboxylase

38
Reductive carboxylation of pyruvate
Reaction is more important for production of NADPH for reductive synthesis (FA, cholesterol)
malic enzyme
(malate dehydrogenase decarboxylating)
C
O
O
H
C
C
H
3
O
C
O
O
NADP
H  +  H
C
O
O
H
C
C
H
2
H
O
H
C
O
O
H
L-malate
NADP

39
Amphibolic character of CAC
•CAC provides important
•metabolic intermediates for
•anabolic processes:
•gluconeogenesis, transamination
Final catabolic pathway:
oxidation of acetyl-CoA to 2 CO2
Also other compounds,
which are metabolized to CAC intermediates, can serve as substrates of the cycle

40
Catabolic processes - entries into the cycle
Leu, Ile
Phe, Tyr, Lys, Trp
oxaloacetate
fumarate
succinyl-CoA
2-oxoglutarate
CC
acetyl-CoA
Phe, Tyr
ureosynthesis
purine synthesis
Ile, Val, Met, Thr
Arg, Glu, Gln, His, Pro
Asp, Asn
pyruvate
Ala, Cys, Gly, Ser, Thr, Trp
fatty acids
glucose
Ile, Val, Met

41
Anabolic processes – intermediates for syntheses
oxaloacetate
succinyl-CoA
2-oxoglutarate
CC
malate
porphyrines, heme
(collector of amino groups)
pyruvate  +  NADPH
aspartate
purine
pyrimidine
phosphoenolpyruvate
g
l
u
co
se
glutamate
citrate
Fatty acids, steroids
Intermediates drawn off for biosyntheses are replenished by anaplerotic reactions

42
CAC and the synthesis of lipids
ATP
TAG
malate
FA synthesis
CAC
mitochondria
citrate
cytosol
citrate
oxaloacetate
acetyl-CoA
malic enzyme



C
O
2




N
A
D
P
H




H
+
+
+
+
pyruvate

43
CAC and transamination
oxaloacetate
2-oxoglutarate
CAC
aspartate
glutamate

44
CAC and vitamins
Vitamin
Reaction in citrate cycle
Riboflavin
Niacin
Thiamin
Pantothenic acid
Complete

45
Relationships in major metabolic pathways
GLYCOGEN
Glucose
FAT STORES
TRIACYLGLYCEROLS
FATTY ACIDS
PROTEINS
Glucogenic AA (non-essent.)
Glucogenic AA (essential)
Ketogenic AA (essential)
  ACETYL-CoA
Citrate cycle
OXIDATIVE PHOSPHORYLATION
ATP
KETONE BODIES
Glycerol
×
Pyruvate
×
×
×
×

46
Interconversions between nutrients
Interconversion
Commentary
Sugars ® lipids
very easy and quickly
Lipids ® glucose
not possible,                                                        pyruvate dehydrogenase
reaction is irreversible
Amino acids ® glucose
most AA are glucogenic
Glucose intermediates ® AA
pyruvate and CAC intermediate provide arbon skeleton for some amino acids
Amino acids ® lipids
in excess of proteins
Lipids ® amino acids
pyruvate dehydrogenase reaction is irreversible
ketogenic AA and most mixed AA are essential
×
×

47
Saccharides are the most universal nutrients –
    the overdose is transformed in the fat stores,
    carbon skelet of non-essential amino acids can originate from saccharides.
Triacylglycerols exhibit the highest energetic yield –
    but fatty acids cannot convert into saccharides or the skelet of amino acids.
Amino acids represent the unique source of nitrogen for proteosynthesis
    that serves as fuel rather when the organism is lacking in other nutrients -
      glucogenic amino acids can convert into glucose,
      a overdose of diet protein may be transformes in fat stores.

The metabolism of nutrients is sophistically controlled with different mechanisms
in the well-fed state (absorptive phase),
          short fasting  (post-absorptive phase), and in prolonged starvation.
It also depends on energy expenditure (predominantly muscular work) –
      either of maximal intensity (anaerobic, of short duration only)
      or aerobic work of much lower intensity (long duration).

48
The tissues differ in their enzyme equipment                            and metabolic pathways
Pathway
Liver
CNS
Kidneys
Muscles
Adipocyte
Ery
CAC
+
+
+
+
+
-
FA β-oxidation
++
-
+
++
-
-
FA synthesis
+++
±
±
±
+++
-
Ketogenesis
+
-
-
-
-
-
KB oxidation*
-
+
+
+++
+
-
Glycolysis
+
+++
+
+++
+
+++
Gluconeogenesis
+++
-
+
-
-
-
* KB = ketone bodies

49
Cellular compartmentation of major metabolic pathways
Nucleus
DNA replication, RNA synthesis (= DNA transcription)
Mitochondria
oxidative decarboxylation of pyruvate, CAC, RCh, FA β-oxidation,               synthesis of KB /
urea / heme / Gln, AST reaction
Rough ER
proteosynthesis on ribosomes (translation of mRNA)
Smooth ER
synthesis of TAG / chol., FA desaturation, hydroxylations of xenobiotics
Lysosomes
non-specific hydrolysis of various substrates
Cell membrane
transport of molecules/ions/information = transporters/channels/receptors
Golgi apparat.
glycosylation of proteins, sorting and export of proteins
Peroxisomes
formation and decomposition of H2O2 and peroxides
Cytosol
glycolysis, gluconeogenesis, glycogen metabolism, pentose cycle, transamination, synthesis of FA /
urea / urate / heme;                                   ethanol ® acetaldehyde

50
Liver
 Glucose phosphorylation
 Glycolysis
¯ Gluconeogenesis
 Synthesis of glycogen
¯ Glycogenolysis
 Synthesis of fatty acids
 Pentose phosphate cycle
Adipose tissue
 Glucose uptake (GLUT 4)
 Glycolysis
 Pentose phosphate pathway
 Ox. decarboxylation of pyruvate
 Hydrolysis of TG in lipoproteins
 Synthesis of TG
¯ Lipolysis
Muscle
 Glucose uptake (GLUT 4)
 Glycolysis
 Synthesis of glycogen
¯ Glycogenolysis
 Synthesis of proteins
Metabolic effects of insulin

51
Liver
¯ Glycolysis
 Gluconeogenesis
¯ Synthesis of glycogen
 Glycogenolysis
¯ Synthesis of fatty acids
 Oxidation of fatty acids
Adipocytes
 Lipolysis (HSL, hormone sensitive lipase)
Metabolic effects of glucagon [not on muscles]

52
Biosynthesis of heme
~
Hemoproteins

53
Heme
Prostetic group of many proteins
(hemoglobin, myoglobin, cytochromes)
Synthesis in the body:
70-80 % in erythroid cells in bone marow - hemoglobin
15 % liver – cytochroms P450 and other hemoproteins
Heme consists of porphyrin ring coordinated with iron cation

54
Biosynthesis of heme
•initial compound for synthesis is succinyl-CoA (intermediate of CAC)
•source of nitrogen is glycine
•reactions are located in mitochondria and cytosol
•regulation: ALA-synthase
•
•
mitochondria
cytosol

55
Synthesis of d-aminolevulinate (ALA)
ALA-synthase
C
H
2
N
H
2
C
O
O
H
H
O
O
C
C
H
2
C
H
2
C
S
O
C
o
A
glycin
succinyl-CoA
H
O
O
C
C
H
2
C
H
2
C
O
C
H
2
N
H
2
H
O
O
C
C
H
2
C
H
2
C
O
C
H
N
H
2
C
O
O
H
2-amino-3-oxoadipate
H
S
C
o
A
-  CO
2
d
-aminolevulinate
(5-amino-4-oxobutanoic acid)
pyridoxalphosphate          is cofactor
mitochondria

56
ALA-synthase is the rate-controlling enzyme             of porphyrine biosynthesis
ALA-synthase
-  is inhibited by heme (allosteric inhibition)
- synthesis of enzyme is repressed by heme
- is induced by some drugs (barbiturates, phenytoin, griseofulvin)
- cytochrome P-450 is needed for biotransformation of drugs/xenobiotics
-
Half-life about 1 hour

57
Condensation to substituted pyrrole
δ-aminolevulinate
cytosol
C
O
O
H
N
O
H
H
H
H
O
N
H
2
C
O
O
H
H
H
- 2 H
2
O
porphobilinogen
N
N
H
2
C
O
O
H
C
O
O
H
H

58
Condensation of porphobilinogen
Porphobilinogen
uroporphyrinogen  I
     (minor product)
uroporphyrinogen  III
   (main product)
Under physiological circumstances, due to the presence of a protein modifier called co-synthase,
uroporphyrinogen III with an asymmetrical arrangement of side chains of the ring D is formed.
Only traces of symmetrical uroporphyrinogen I are produced

59
Condensation of porphobilinogen
N
N
H
2
C
O
O
H
C
O
O
H
H
porphobilinogen
4
N
H
3
4
H
N
H
N
N
N
C
O
O
H
H
O
O
C
C
O
O
H
H
O
O
C
H
O
O
C
H
O
O
C
C
O
O
H
C
O
O
H
H
H
uroporphyrinogen III
methylene bridge
A
B
C
D
cytosol

60
Decarboxylation of four acetates – formation of methyl groups
H
N
H
N
N
N
C
O
O
H
H
O
O
C
C
O
O
H
H
O
O
C
H
O
O
C
H
O
O
C
C
O
O
H
C
O
O
H
H
H
H
N
H
N
N
N
C
H
3
H
3
C
C
O
O
H
H
O
O
C
H
3
C
H
O
O
C
C
H
3
C
O
O
H
H
H
4 CO
2
uroporphyrinogen III
coproporphyrinogen III
cytosol

61
Formation of vinyl groups from two propionates
H
N
H
N
N
N
C
H
3
H
3
C
C
O
O
H
H
O
O
C
H
3
C
H
O
O
C
C
H
3
C
O
O
H
H
H
coproporphyrinogen III
-  4H
- 2 CO
2
H
N
H
N
N
N
C
H
3
H
3
C
C
O
O
H
H
O
O
C
H
3
C
C
H
3
H
H
protoporphyrinogen IX
mitochondria

62
Formation of conjugated system
colourless
red
H
N
H
N
N
N
C
H
3
H
3
C
C
O
O
H
H
O
O
C
H
3
C
C
H
3
H
H
protoporphyrinogen IX
-  6 H
N
N
N
N
C
H
3
H
3
C
C
H
3
H
3
C
C
O
O
H
H
O
O
C
H
protoporphyrin  IX
H
methyne (methenyl) bridge
mitochondria

63
Heme is coloured chelate with Fe2+
protoporphyrin  IX
F
e
heme
H
N
N
N
N
C
H
3
H
3
C
C
H
3
H
3
C
C
O
O
H
H
O
O
C
H
ascorbate
F
e
3+
- 2H
+
N
N
N
N
C
H
3
H
3
C
C
H
3
H
3
C
C
O
O
H
H
O
O
C
F
e
2+

64
CO and bilirubin are formed by the degradation of heme
oxidative cleavage (heme oxygenase)
CO +  biliverdin
bilirubin
carbonyl-hemoglobin
3 O2
3 NADPH+H+
more details                             in the 4th semester

65
Porhyrias are caused by partial deficiency                                   of one of the heme
synthesizing enzymes
•Primary (genetic)
•Defective enzyme of heme biosynthesis
•Overproduction and accumulation of intermediates (ALA, PBG)
•Porphyrinogens in skin - photosensitivity
•
•
•Secondary
•Inactivation of enzymes as a consequence of disease or poisoning
•Similar symptoms

66
Hemoproteins
Protein
Redox state of Fe
Function
Hemoglobin
Myoglobin
Catalase
Peroxidase
Cytochroms
Cytochrom P-450
Desaturases of FA
Fe2+
Fe2+
Fe2+ D Fe3+
Fe2+ D Fe3+
Fe2+ D Fe3+
Fe2+ D Fe3+
Fe2+ D Fe3+
Transport of O2 in blood
Deposit of O2 in muscles
Decomposition of H2O2
Decomposition of peroxides
Components of resp. chain
Hydroxylation
Desaturation of FA

67
Oxidation number of Fe in various hemes
•Does not change
•Fe2+
•prosthetic group for O2 transport
•hemoglobin, myoglobin
•heme is hidden in                          hydrophobic pocket of globin
•oxidation of Fe2+ means the loss of function
•Does change
•Fe2+ D  Fe3+
•cofactor of oxidoreductases
•cytochromes, heme enzymes
•heme is relatively exposed
•reversible redox change is the primary function = the transfer of one electron

68
Hemoglobin and myoglobin bind O2
•Hemoglobin
•transports O2 from lungs to tissues
•from tissues to lungs, it transports some H+ and CO2 (carbamino-Hb)
•tetramer Þ sigmoidal saturation curve
•two conformations: T-deoxyHb(2,3-BPG), R-oxyHb
•binding O2 in lungs ® release of H+
•binding H+ in tissues ® release of O2
•buffer system in erythrocytes (His)
•Myoglobin
•muscle hemoprotein – deposition (reserve) of oxygen
•monomer Þ saturation curve hyperbolic = stronger binding O2
Bohr effect
!

69
Quaternary structure of hemoglobin
α2
α1
β2
β1
4 O2
α1
α2
β1
β2
deoxygenated hemoglobin
(2,3-bisphosphoglycerate)
T-conformation
oxyhemoglobin
R-conformation
α1O2
α2O2
β1O2
β2O2
4 O2
2 H+
2 H+

70
Derivatives of hemoglobin
•Carbonylhemoglobin
•CO has great affinity to Fe2+ in heme
•physiological level: 1 - 15 % from total Hb (environment, smokers etc.)
•Glycated hemoglobin
•non-enzymatic reaction with free glucose, –NH2 group of Hb                          (N-terminus,
Lys) and aldehyde group of glucose
•physiological level: 2.8 – 4.0 % (from total Hb)
•Methemoglobin (hemiglobin)
•oxidation of heme iron, Fe2+ ® Fe3+, physiol. level: 0.5 - 1.5 %
•oxidation agents: nitrites, alkyl nitrites, aromatic amines, nitro compounds
•Hb mutation: hemoglobin M (HbM), the replacement of F8His®Tyr
•deficit of methemoglobin reductase

71
Language note: Methemoglobin
•it has nothing to do with methyl group !!!
•abbreviated from metahemoglobin
•the prefix meta- (from Greek) indicates                                                    change,
transformation, alteration
•other examples with the prefix
meta:                                                                            metabolic (=
catabolic + anabolic)
•    metamorphosis, metazoan ...

72
Linguistic note:
How to express two redox states of iron
Fe2+
Fe3+
Infix
-o
-i
Biochemical names
hemoglobin,                       ferroportin,                      ferroxidase
hemiglobin = methemoglobin, ferritin, transferrin, lactoferrin, gastroferrin, ferric reductase
Chemical names
Latin
ferrosi chloridum
ferri chloridum
Old English
ferrous chloride
ferric chloride
New English
iron(II) chloride
iron(III) chloride

73
Heme as cofactor of oxidoreductases transfers one electron
Examples of heme enzymes
Catalase: H2O2      ®   ½ O2  +  H2O
Myeloperoxidase: H2O2  +  Cl-  +  H+  ®  HClO  +  H2O

74
Cytochrome P450 (CYP)
•superfamily of heme enzymes (many isoforms)
•catalyze mainly hydroxylation of various substrates
•exhibits wide substrate specifity (advantage for the body)
•can be induced and inhibited by many compounds
•occurs in most tissues (except of muscles and erythrocytes)
•the highest activity in the liver (ER)
Abbreviation: P = pigment, 450 = wave length (nm) of a absorption peak after binding CO

75
Hydroxylation by CYP 450 occurs                                          in endogenous and
exogenous substrates
•Endoplasmic reticulum:                                                 squalene, cholesterol, bile
acids, calciol,                               FA desaturation, prostaglandins, xenobiotics
•Mitochondria:                                                                   steroidal hormones
•

76
Mechanism of hydroxylation
•the formation of hydroxyl group
•monooxygenase: one O atom from O2 molecule is incorporated into substrate between C and H (R-H ®
R-OH )
•the second O atom and 2H from NADPH+H+ produce water
R-H + O2 + NADPH + H+  ®  R-OH  +  H2O  + NADP+
2 e- + 2 H+

77
Desaturation of fatty acids
∆9 desaturase
H
3
C
C
O
O
H
C
O
O
H
H
3
C
C
O
O
H
C
H
3
9-10 desaturation (humans)
12-13 desaturation (plants)
stearic acid
        18:0
oleic acid
     18:1 (9)
linoleic acid
   18:2 (9,12)

78
Desaturation of FA requires cytochrome b5
hydroxylation at C-10
dehydration