C-565 & CH-665

ADVANCED BIO-INORGANIC CHEMISTRY

 Two types;
[Link] Derivative
[Link]-Heme Derivative

HEME Derivatives

• Consists of Porphyrin ring bound with an Iron atom

• Porphyrin: Four pyrrole unit linked with four meso carbons

• Porphyrin unit accepts two hydrogens in the presence of acid
to form dication and Also donates two proton and become
dianion, which binds with metal ions to form metalloporphyrin
complex (structure is shown in the next slide)
 HEME Derivatives
CH3
R1 CH3
H3C
N N H3C R2
N N
Fe(II)
Fe(II)
N N
H3C CH3 N N
H3C CH3

-
O2C CO2- -
O2C CO2-
Heme Unit

Type R1 R2 Complex

Heme A -CH=CH2 C18H30OH Cytochrome ‘a’

Heme B -CH=CH2 -CH=CH2 Hemoglobin, Myoglobin,

Peroxidase, Cytochrome ‘b’
Heme C -CH(CH3)S-protein -CH(CH3)S-protein Cytochrome ‘c’

Chloroheme -CHO -CH=CH2 Chlorocruorin

HEMOGLOBIN & MYOGLOBIN
 MYOGLOBIN (Mb)
• M. Wt.: 17,000

• Protein with 153 aminoacid and single heme group

• Fe is with 5 coordinate center: (a) four coordination from porphyrin pyrrole Ns;
(b) fifth one from imidazole N-Histidine & (c) sixth position is vacant

• Central metal: Fe2+ HS with pseudo Oh environment [(SqPy) arrangement of

Heme in Hb and Mb may be considered an Oh with sixth ligand removed)]

• Fe2+ lies above the porphyrin plane with the distance of 0.42Å

• When O2 binds, HS-Fe2+ (deoxy form) becomes LS-Fe2+ (oxy form)

[Ionic radius: 0.92 Å (HS Fe2+) and 0.75 Å (LS Fe2+)], where Fe atom shrinks
and fit inside the porphyrin plane upon oxygen binding

• Function: O2 reservoir – organism can draw during increased metabolism

 HEMOGLOBIN (Hb)

• Tetramer of Myoglobin contains 4 heme units + 4 protein chains

(two α + two β chains); α – chain contains 141 amino acids, where β chain
146 amino acids. Overall, M. Wt.: 64,500

• Function: Transfer O2 from Lungs to Tissue.

• Deoxy state: ‘T’ state (Tense state) and Oxy form: ‘R’ state (Relax state)

• Deoxy form [HS-Fe2+: t2g4eg2] & oxy form [LS-Fe2+: t2g6eg0]

 HEMOGLOBIN (Hb): O2 binding Models
N N

N N
His-H2C H His-H2C H
O O
O O

N N N N N N
Fe(II) Fe(II) Fe(III)
N N N N N N

N N N

N CH2-His N CH2-His N CH2-His

H H H
: Globin chain

OXY Form DEOXY Form OXY Form

Pauling Model (HS Fe2+) Weiss Model
(LS Fe2+) (LS Fe3+)
 HEMOGLOBIN (Hb): O2 binding Models
PAULING MODEL DEOXY WEISS MODEL
(OXY Form) form (OXY Form)
LS-Fe2+ HS-Fe2+ LS-Fe3+
t2g6eg0 t2g4eg2 t2g5eg0
Relax state Tense state Relax state
Ionic Radius: 0.75Å 0.92 Å 0.68 Å
LS-Fe2+ fits at the middle LS-Fe3+ slightly deviate from the core;
of the core HS-Fe2+ is reversibly oxidized to LS-
Fe3+ & O2 is reduced to O2-
Suits for other π-bonding O2- can easily be replaced by Cl- ligand
ligands like: CO, NO etc
Fe-O-O linkage: Bent Fe-O-O linkage: Bent
νo-o : 1100 cm-1 1100 cm-1

Mossbauer confirms: LS-Fe3+

An antiferromagnetic coupling between
unpaired electron of LS-Fe3+ &
unpaired electron of superoxide.
 HEMOGLOBIN (Hb)
100
Partial pressure of O2 (ppO2) is high in Lungs,
80
Lesser in capillaries and very low in Tissues,
60
ppO2 •O2 concentration is higher in Lungs &
40 Lower in tissues.
20
•Means, pp (partial pressure) is high in lungs –
Hb picks up O2 and the pp is low in tissues –
Hb releases O2
Lungs Capillaries Tissues

100

80 O2 binding in Myoglobin:

60 •For low O2 PP (30 mmHg), myoglobin is 97%

saturated in tissues.
40
•Hence, O2 binds with myoglobin in
Hyperbolic fashion.
20

20 40 60 80 100
ppO2 mmHg
 O2 binding in Hemoglobin
100 100

80 80

60 60

40 40

20 20

20 40 60 80 100 20 40 60 80 100
ppO2 mmHg ppO2 mmHg
Strong Binding transport protein Weak Binding transport protein

•At high ppO2: Efficient in binding •At high ppO2: Inefficient in binding
with O2 in Lungs with O2 in Lungs
•At low ppO2: Inefficient in releasing •At low ppO2: Efficient in releasing
the O2 in the tissues. the O2 in the tissues.
So, the transport protein should be:
•At high ppO2: Effectively binds with O2 in Lungs
•At low ppO2: Effectively releases the O2 in the tissues
 O2 binding in Hemoglobin
100

80

60

40

20

20 40 60 80 100
ppO2 mmHg

Ideal transport protein is:

•At high ppO2: Effectively binds with O2 in Lungs

•At low ppO2: Effectively releases the O2 in the tissues

•Hence, in Hemoglobin O2 binds in Sigmoidal Fashion

CARBONIC ANHYDRASE
 CARBONIC ANHYDRASE (CA)
H2O + CO2 H+ + HCO3-
• In the absence of CA, reaction is slow, where k: 10-11 s-1
In the presence of CA, reaction is fast, where k: 104 s-1

• Active site contains Zn2+ which lowers the pKa value and allows nucleophilic
attack on the CO2

• Maintain pH balance and transporting CO2 from tissues to lungs

• Zn2+ is in distorted Td geometry, which contains three imidazole-Histidine units and

a water molecule with M. Wt. of ~30,000 unit

• Carbonated drink doesn’t degas instantly when opening the container, however, it
will rapidly degas in the mouth when it comes to contact, because CA is there in
the saliva H H
O
II
Zn
119 His His
94
His
96
Mechanism
1. Binding of Zn lowers the pKa
H H H of water from 15.7 to 7,
O O generating a hydroxide ion
H
Zn
II II (OH-) to attack CO2. Zn
Zn
119 His His 119 His His
94 1 94 releases H+ to generate OH-
His His ion.
96 96

O 2. CO2 substrate binds to the

HCO3
enzyme active site and is
4 2 C
positioned for optimal
H2O O interaction
O
O
H C H
3. OH- attacks CO2 to generate
C
O O O bicarbonate ion by
II
Zn
3 II O Nucleophilic attack
119 His His Zn
119 His His
94 94
His His 4. Enzyme regenerated and
96 96 bicarbonate ion is released.

H+ ion in step 1: temporarily

resides in amino acid side
chain
 CO2 transport
CA
H2O + CO2 H2CO3

In Tissue In Lungs
• Partial pressure of O2 is low • Partial pressure of O2 is high
• pH is reduced due to formation of • Deoxy-Hb.H+ undergoes
+ -
oxygenation
Deoxy-Hb.H + HCO3
carbonic acid & Lactic acid
O2
• Oxy Hb releases O2 in tissue &
generate deoxy-Hb which is Oxy-Hb.H+ + HCO3-
stabilized by H+
H2CO3 + Oxy-Hb
• Oxy-Hb.H+ is stabilized by releasing
H+ which reacts with HCO3- to form
H2CO3 + Deoxy-Hb + O2 H2CO3 releases O2 & generate
deoxy Hb which+is stabilized
- by H+
Oxy-Hb.H + HCO3
Deoxy-Hb.H+ + HCO3-

Oxy-Hb + H2CO3

CA
H2O + CO2 H2CO3
 CO2 transport – Overall Mechanism
H2O + CO2 H2O + CO2
CA CA

H2CO3 H2CO3

Oxy-Hb
HCO3- H + H+ + HCO3-
(R-state)
+
H loading Stimulates H+ unloading Stimulates
releasing Oxygen from Hb loading Oxygen to Hb
O2 Deoxy-Hb O2
+ H+
In Peripheral Tissues (T-state) In Lungs

Böhr Effect: Deoxy-Hb binds tightly with H+ ion than Oxy-Hb. Hence, H+
loading (in tissues) and unloading (in Lungs) stimulate O 2 unloading (in
Tissues) and O2 loading (in Lungs), respectively

Haldane Effect: Deoxy-Hb shows higher affinity with CO2 than Oxy-Hb.
Hence, CO2 loading (in tissues) and unloading (in Lungs) stimulate O 2
unloading (in Tissues) and O2 loading (in Lungs), respectively
 -
BPG
O O
C • 2,3-Bisphosphoglycerate

H C OPO32-
• Synthesized by red blood cells
• Binds tightly with deoxy Hemoglobin. BPG
H C OPO32- housed at the central cavity of Deoxy-Hb
and experiences salt bridge interaction
H with Lysine, Histidine and N-terminal
amino groups of β1 and β2 chains.

• Facilitates release of O2 in capillaries

• Responsible for high altitude adaptation,
where cells make BPG, so Hb can release
more O2 in capillaries.
Deoxy-Hb-A + BPG Deoxy-Hb-A-BPG
KA >> KF
Deoxy-Hb-F + BPG Deoxy-Hb-F-BPG

A: Adult F: Fetal

• In the above equation, BPG binds tightly with Hb-A than to Hb-F. Mainly due to
histidine in the β chain (α2β2) of Hb-A is replaced by serine residue in the γ chain
(α2γ2) of Hb-F. Thus, the BPG mediated salt-bridge interaction in Hb-F is relatively
weak, however, has a high O2 affinity compared to Hb-A and this enables the
transfer of O2 from mother to fetus.
SICKLE CELL Anemia:

•Amino acid sequence of the apoprotein is very important for the structure &
Function of Hemoglobin. During R to T conversion Hb polymerizes and distorts the
red blood cell into the shape of Sickle.

Mainly due to the mutation occur at the 6th position of β-chain,

where ‘Glutamic acid (glu)’ is replaced by ‘Valine (Val).’
Val – His – Leu – Thr – Pro – Glu – Glu – Lys

Val – His – Leu – Thr – Pro – Val – Glu – Lys

Glutamic acid: negatively charged Amino acid

Valine: Hydrophobic side chain

•The side chain of valine is nonpolar, whereas the side chain of glutamate is
highly polar. It drastically reduces the solubility of deoxy-Hb and forms fibrous
precipitate that deforms the red blood cells to give the sickle shape or crescent
shape. These aggregated red blood cells may clog the small capillaries to impair
the blood circulation.
HEMERYTHRIN & HEMOCYANIN
 HEMERYTHRIN
His73 His73
His101 His77 His101 His77
Fe Fe
II III
O O O O
O2
C C O H C C O
Glu58 Glu58 H
Asp106 O Asp106 O
O O O
II III O
Fe Fe
His25 His54 His25 His54

Deoxyhemerythrin oxyhemerythrin
Colourless Violet-red

• Non-Heme O2 binding pigment utilized by Four Phyla of marine invertebrates

• [Link].: 108,000; There is an octameric form which transports dioxygen in the blood

• In the tissues are lower M. Wt. monomer, dimer, trimer / tetramer

• Two Fe atoms are approximately Oh Coordination bridged by aspartate (Asp-106),
glutamate (Glu58) ligands and an Oxygen atom (from H2O / hydroxo / oxo unit).
Three Histidine (His101, His73 & His77) residues in one Fe atom &
Two Histidine (His25 & His54) residues in another Fe atom
 His73
HEMERYTHRIN His73
His101 His77 His101 His77
Fe Fe
II III
O O O O
O2
C C O H C C O
Glu58 Glu58 H
Asp106 O Asp106 O
O O O
II III O
Fe Fe
His25 His54 His25 His54

Deoxy-hemerythrin Oxy-hemerythrin
• In deoxy-hemerythrin a (μ-hydroxo)bis(μ-carboxylate)diiron(II)
Where vacant terminal coordination of one of the Fe atoms (bottom one) binds
with O2 & transfers two electrons and a proton from the bridging –OH group

• The resulting oxy-hemerythrin has a terminal hydroperoxy group bound to Fe atom

• A H-bond is formed between the proton of the hydroperoxyl group (the donor)
& μ-oxo group (the acceptor)

• The oxidative addition of O2 to the dimetallic may occur by two sequential 1

electron transfer steps involving mixed valence (Fe2+ / Fe3+) intermediate

• H-atom can’t be located by single crystal X-ray resolution & the H-atom shown is
merely one suggestion for the possible stabilization of the peroxide ion.
 HEMERYTHRIN H
H H O O

O + O2 O O O O
Fe Fe Fe Fe Fe Fe
II II III III III III

II II o III III o

Fe Fe : 3.32 A Fe Fe : 3.27 A

O III O III
II II Fe Fe
Fe o Fe o
111 125
II o III o

Fe O-H : 1.98 A Fe O : 1.82 A

νo-o : 845 cm -1

• LMCT O2- to Fe(III) : 330 nm and

shoulder at 360 nm

• LMCT O22- to Fe(III) : 500 nm

(responsible for visible colour change)
 HEMOCYANIN
224 344 224 344
His His His His
O2 II O
I I II
His Cu Cu His His Cu Cu His
198 384 198 384
O
His His His His
194 348 194 348

Deoxy-hemocyanin Oxy-hemocyanin
[Cu(I)……Cu(I): 4.6 Å] [Cu(II)…Cu(II): 3.6 Å]
Colourless Blue Colour
Geometry: Distorted Trigonal Planar Geometry: Distorted Square Pyramidal
νo-o : 800 cm-1
• Oxygen containing pigment – neither heme group nor cyanide ion

• Hemocyanin means Blue Blood

• Found in Mollusca (Snails, Octopi etc): Molluscan-HC: M. Wt: 53,000
Arthropoda (Lobsters, Scorpions etc): Arthropodan-HC: M. Wt: 77,000

• Oxygen molecule oxidized each Cu(I) to Cu(II) & in turn reduced to the
peroxide ion (O22-) with μ-η2:η2 bonding mode

• LMCT O22- to Cu(II) at 340 nm and 580 nm as shown in next page

 LMCT O22- to Cu(II)

LMCT O22- to Cu(II)

• LMCT O22- to Cu(II) at 340 nm and 580 nm

 Properties of Protein O2 Carriers
Properties Hemoglobin Hemerythrin Hemocyanin
Metal Fe Fe Cu
Oxidation state HS Fe(II) HS Fe(II)/Fe(II) Cu(I)/Cu(I)
of Deoxy level
Oxidation state LS Fe(II) –Pauling HS Fe(III)/Fe(III) Cu(II)/Cu(II)
of Oxy level LS Fe(III) -Weiss
Color, Red Violet-Pink Blue
Oxygenated
Color, Red-Purple Colourless Colourless
Deoxygenated
Geometry deoxy Sq. pyramidal / Distorted Oh Distorted
Pseudo Oh Trigonal planar
Geometry Oxy Oh Oh Distorted Sq.
Pyramidal
Molecular 65,000 108,000 53,000 – 77,000
Weight
Function Oxygen Oxygen Oxygen
transport transport transport
 Properties of Protein O2 Carriers
Properties Hemoglobin Hemerythrin Hemocyanin
Metal:O2 ratio 4:4 2Fe : 1 O2 2Cu : 1 O2
(4 heme : 4 O2)
Chemical state of O2 – bent HO2- O22-
O2 Superoxide (O2-) (Hydroperoxo) (peroxide ion)

νo-o 1100 cm-1 845 cm-1 800 cm-1

Electronic State 410 nm (π – π*) LMCT O2- to Fe(III) LMCT O22- to Cu(II)
500 – 600 nm (π – π*) 330 & 360 (sh) nm 340 & 580 nm

LMCT O22- to Fe(III)

500 nm
 NON-HEME Derivatives

• Non-Heme Fe-S Proteins involved in e-transfer

• Commonly known as Ferredoxins
(Fer: Iron; redoxin: Redox center)
• Contains distinct Fe-S cluster composed of
(a) Fe atoms
(b) Cysteinyl sulfur
(c) Inorganic Labile sulfur
Where, labile sulfur can +easily removed by8+washing with acids
(RS)4Fe4S4 + 8H (RS)4Fe4 + 4H2S

• Play vital roles in Photosynthesis, mitochondrial respiration,

nitrogen fixation
 TYPES

Common Formula: nFe – mS or Fen - Sm

(n: Number of cations &
m: Number of Inorganic Labile Sulfur)

•Rubredoxin: Fe1S0 cluster

•Ferredoxin: Fe2S2 cluster

•Ferredoxin: Fe3S4 cluster

•Ferredoxin: Fe4S4 cluster

•Ferredoxin: Fe8S8 cluster

 RUBREDOXIN
S-Cys

Fe
Cys-S S-Cys

Cys-S

• Fe1S0 Cluster
• Bacterial Protein
• M. Wt.: 6000
• Fe: distorted tetrahedral geometry surround by four
Cysteine ligands
• Mainly involved 1 electron transfer
• Remain High spin complex
• Oxidized state is reddish
(due to ligand to metal charge transfer)
• Reduced state is colourless
 FERREDOXIN : Fe2S2
RIESKE Centre
Cys-S S S-Cys
III III Cys-S S N-His
III III
Fe Fe
Fe Fe
Cys-S S S-Cys
Cys-S S N-His

• 2Fe – 2S or Fe2S2 Cluster contains two bridging inorganic labile sulfur and
two Fe center.
• 2 Fe centers act as one electron transfer process
• In the oxidized form, both the Fe are in +3 state and in the reduced form one
is in +3 state and the another one is in +2 state.
• Both are involved in electron transfer in Photosynthesis of higher plants

Cys-S S S-Cys Cys-S S S-Cys

III III +e III II
Fe Fe Fe Fe
Cys-S S S-Cys Cys-S S S-Cys

+e [(Cys-S)2-Fe(III)-(S2-)2-Fe(II)-(S-Cys)2] 3-
[(Cys-S)2-Fe(III)-(S2-)2-Fe(III)-(S-Cys)2]2-
(Oxidized form) (Reduced form)
 FERREDOXIN : Fe4S4
S-Cys
Cys-S • Cubane like cluster
Fe S • Contains;
(a) Four Fe atoms
S Fe (b) Four labile ‘S’ atom
(c) Each Fe center is tetrahedrally
S Fe
Coordinated by cys-S and three
S-Cys
Fe S labile ‘S’ sites
Cys-S H (d) Involved in one electron transfer process,
CV studies indicate, it exists in three forms.
(i) [(FeIII)3-(FeII)]
(ii) [(FeIII)2-(FeII)2]
(iii) [(FeIII)-(FeII)3]
Mostly exists in: [(FeIII)2-(FeII)2] (Oxidized form) and [(FeIII) -(FeII)3] (Reduced form)

[Fe(III)2-Fe(II)2-(S2-)4(S-Cys)4]2- +e [Fe(III)-Fe(II)3-(S2-)4(S-Cys)4]3-
(Oxidized form) (Reduced form)

Bacterial Ferredoxin mainly involved in anaerobic metabolism

 FERREDOXIN : Fe4S4
HiPISP (High Potential Iron Sulfur Protein)
[Fe(III)3-Fe(II)-(S2-)4(S-Cys)4]- +e [Fe(III)2-Fe(II)2-(S2-)4(S-Cys)4]2-
(Oxidized form) (Reduced form)

• Considering the previous page equation and the above equation,

the overall electron
2-
transfer process
-
goes like this:
[Fe(III)3-Fe(II)-(S )4(S-Cys)4]
(Oxidized form)

+e • Different Fe centers are designated

2- 2- as Fe(III) and Fe(II), but the
[Fe(III)2-Fe(II)2-(S )4(S-Cys)4] experimental evidence indicate that
(Reduced form-1) these Fe centers are equivalent with
same average oxidation state.
+e

[Fe(III)-Fe(II)3-(S2-)4(S-Cys)4]3-
(Reduced form-2)
 FERREDOXIN : Fe3S4
Cys-S
S-Cys Fe S
“Void Cubane”
S
Fe S-Cys
S S or
S S Fe
Fe Fe “Fe-Depleted Cubane”
Cys-S S-Cys Fe S
S
Cys-S

FERREDOXIN : Fe8S8
8 S-Cys 42 S-Cys
Cys-S 45 Cys-S 38
Fe S 45 Fe S

S Fe S Fe

S Fe S Fe
S-Cys S-Cys
Fe S 14 Fe S 35
Cys-S Cys-S
18

2[Fe(III)2-Fe(II)2-(S2-)4(S-Cys)4]2- + 2e 2[Fe(III)-Fe(II)3-(S2-)4(S-Cys)4]3-
(Oxidized form) (Reduced form-2)

• Mainly involved for 2 electron transfer process

 MÖSSBAUER Spectroscopic Results
Rubredoxin: (Fe1S0)

RUBREDOXIN (Fe1S0)
HS Fe3+ HS Fe2+
e2t23 e3t23
Symmetric Asymmetric
Quadrupole splitting (ΔEQ) Quadrupole splitting (ΔEQ)
is Less is high
Isomer Shift Value: Isomer Shift Value:
δ: 0.25 mm/s δ: 0.65 mm/s
One Signal: Fe3+ (oxidized) One Signal: Fe2+ (reduced)
i.e.: one small doublet i.e.: one large doublet
 FERREDOXIN: (Fe2S2)

FERREDOXIN (Fe2S2)
HS Fe3+/Fe3+ HS Fe3+/Fe2+
Oxidized Reduced
One Signal: 2Fe3+ Two Signals: Fe3+/Fe2+
i.e.: one small doublet one small inner doublet for Fe3+
one larger outer doublet for Fe2+
Isomer Shift Value: Isomer Shift Value:
δ: 0.26 mm/s (for 2Fe3+) δ: 0.25 mm/s (for Fe3+) &
0.55 mm/s (Fe2+)
 FERREDOXIN: (Fe3S4)

FERREDOXIN (Fe3S4)
HS Fe3+/Fe3+/Fe3+ HS Fe3+/Fe2+ & Fe3+
Oxidized Reduced
One Signal: 3Fe3+ Two Signals: Fe3+/Fe2+
i.e.: one small doublet one small inner doublet for Fe3+
one larger outer doublet for Fe2+
Isomer Shift Value: Isomer Shift Value:
δ: 0.27 mm/s (for 3Fe3+) δ: 0.29 mm/s (for Fe3+) &
0.45 mm/s (Fe2+ & Fe3+)
 FERREDOXIN: (Fe3S4)

HS Fe3+/Fe2+ & Fe3+

Oxidation state of Fe2+ & Fe3+ is: 2.5+; means: 2Fe2.5+
(Fe2+ & Fe3+ delocalized couple with average oxidation state of 2.5 per metal ion)
3+
+e
3Fe Fe3+ + Fe2+ & Fe3+
Change to

3+
+e
3Fe Fe3+ + 2Fe2.5+

Isomer shift value of Fe2+ & Fe3+ is calculated:

For Fe2+ : δ: 0.25 mm/s & for Fe3+: δ: 0.65 mm/s

Hence, for 2Fe2.5+: 0.25 + 0.65 / 2
: 0.90 / 2
: 0.45 mm/s
Here, the electron exchange between Fe2+ & Fe3+ is
slow on the Mössbauer time scale, hence, able to see
the signal for Fe3+/2Fe2.5+
 FERREDOXIN: (Fe4S4)

• Single signal

• Electron Delocalized between Fe3+ & Fe2+. Hence, no fixed oxidation state.
Calculated average oxidation state is shown in the next slide

• Similarly, the calculated and observed Isomer shift value is shown in the
next slide

• Here, the electron exchange between 4 Fe ions are faster on the Mössbauer
time scale. Hence, able to see only the averaged spectral lines.
 FERREDOXIN: (Fe4S4)

Oxidation state:
[Fe(III)3-Fe(II)-(S2-)4(S-Cys)4]- + e [Fe(III)2-Fe(II)2-(S2-)4(S-Cys)4]2- +e
[Fe(III)-Fe(II)3-(S2-)4(S-Cys)4]3-
(Oxidized form) (Reduced form-1) (Reduced form-2)

3Fe3+ + 1Fe2+ 2Fe3+ + 2Fe2+ 1Fe3+ + 3Fe2+

9 + 2: 11 / 4: 2.75, hence 6 + 4: 10 / 4: 2.50, hence 3 + 6: 9 / 4: 2.25, hence
Oxidation state is: 4Fe2.75+ Oxidation state is: 4Fe2.5+ Oxidation state is: 4Fe2.25+

Isomer shift value (δ):

Calculate value
3Fe3+ + 1Fe2+ 2Fe3+ + 2Fe2+ 1Fe3+ + 3Fe2+

3 x 0.25 + 1 x 0.65 2 x 0.25 + 2 x 0.65 1 x 0.25 + 3 x 0.65

4 4 4
: 1.40 /4 : 1.80 /4 : 2.20 /4
: 0.35 mm/s : 0.45 mm/s : 0.55 mm/s
(Oxidized form) (Intermediate form) (Reduced form)

Observed value
0.32 mm/s 0.42 mm/s 0.57 mm/s
CATALASE & PEROXIDASE
 CATALASE
• Is an enzyme contains – tetramer of Four Polypeptide chain – each containing 500
amino acids and also four Heme porphyrin unit which allow enzyme to react with
H2O2 to form H2O and O2

• Involved in disproportionation of H2O2

Catalase
2H2O2 2H2O + O2

• One molecule of catalase can convert millions of molecules of H2O2 into H2O & O2
per second. Enzymatic reaction is of very fast: 107 dm3mol-1s-1
ACTIVE SITE

OH2

N N • Porphyrin unit with HS Fe3+

Fe (III)
N N • Fifth position by Tyrosinyl oxygen
• Sixth position by water molecule replaced by
O
substrate H2O2
Mechanism H2O2 + Fe(III)-E H2O + O=Fe(IV)-E( )
O=Fe(IV)-E( ) + H2O2 H2O + Fe(III)-E + O2

• Fe(III)-E: Fe-Heme attached to the Enzyme

• O=Fe(IV)-E (•+) is the mesomeric form of Fe(V)-E; means, Fe is not completely

oxidized to +V, however, receives some supporting electrom from heme ligand
which generate radical
H O cation (•+).
+ Fe(III)-E H O + O=Fe(V)-E
2 2 2

O=Fe(V)-E + H2O2 H2O + Fe(III)-E + O2

Greying & Whitening of Hair with Ageing and Catalase Activity :

• Pigment “MELANIN” (is produced by the enzyme activity of Tyrosinase) gives the
normal black colour of the hair

• Catalase activity declines due to ageing. Thus, H2O2 level increases.

• Increased H2O2 bleaches the melanin pigment of hair and turns from black to grey
and end up with white.

• Upon ageing, Melanin synthesis is also reduced due to decline in tyrosinyl activity.

• Reduced / decreased activity of Catalase and Tyrosinase enzymes with ageing

leads to greying & whitening of Hair.
 PEROXIDASE

• Oxidation of wide variety of organicPeroxidase

and Inorganic substrates by H2O2
H2O2 + AH2 2H2O + A

ACTIVE SITE • Porphyrin unit with HS Fe3+

OH2
• V position by Imidazolyl-His-N unit.
N N
Fe (III)
• VI position by H2O molecule
N N

O • His-Imidazolyl-NH is in hydrogen bonding interaction

with neighbouring carboxylate group of Asp to enhance
the electron donor capacity of imidazolyl unit, thus
provides HS-Fe3+ state. OH2 OH2

N N N N
Fe (III) Fe (III)
N N N N

N N

N N
H
O2C-Asp
Mechanism H2O2 + Fe(III)-E H2O + O=Fe(IV)-E( )

O=Fe(IV)-E( ) + AH2 O=Fe(IV)-E + H+ + HA

O=Fe(IV)-E + AH2 O=Fe(IV)-E + OH- + HA

HA + HA HA-AH or A + AH2

O2 H2O2
CATALYTIC CYCLE
( )
OH2 O CATALASE

N N H2O2 H2O N N
Fe (III) Fe (IV)
N N N N

L L

AH2 PEROXIDASE
- O
HA + OH

AH2 N N
HA + H+
Fe (IV)
N N

HA + HA HA-AH or A + AH2
CYTOCHROME P-450
 CYTOCHROME P-450
Active Site

OH2

N N
Fe (III) • Heme containing enzyme that oxygenate wide variety of
N N substrate with [Link].: 50,000 dalton
S
• 4 positions are with Heme ‘b’ unit
Cys
LS-Fe(III) • 5th Position by Thiolate side chain (S-Cys) which is better
electron donor as compared to His-N in Hb / Mb
(Hemoglobin / Myoglobin)
RH
• 6th position by H2O molecule
N N
Fe (III) • Low spin Fe3+ in the resting state. Binding with
N N substrate (RH) changes to High spin Fe3+

S
Cys
HS-Fe(III)
 ACTIVE SITE

OH2 OH2 OH2 OH2

N N N N N N N N
Fe (III) Fe (III) Fe (III) Fe (II)
N N N N N N N N

S O N N
Cys
N N
H
LS-Fe(III) HS-Fe(III) HS-Fe(III) HS-Fe(II)

CYTOCHROME P-450 CATALASE PEROXIDASE HEMOGLOBIN

(Pauling Model)
 CYTOCHROME P-450
Types of reaction catalyzed Cytochrome P-450

1. Aliphatic hydroxylation:
5. Amine oxidation to Amine oxides:
Cyclohexane Cyclohexanol

N N O
2. Aromatic hydroxylation:

Benzene Phenol
6. Sulfide oxidation to sulfoxides:

3. Alkene Epoxidation:
O S S O

7. Reductive dehalogenation:

4. Oxidative dealkylation of heteroatoms: C6H5CH2Br C6H5CH3

8. Oxidative demination:
NH CH3NH2 + HCHO
(CH3)2CHNH2 (CH3)2CO + NH3

C6H5OCH3 C6H5OH + HCHO

 CYTOCHROME P-450
STEP-1

• Substrate (R-H) binds the active site of the enzyme - close proximity to the
heme – on the side opposite to –S(Cys) unit
OH2
R-H
430
390
N N N N
R-H
Fe (III) Fe (III)
1
N N N N
S S
Cys Cys

• Change in the conformation of the active site

• Displacing H2O molecule leads to change from LS-Fe(III) to HS-Fe(III)
• Substrate shows band at 390 nm (Type-1) and some substrate appears at
430 nm (Type-II)
 CYTOCHROME P-450
STEP-2
• Change in the electronic state of the active site favours transfer of an electron
NAD(P)H, thus, HS-Fe(III) changes to HS-Fe(II) O

R-H NADPH NADP+ + H+ R-H R-H C

N N N N N N
Fe (III) e Fe (II)
CO
Fe (II)
N N 2 N N N N
S S S
Cys Cys Cys
Fe(III) Fe(II)

NAD(P)H NADP+ + H+

CO - Binding
450

• CO binds reduced P-450, thus catalytic cycle is

interrupted and shows a Soret band at 450 nm,
thus, the name is: P-450. Also changes HS-Fe(II)
to LS-Fe(II)
STEP-3
• O2 binds covalently to the vacant axial coordination position
• Electron transfer converts O2 into negatively charged peroxo which is the
short lived intermediate group O
R-H R-H O
N N N N
O2
Fe (II) Fe (III)
e 3
N N N N

S S
Cys Cys
Fe(II) Fe(III)
FAD ox FAD red
STEP-4
• The peroxo group protonated twice by local transfer from surrounding amino acid
side chain and releasing one molecule of H2O and also forming a highly active
intermediate Fe(V)-Oxo specie O
R-H O O
R-H
N N N N
Fe (III) 2H
Fe (V)
-H2O
N N N N
4
S S
Cys Cys
 STEP-5
• Final step is the hydroxylation step R-H O
R-H
OH2

N N N N
H2O
Fe (V) Fe (III) + R-OH
5
N N N N
S S
Cys Cys

PEROXIDE SHUNT
• An alternative route for mono-oxygenation by single oxygen donors can lead
to the formation of Fe=O(V) intermediate which completes the catalytic cycle.

• Alkyl hydroperoxides (RCO3H) and single oxygen atom donors such as

iodosylbenzene (PhIO) and Periodates (IO4-) are capable for donating a
single oxygen atom
OH R-H
O OH2
2
R-H R-H R-H
N N "Peroxide N N N N
N N N N
R-H e Shunt" H2O
Fe (II) Fe (V) Fe (III) + R-OH
Fe (III) Fe (III)
-H2O RCO3H /
H2N N N N N N
N N N N PhIO / IO4
S S S
S S
Cys Cys Cys
Cys Cys
Fe(III) Fe(II)
NAD(P)H NADP+ + H+
CYTOCHROME P-450 - MECHANISM
OH2
R-OH R-H
N N -H2O
Fe (III)
R-H
5 N N 1
N N
O S
R-H Fe (III)
Cys
N N RCO2H N N
Fe (V) RCO3H
S
N N Cys
e
S 2
Cys
O
R-H
R-H C
N N
CO N N
-H2O 4 O Fe (II)
Fe (II)
R-H O N N
2H H2N N
N N S
3 Cys S
Fe (III) O2 Cys
N N e
S
Cys
BLUE COPPER PROTEINS
 BLUE COPPER PROTEINS
• Depending on the spectral and structural properties, Blue copper proteins are
classified as: (i) Type - 1; (ii) Type – 2; (iii) Type -3
Type – 1: Type – 2:
• Has three electronic bands at 450, • Similar to Type-1 Cu proteins
600 and 700 nm, where 600 nm associated / presence of other metal
band is highly intense ion in the system

• Unusual EPR spectrum with very Example: Cu-Zn SOD

small hyperfine coupling constant

• Contains only one Cu and the

geometry is unusual.

• Mainly involved for electron

transport in Photosynthesis.

Ex: Plastocyanin,
Stellacyanin
Azurin
 BLUE COPPER PROTEINS
Type – 3:
• Contains multicopper protein

• Cu centers occur in pairs and they are close to each other to experience the spin
pairing interactions between adjacent d9 centers by “Antiferromagnetic
coupling” results in Diamagnetic and EPR inactive

• Two electron transfer agent. Can also act as one electron transfer agent.

Example: Tyrosinase, Ceruloplasmin

Hemocyanin – Respiratory protein
TYPE – 1 PLASTOCYANIN
S-Met
ACTIVE SITE 92
o
Cu - N-His (87): 2.10 A
(II) o
Cu Cu - N-His (37): 2.04 A
His-N o
S-Cys Cu - S-Cys (84): 2.13 A
His-N 84 o
Cu - S-Met (92): 2.90 A
37
• Contains single Cu center – mainly involved in electron transport in
Photosynthetic electron transport chain.

• Three short bonds with almost “Trigonal Planar arrangement, with 4th out of
plane axial longer band to provide “Trigonally distorted Td arrangement or
distorted Td geometry.

• Bond angle varies from 85° to 132° and the Tetrahedron is substantially
compressed towards a Trigonal arrangement

• Bands at 450, 600 and 700 nm with intense band at 600 nm which is mainly due
to Cys-S Cu LMCT band

• The coordinated imidazole group of N-His (87) is exposed to the surrounding

medium and plays an important role for outer-sphere electron transfer process.
 TYPE – 1
STELLACYANIN AZURIN
• Contains; • Contains;
His-N His-N: 1.97 Å
His-N His-N: 1.97 Å
Cys-S Cys-S: 2.10 Å
These three positions are Same as These three positions are Same as
Plastocyanin Plastocyanin; three short bonds are
in trigonal plane
• 4th position Met-S is absent
• In addition, two axial bonds:
• Cu-X (from spectral EPR analysis) – Met-S
as a Fourth bond is relatively shorter Gly-O
and flattening of the geometry is Both are longer bond with 3.00 Å.
more towards to Square Planar. Combine together to form Trigonal
bipyramidal geometry
His-N X
(II) • Sometime, Gly-O may not be there,
Cu thus geometry is distorted Td.

His-N S-Cys
 ACTIVE SITE
PLASTOCYANIN STELLACYANIN

S-Met
His-N X
92
(II)
(II)
Cu Cu
His-N
S-Cys His-N S-Cys
His-N 84
37

AZURIN
S-Met S-Met
His-N (II)
(II)
Cu S-Cys Cu
His-N
His-N S-Cys
O-Gly His-N
TYPE – 2 Superoxide Dismutase
• Type-2 Blue Copper proteins

• Partial reduction of O2 may lead to generate the toxic substances such as

Peroxide ion (O22-)and superoxide ion (O2-)

• Cytochrome C-oxidase and other enzymes can convert O2 to H2O without

formation of toxic intermediates.

• Similarly, H2O2 disproportionate by Catalase enzyme and convert it into to H2O

and O2.

• Hence, efficient mechanism is necessary to prevent accumulation of O 2- and O22-

ions in the biological system.
+
-- - 2H
• Here, superoxide O
(O22 ) +dismutate
O2 H2O2 by
/ disproportionate + OSOD
2 and convert it into
H2O2 and O2. SOD

• Disproportionation reaction with Cu-Zn SOD, the rate is 104 times faster than
uncatalyzed reaction:
kuncat: 2 X 105 M-1s-1 k Fe-SOD: 3 X 108 M-1s-1
9 -1 -1 7 -1 -1
 Cu-Zn Superoxide Dismutase
• Usually exist in dimeric form

• Monomeric unit contains two subunits. Each subunit contains Cu & Zn – both are
bridged by deprotonated Histidine imidazole unit [His-N (61)]
Zn Cu
• His-N (78) • His-N (46)
• His-N (69) • His-N (118)
• ASP-Carboxylate - O (81) • His-N (44)
• bridging His-N (61) • bridging His-N (61)
• Tetrahedral geometry • V position by weakly bound H2O
His
61
• Distorted Square pyramidal geometry
81
Asp-O OH2 118
N N N-His
(II) Zn Cu
(II)
His-N N-His
His-N N-His 44 46
78 69
 Cu-Zn Superoxide Dismutase
Active Site: His
61
81
Asp-O OH2 118
N N N-His
(II) Zn Cu
(II)
His-N N-His
His-N N-His 44 46
78 69

Role of Zn:
• Removal of Zn does not decrease SOD activity

• Replace Zn by other metal ions [M(II)] – does not increase SOD activity

• Zn depleted protein lacks thermal stability and it denatures at lower temperature

• Thus CuII is essential and Zn is only a mere spectator.

 Cu-Zn Superoxide Dismutase
Mechanism:

His His
61 61
O2

(II) - H2O (II) (II) 1. O2- binds to CuII center and

Zn N N Cu Zn N N Cu
1 release H2O molecule
OH2 O2
Arg
H2O2 4 2. (i) Arg and Lys – a positively
H
2 charged residue forms a
H2O
H O2 channel, where O2- ions
passes through. (ii) CuII
His reduced to CuI; (iii) CuI ion
61 His
O2 61 breaks the bridging with His-N-
(II) (II) 3 61 and remain connected with
Zn N N Cu (II) (I)
Zn N NH Cu ZnII and (iv) deprotonated
O O imidazole is protonated and
H retain neutral form

3. Second O2- oxidizes CuI to CuII and itself reduced to HO2-

4.HO2- further protonated to release H2O2 and H2O coordinated with CuII ion and
retain the structure, where ZnII plays a supportive role and helps stabilization of
CuII active site.
 Cu-Zn Superoxide Dismutase
Mechanism:

His His
61 61
O2
Overall, the Cu center is
- H2O
Zn
(II)
N N Cu Zn
(II)
N N
(II)
Cu reversibly reduced and oxidized
1
by superoxide ions to produce
OH2 O2
Arg O2 and H2O2
H2O2 4
H

H2O
2 The overall reaction is:
H O2
O2 O2 + e

His O2 + 2H + e H2O2
61 His
O2 61

(II) (II) 3 O2 + O2 + 2H O2 + H2O2

Zn N N Cu (II) (I)
Zn N NH Cu
O O

This mechanism is called “PING – PONG” mechanism: The enzyme bounce back and
forth from an intermediate state to its standard state.
 M: Fe or Mn - Superoxide Dismutase

OH OH
O2 H2O
Asp-O N-His Asp-O N-His
M (III) M (III)
His-N OH2 His-N O2
N-His N-His

H2O2 H+ + H2O

H+ + H2O O2

OH OH2
H2O O2
Asp-O N-His Asp-O N-His
M (III) M (II)
His-N O O His-N OH2
N-His H N-His
TYROSINASE
 TYROSINASE
• Type-3 Blue Copper proteins

• Contains multicopper ions, here, it contains two Cu metal ions.

• Cu plays an important role to protect the body from UV radiation from the Sun

• “Malanocyte” cells contains Cu-containing enzyme “Tyrosinase” – located

beneath the Epidermis.

• Exposure of sunlight accelerates the activity of “Tyrosinase” and increases

Melanin production.

• “Tyrosinase” catalyses conversion of amino acid “tyrosine” to “Melanin” pigment

H O
CO2H N
HO CO2H O CO2H
O2 O2
NH2 Tyrosinase NH2 Tyrosinase O
HO HO N O
O H2
L-Tyrosine L-DOPA
5,6-dihydroxy L-Dopaquinone
O N
phenylalanine H
MELANIN
 TYROSINASE
MELANIN SYNTHESIS

CO2H HO CO2H O CO2H

O2 O2

NH2 Tyrosinase Tyrosinase NH2

HO HO NH2 O
L-Tyrosine L-DOPA L-Dopaquinone
5,6-dihydroxy
phenylalanine

HO O HO

CO2H CO2H
N N N
HO H O H HO H
5,6-dihydroxy indole Dopachrome Leucodopachrome

H O
N
O
Polymerization O
O
N
O H
5,6-indoloquinone N
O H
MELANIN
 TYROSINASE
Active Site:

38 194 38 194
His His His His
N N N N
(I) (I) O2 (II) O (II)
His-N Cu Cu N-His His-N Cu Cu N-His
54 190 54 O 190
N N N N
His His His His
63 216 63 216
DEOXY OXY

• Two Cu atoms, each connected with three Histidine-N atom in the deoxy form,
where the Cu is in +1 state. Upon O2 binding, it forms oxy form and Cu(I)

changes to Cu(II), where the O2 is in peroxo bridged form with μ-η2:η2 linkage.
The lone electron in one of the Cu(II) combine with another electron in Cu(II) to
form an antiferromagnetic coupling, thus leads to EPR inactive and
diamagnetic
 R
R
TYROSINASE
MONOPHENOLASE
2H+
DIPHENOLASE
N N HO OH N O O N
O O
N Cu Cu N N Cu C N
(II) u
(II
(II) O (II) O
N N N ) N
OXY "DI"
R 3H+
R
R O
O2

HO
H+ O
N O N
N N O N N
(I) (I) N Cu Cu N (II) (II)
N Cu Cu N (II)O (II) N Cu Cu N
N N O
N N "MONO"
N N
Deoxy H

R
R
O R

O
N O O N HO OH
H+ 2H+
N Cu Cu N
(II) O (II)
N N
H
 TYROSINASE
MECHANISM

• In the oxy form, the μ-η2:η2 bridging linkage activates the hydroxylation of
monophenol to Catechol.

• Due to σ-donation from the μ-η2:η2 linkage to Cu, this peroxide is more
electrophilic than the peroxide bound in an η1 end-on fashion. Thus, in the
MONO form where the monophenolic substrate is present, donation of π-
electron cloud of the aromatic substrate into electrophilic peroxide linkage breaks
down the O-O bond and initiates the hydroxylation process.