Bioinorganic Chemistry

Essentials and trace elements of life; basic reactions in the

biological systems and the role of metal ions, especially
Fe2+, Fe3+, Cu2+ and Zn2+; structure and function of
hemoglobin and myoglobin and carbonic anhydrase.

By
SHILPENDU GHOSH
 Average human Fe distribution

Oxidation State of
Protein Function Amount of Fe (g) Percent of Total
Fe
Hemoglobin Plasma O2 transport +2 2.6 65

Myoglobin Muscle O2 storage +2 0.13 6

Transferrin Plasma Fe transport +3 0.007 0.2

Ferritin Cell Fe storage +3 0.52 13

Hemosiderin Cell Fe storage +3 0.48 12

Catalase H2O2 metabolism +2 0.004 0.1

Cytochrome c Electron transport +2/3 0.004 0.1

Oxidases, other
Other 0.14 3.6
enzymes, etc.
 Protoporphyrin IX and Heme

porphin Protoporphyrin IX Fe(II)-protoporphyrin IX complex

Heme-B
❖Type A hemes are found in cytochrome a, Type B hemes are found in
hemoglobin; myoglobin ,peroxidase , catalase, and cytochrome b
;Type C hemes are found in cytochrome c.
 Metalloenzymes = Protein + Cofactor
A cofactor is a non-protein chemical compound that is bound to a protein and is required for
the protein's biological activity. These proteins are commonly enzymes. Cofactors are either
organic or inorganic. They can also be classified depending on how tightly they bind to an
enzyme, with loosely-bound or protein-free cofactors termed coenzymes and tightly-bound
cofactors termed prosthetic groups.

➢Porphyrins with iron is a common prosthetic group in bioinorganic

chemistry

Cytochrome C Myoglobin Catalase Cytochrome P-450 Peroxidase

 JAM 2017
1. The 'heme’ containing protein(s) is/are

✓(a) Cytochrome c
(b) Hemocyanin
(c) Hemoerythrin

✓(d) myoglobin
 Inorganic Active site / Prosthetic group

In molecular biology the

active site (prosthetic
group) is part of an
enzyme where substrates
bind and undergo a
chemical reaction. It can
perform its function only
when it is associated with
the protein unit

Ferredoxin (e transfer)
Heme in Myoglobin (O2
storage)

Nitrogen Fixation
 Metalloprotein Ring name

❖Other Posthetic groups: Chlorophyll chlorin

Hemoglobin porphyrin

Vitamin B12 corrin

Coenzyme B12 Chlorophyll

 GATE 2011
2. A well known naturally occurring organometallic
compound is

✓ (a) vitamin B12 coenzyme

(b) chlorophyll
(c) Cytochrome P-450
(d) myoglobin
Hemoglobin and Myoglobin: structure

Myoglobin(monomer) Hemoglobin(tetramer)
 Changes at the active site during oxygenation of Myoglobin
DEOXYMYOGLOBIN OXYMYOGLOBIN
Distal
N histidine
N
N Protein
H N H H N Protein
N H
N N
N Fe
N Fe O
N N O
Protein Protein
Proximal
histidine

Fe2+ t2g4eg2, HIgh spin, radius 92 pm Fe3+ t2g5eg0, Low spin, radius 75 pm

Paramagnetic Diamagnetic

Fe 42 pm outside porphyrin plane Fe fits inside the porphyrin plane

Role of distal histidine: Makes O2 to bind in a bent fashion and makes it difficult for
CO to bind in a linear fashion.
▪ An isolated heme binds CO 25000 times as strongly as O2 in solution. In the living
system binding affinity for oxygen is reduced considerably. For CO to bind strongly, it
has to bind linearly which is made difficult by distal histidine
 GATE 2011
3. The red colour of oxyhemoglobin is mainly due to

(a) d-d transition

(b) metal to ligand charge transfer transition
(c) Ligand to metal charge transfer transition

✓ (d) inteeligand Pi to pi* transition

MSQ
4. Which of the following statement(s) is/are true about deoxy-
hemoglobin

✓(a) Fe present in deoxy-hemoglobin have +2 oxidation state

✓(b) Deoxy-hemoglobin is high spin complex
(c) The 6th coordination site is vacant or occupied by O2
✓(d) Deoxy-hemoglobin is a O2 carrier
 Can the prosthetic unit part of a metalloprotein perform its
normal function without the protein unit around it ?

Fe2+ + O2 Fe2+ O
O
Free Heme
Ferryl complexe
4+
Fe2+ O + Fe2+ 2 Fe O
O

Fe4+ O + Fe2+ Fe3+ O Hematin

Fe3+

❖ Without the globin protein ,Fe (II) will be

irreversibly oxidised . The oxide form of hemoglobin
and myoglobin contaion Fe(III) are described as
methemoglobin and metmyoglobin respectively.
Sickle Cell Anemia( Hemoglobin S ) and Thalassemia

❖ Sickle-cell anemia is caused by a mutation in the β-globin chain of hemoglobin, causing a

hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid
valine.
❖ In thalassemias, the alpha and beta chain are wrongly produced. Alpha- thalassemias are
caused by an erratic synthesis or total absence of alpha –globin chain of hemoglobin
.Similarly the beta-thelassemias are caused from the defect in the beta-globin chains of
hemoglobin .
Oxymyoglobin and oxyhemoglobin: Evidence for Fe3+ O2−

 O-O of oxyhemoglobin, 1107 cm-1

O2
is closer to the
 O-O O2− value of 1145 cm-1 than
 O-O O2 value of 1550 cm-1

N N
Fe3+

This difference suggests the formation of O2− which

is a spin ½ ion in combination with low spin Fe3+
which is also spin ½ and these two spins can pair by
N

N what is well known as antiferromagnetic coupling

and will be diamagnetic
 NET JUNE- 2015
5. The resonance Raman streaching frequency ( O-O ) for
O2 in bound oxy-hemoglobin is close to

(a) 1600 cm-1

(b) 1900 cm-1
(c) 800 cm-1

✓ (d) 1100 cm-1

 JAM 2015
6. Low spin iron(III) center is present in

(a) deoxy form of hemoglobin

✓(b) Oxy form of hemoglobin
(c) hemocyanin
(d) Carbonic anhydrase
 GATE 2013
7. Oxyhemoglobin Hb(O2) and oxymyoglobin Mb(O2),
respectively are

(a) Paramagnetic and paramagnetic

✓(b) Diamagnetic and diamagnetic
(c) Paramagnetic and diamagnetic
(d) Diamagnetic and paramagnetic
  141 Amino acid
Hemoglobin  146 Amino acid
Mb 153 Amino acid
Hb is not an exact tetramer of Mb

Four units of Hb

3 major types of Hb
Hb A (Adult)
Hb F ( Fetal)
Hb S (Sickle cell)
 DEOXYMYOGLOBIN OXYMYOGLOBIN
Distal
N histidine
N
N Protein
H N H H N Protein
N H
N N
N Fe
N Fe O
N N O
Protein Protein
Proximal
histidine

Fe2+ t2g4eg2, HIgh spin, radius 92 pm Fe3+ t2g5eg0, Low spin, radius 75 pm

Paramagnetic Diamagnetic

Fe 42 pm outside porphyrin plane Fe fits inside the porphyrin plane

➢ Basics of oxygenation remains same for Hb and Mb.

But there are some differences in the way the four
units get oxygenated. This begins with pulling of the
proximal histidine when Fe gets inside the plane of
the porphyrin ring upon oxygen binding.
 8. Hemoglobin is a oxygen carrying protein . The correct GATE 2012
statement about oxy-hemoglobin is that

✓(a) The metal is low spin +3 oxidation state while dioxygen is

in O2- form
(b) The metal is high spin in +3 oxidation state while
dioxygen is in O2- form
(c) The metal is in low spin in +3 oxidation state while
dioxygen is in neutral form
(d) The metal in high spin in +3 oxidation state while
dioxygen is in neutral form
 Hemoglobin: Tense (T) and Relaxed (R) States; Deoxy versus Oxy:
The cooperative effect

Binding of O2 to Hb is cooperative. The presence of bound

oxygen favor addition of more O2. The Hb molecule goes from a
tense to a relaxed state. Pockets of heme gets more easy for
the following O2 units to access due to breaking of some weak
interactions.
This happens like chain and pulley. The pulling of the proximal
histidine along with the activity of Fe getting into the plane of
the porphyrin triggers this activity

Removing the first stamp

requires more effort

Deoxy Hb Oxy Hb
 Hemoglobin; An allosteric protein
▪ An allosteric protein does not have fixed properties. Its functional characteristics of
are regulated by specific molecule present in its environment. Hemoglobin is an
allosteric protein while Myoglobin is not.
▪ Function of Hemoglobin in the living system is regulated by oxygen partial pressure,
H+ concentration and 2, 3 biphosphoglycerate presence (BPG)

O2
The Physiology of Hemoglobin and Myoglobin

➢ Hemoglobin has
relatively high affinity
for dioxygen at high
partial pressure of
dioxygen where
myoglobin has
relatively high affinity
for dioxygen At lower
partial pressure of
dioxygen.
 GATE 2016
9. At pH 7.2 and 10 Torr oxygen partial and extent of O2
binding is

(a) high for both hemoglobin and myoglobin

(b) High for hemoglobin and low for myoglobin

✓(c) High for myoglobin and low for hemoglobin

(d) Low for both hemoglobin and myoglobin
 The Bohr Effect
Christian Bohr, father of Niels Bohr discovered this effect. An increase
in concentration of protons(decrease in PH) and/or carbon dioxide will
reduce the oxygen affinity of hemoglobin

CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3-

▪ The chemical basis for the Bohr effect is due

to the formation of two salt bridges of the
quaternary structure. One of the salt bridges
is formed by the interaction between
Histidine 146 and Lysine 40. This connection
will help to orient the histidine residue to also
interact in another salt bridge formation with
the negatively charged aspartate 94. The
second bridge is formed with the aid of an
additional proton on the histidine residue.
 GATE 2015
10. Among the given pH values , the O2 binding
efficiency of hemoglobin is maximum at

(a) 6.8
(b) 7.0
(c) 7.2

✓(d) 7.4
 2, 3 biphospho glycerate (BPG)

2,3- Biphosphoglycerate

➢ The organic compound 2, 3 biphospho glycerate (BPG) binds to

hemoglobin A and reduces its O2 affinity by a factor of 26. This at the
first instance will make one wonder why?
▪ Interestingly, this increases the oxygen-binding affinity of fetal
hemoglobin (Hb-F) relative to that of maternal (Hb-A) hemoglobin. This
difference in oxygen affinity allows oxygen to be effectively transferred
from maternal to fetal red cells, the transport of oxygen from mother to
fetus.
 JAM 2020
11.

✓
CURIE LAW:the susceptibility of paramagnetic materials is
inversely proportional to their temperature, i.e. that materials
become more magnetic at lower temperatures.

where
is the (volume) magnetic susceptibility,
is the magnitude of the resulting magnetization in amperes /meter (A/m),
is the magnitude of the applied magnetic field (A/m),
is absolute temperature, measured in kelvins (K),
is a material-specific Curie constant (K).
 Other Biological Dioxygen Carrier: Hemerythrin
It is a non-Heme iron containing protein found in marine invertebrates. It contain 8 subunit but no
cooperativity between the subunits.
Deoxy Form: Oxy form:
I. Active site has two Fe in +2 oxidation state I. Both Fe in low spin +3 oxidation state
II. Diamagnetic(antiferromagnetically coupled) II. Diamagnetic(antiferromagnetically coupled)(EPR inactive)
III. colourless III. Violet-pink
IV. ( O-O )=845 cm-1
V. HO2- form
 GATE 2016
12. During the oxygen transport by Hemerythrin , oxygen
is bound as

(a) O2- to one Fe(III) only

✓ (b) HO2
- to one Fe(III) only

(c) O22- to one Fe(II) and one Fe (III)

(d) O22- to two Fe(II)
MSQ
13. The change(s) which occurs when O2 binds to hemoerythrin
is/are

(a) One iron atom is oxidised

✓(b) Both of the iron atoms are oxidised
✓(c) O2 bonded to one iron atom
(d) O2 binds to both the iron atoms and is also hydrogen bonded
 GATE 2013
14. The number of oxygen molecule(s) that a subunit of
Hemerythrin can transport is

✓ (a) One
(b) Two
(c) There
(d) Four
 Hemocyanin
▪ It is Cu containing oxygen carrier in invertebrates , Mollusca , orthopoda .

Deoxy Form: Oxy form:

I. Active site has two Cu in +1 oxidation state I. Both Cu in +2 oxidation state
II. Diamagnetic(d10 system) II. Diamagnetic(antiferromagnetically coupled)(EPR inactive)
III. colourless III. Blue colour
IV. O2- ( O-O )=750 cm-1
 horseshoe crab (Limulus polyphemus)
The blood of horseshoe crabs contains one type of blood cell, the amebocytes. These play an important role in
the defense against pathogens. Amebocytes contain granules with a clotting factor known as coagulogen; this
is released outside the cell when bacterial endotoxin is encountered. The resulting coagulation is thought to
contain bacterial infections in the animal's semi closed circulatory system.
 GATE 2015
15. Identify the function of hemocyanin and the metal
responsible for it

(a) O2 transport and Fe

✓(b) O2 transport and Cu
(c) election transport and Fe
(d) Election transport and Cu
 JAM 2019
16. The correct option for the metal ion present in the
active site of myoglobin, hemocyanin and vitamin B12
respectively is

(a) iron,iron and zinc

(b) molebodynum, iron and copper

✓(c) iron, copper and cobalt

(d) molebodynum, copper, cobalt
 17. In oxy-hemocyanin , the oxidation state of copper
and total number of histidine ligands respectively are

(a) 1,3
(b) 2,4

✓(c) 2,6
(d) 1,6
 GATE 2006
18. In the biological systems, the metal ion involved in
the dioxygen transport besides Fe is

(a) Co
(b) Zn
(c) Mg
✓(d) Cu
 NET DEC- 2015
19.

✓
 Metalloenzymes: Carbonic Anhydrase
A single polypeptide chain ( M = 29,000) complexed to one Zn2+ ion.
The zinc prosthetic group in the enzyme is coordinated in three
positions by histidine side-chains. The fourth coordination position
is occupied by water. This causes polarization of the hydrogen-
oxygen bond, making the oxygen slightly more negative, thereby
weakening the bond. A fourth histidine is placed close to the
substrate of water and accepts a proton. This leaves a hydroxide
attached to the zinc. The reaction catalyzed by carbonic anhydrase
is given below which occurs 5000 times faster in presence of the
enzyme:

in tissues- high CO2 concentration)

Carbonic anhydrase has one of the highest overall rates of reactions of any
enzymes. This is expressed in terms of turnover number of a catalyst (number of
substrate molecules converted per molecule of the enzyme per second; same
as TOF in organometallic catalysis). For human carbonic anhydrase it is 400,000
to 600,000 per second.
Most efficient catalytic reaction known so far
!!
Reaction increases acidity in the tissues
Metalloenzymes: Carbonic Anhydrase

Why Zinc?
I. A good Lewis Acid
II. Only one stable oxidation state
III. Complexes are labile than other
divalent metals
IV. Favors tetrahedral geometry
The active site also contains specificity pocket for carbon dioxide,
bringing it close to the hydroxide group. This allows the electron rich
hydroxide to attack the carbon dioxide, forming bicarbonate

Carbonic anhydrase increases acidity in the vicinity..With enough

carbonic anhydrase enzymes present, therefore, carbon dioxide can
cause a decrease in the pH of the solution due to all the protons
produced from its reaction with water.
 JAM 2018
20. Carbonic anhydrase is an example of

✓(a) Hydrolysis enzyme

(b) Redox enzyme
(c) O2 transport protein
(d) Heme protein
 GATE 2005
21. The metal present at the active site of the protein
carboxypeptidase A is

✓(a) Zinc
(b) molybdenum
(c) magnesium
(d) cobalt
 JAM 2014
22. The metal ion of an enzyme involved in the
hydration of CO2 is

(a) Cu(II)
(b) Fe(II)
(c) Mg(II)

✓(d) Zn(II)
 Redox intermediates in electron
transfer
S(Cys) Protein

Cytochrome C
protein S(Cys) Protein

N N
N N CH3
H Fe S
Protein chain has 103 amino
acid units in some fish, 104 N N
methionine
residue of
units in terrestrial vertebrates protein
and 112 in plants

OH
HO O
O

The most structurally well understood cytochrome. The heme active site is hexa
coordinated with N from a histidine residue and S from a methionine residue.
Present in photosynthesis and respiration chains- one of the oldest chemicals
present in biological processes
 GATE 2008
23. In biological systems , the metal ions involved in
electron transport are

(a) Na+ and K+

(b) Zn2+ and Mg2+
(c) Ca2+ and Mg2+

✓ (d) Cu2+ and Fe3+

 GATE 2010
24. Among the following pair of metal ions present in
nature .The first one functions as an electron transfer
agent and the second one catalyses the hydrolysis
reaction . The correct pair is

✓ (a) Fe and Zn
(b) Mg and Fe
(C) Co and Mo
(d) Ca and Cu
 GATE 2005
25.

✓
Active site of Cytochrome c oxidase
The last enzyme in the respiratory
electron transport chain and is located
in the mitochondrial membrane. It
receives an electron from each of four
cytochrome c molecules, and transfers
them to one oxygen molecule,
converting molecular oxygen to two
molecules of water.

The Fe is pentacoordinated and binds

O2 (along with the Cu) before
reducing it.
This is also the site which CN- binds
during cyanide poisoning [stabilizing
the Fe3+ state and preventing its
redox (Fe2+/ Fe3+) activity] (Cyanide
is a very strong ligand) Cyanide
Summary reaction:
4 Fe 2+cytochrome + 8 H+in + O2 → 4 Fe 3+cytochrome + 2 H2O + 4 H+out
 JAM 2013
26. For each of the following metalloproteins identify
the metal ion at the active site and the function of the
proteins: deoxy-hemoglobin, deoxy-myoglobin, oxy-
haemoglobin, cytochrome- c and carbonic anhydrase.
 GATE 2006
27. Iron sulpher clusters in biological systems involved in

(a) proton transfer

(b) Atom transfer
(c) Group transfer

✓(d) Election transfer