If you find it difficult to reconcile the structure of heme with the usual concept of

ferrous iron as a divalent ion, remember that iron, in common with certain other
metals, is capable of forming two types of bonds, ionic and covalent. Often our
attention is directed only to the ionic bonding, so that Fe++ would be considered to have
a valency of two. But a ferrous ion can also form up to six covalent bonds; in fact, in
water solution the ion is not Fe++ but Fe(H2O)6++. Such ions are called complex ions,
and are of major importance in biological systems in the binding of metals to proteins
and other substances. The structure of heme, in simplified form, is shown in Figure
2.2. The N's representing the nitrogen atoms of the porphyrin ring system. Heme is a
neutral molecule in that two of the bonds of the dispositive iron are to nitrogen atoms
which can be viewed as having negative charges.
Heme-proteins are characteristic of aerobic life. Hb is important in O2-binding and its
transport and delivery to tissues which is required for metabolism.
Heme is a Fe-porphyrin compound. The porphyrins are complex compounds with a
"tetra-pyrrole" structure, each pyrrole ring having the following structure;

Four such pyyroles called I to IV, are combined through CH=bridges, called as
"methyne" or "methylidene" bridges to form a porphyrin nucleus.
Heme may be represented by following structural formula schematically, with its
attachment to globin (Fig. 2.2). The outer carbons of the four pyrrole rings, which
are not linked with the methylidene-bridges, are numbered 1 to 8. The methylidene
bridges are numbered 1 to 8. The methylidene bridges, are numbered 1 to 8. The
methylidene bridges are referred to as ,,  and respectively. The two hydrogen
atoms in the – NH groups of pyrrole rings (II and IV) are replaced by ferrous iron (Fe+
+
) which occupy the centre of the compound ring structure and establish linkages with
all the four nitrogens of all the pyrrole rings.

Fig. 2.2: Structure of Heme

The Fe, besides its linkages to four nitrogens of the pyrrole rings, is also linked
internally (5th linkage) to the nitrogen of the imidazole ring of histidine (His) of the
polyeptide chains ("heme-linked" group). It is considered to have a valance of six as in
ferrocyanide H4Fe(CN)6 and the sixth valence is directed outwards from the molecule
and is linked to a molecule of H2O in deoxygenated Hb. When Hb is oxygenated, the
H2O is displaced by O2.

Hb.H2O + O2  Hb.O2 + H2O

The propionic acid COOH groups of 6 and 7 positions of heme, of III and IV pyrrole,
are also linked to the basic groups of amino acids Arg and Lys of the polypetide
chains.

2.1Structure & Function of Hemoglobin

Hemoglobin, a conjugated type of protein, is the red colouring matter of the blood. Its
normal concentration is 14-16 gm./100 ml. of blood. Hemoglobin is .found to be
present in special cells, known as red blood corpuscles (RBC). This blood protein
plays an important function in the phenomenon of respiration as it carries oxygen from
the lungs to the tissues and carbondioxide from tissues to lungs. It is chromoprotein
(type of conjugated protein), the protein part is globin (94%) and prosthetic group is
heme which is an iron (ferrous) complex of protoporphyrin.

Heme, the coloured component of hemoglobin, is an iron (ferrous) complex of

protoporphyrin The protoporphyrin nucleus in turn is composed of four  substituted
pyrrole nuclei linked by means of methine (= CH) groups on the -positions.

Haemoglobin is a red pigment of blood. It has two part: globin and a histidine group
(Fig. 2.3). As has been mentioned above in hemoglobin, heme nucleus remains
attached via its iron with the histamine residues of the globin molecule by means of
coordinate linkage.
The giobin molecule (an example of histones) consists of four peptide units, arranged
in tetrahedral configuration; it is the nature of the four peptides which differentiate the
different types of hemoglobins. Each polypeptide chain has one heme molecule which
is suspended between two histidine residues. One of the histidine residues (number 87
in the -chain and 92 in the -chain) is linked directly to the iron atom of heme, while
the other histidine residue (58 in the -chain and 63 in the -chain) is linked with the
iron atom but with a gap between them into which the oxygen molecules are
introduced during the formation of oxyhemoglobin. Thus each globin molecule
containing four polypeptide chains possess four heme molecules to form hemoglobin.
Thus, it is a globular protein which is composed of polypeptide chains. These chains
are arranged in a regular tetrahedral form and are attached with four rings of pyrole
(Fig. 2.2).

Functions:

(i) It is essential for O2 carriage.

(ii) It plays important part in CO2 carriage.

(iii) It is important buffering system of blood.

(iv) Various pigments of bile, stool and urine are formed from it.
In has been indicated that the outstanding property of the hemoglobins is their
reversible interaction with oxygen. This can be visualized in simplified fashion as
occurring in the following way:

The combination occurs at the unpaired electrons of iron in the heme portion. Now
since each hemoglobin molecule has four heme molecules, one hemoglobin molecule
can combine with four molecule of oxygen. In hemoglobin at least one, and probably
both, of the water molecules of the heme structure are replaced by groups of the
protein, in all likelihood nitrogen atoms of histidine side-chains. The combination of
hemoglobin with oxygen results in the replacement of one of these groups by the
oxygen molecule. The oxygenated form is usually called oxyhemoglobin; the
deoxygenated form, simply hemoglobin. A diagrammatic version of oxyhemoglobin
appears in Figure. 2.4. In haemoglobin, the globin part is in combination with histadine
nitrogen of protein, through Fe(II) of hem group. Thus, there are five nitrogen in the
coordination sphere of iron (four from porphyrin ring and the fifth from histamine),
and the sixth coordination position is available for O2 or H2O.

Fig. 2.4: Formation of oxyhaemoglobin

2.2Myoglobin
The red color of many animal tissues as seen in a meat market is not due to the
hemoglobin of blood but to a related compound called myoglobin. This compound is
located within the various cells and therefore is not involved in oxygen transport from
place to place in the organism. Rather it seems to serve as an emergency store of
oxygen. Generally, those cells which are more active in metabolic transformation have
higher myoglobin contents.
Myoglobin has a molecular weight of about 17,000 and contains only one heme unit
per molecule. It combines more strongly with oxygen than does hemoglobin, and
releases the oxygen only when the free oxygen concentration drops to rather low
levels. The brown color of meat which has been unduly exposed to oxygen, as in long
storage or in cooking is due to the oxidation of the iron to the ferric stage, giving
metmyoglobin. Similar to haemoglobin, its sixth coordination is also vacant; hence
dioxygen molecule can coordinate at this position reversible. At high pressure, like
haemoglobin, myglobin also binds O2 efficiently. However, at low pressure it binds O2
at relatively faster rate. Due to utilization of oxygen, the pressure of oxygen in
muscles reduces, resulting in assimilation of CO2 in tissues. This also reduce pH. As a
result removal of oxygen from haemoglobin and getting into myoglobin becomes
easier. The mechanism of linking with O2 is analogous to haemoglobin and the
molecular state of dioxygen in oxymyoglobin is similar to that in oxyhaemoglobin.

Physiology of Myoglobin and Haemoglobin:

In vertebrates when blood enters in to lungs (or gills), the partial pressure of dioxygen
there is relatively high. When it reaches in the tissues of red blood cells, the partial
pressure is quite less there; hence the following reactions take place:

In lungs : Hb + 4O2  Hb (O2)4

In Tissues : Hb(O2)4 + 4Mb  4Mb(O2) + Hb

Thus, haemoglobin is ambivalent here, it strongly combines with dioxygen and

transfers it to tissues as far as possible. During this process it is easily accepted by
myoglobin, which stores it for oxidation of food. This transfer is due to higher affinity
of myoglobin for dioxygen, as compared to that of haemoglobin. The stability constant
of myoglobin-dioxygen complixation may be given as-

In cells concentration of oxygen is quite low and myoglobin can combine with it, even
in low concentrations of oxygen, resulting in sufficient quantity of oxymyoglobin. As
this is not possible in case of haemoglobin, it becomes saturated with doixygen in
lungs and becomes deoxygenated in capillaries. Haemoglobin shows dependence on
pH, but myoglobin does not. Thus, the difference in the behaviors of haemoglobin and
myoglobin towards dioxygen is related with the structure and mobility of four chains.

2.3Hemoglobin and Haemerythin

Haemocyanins are iron proteins which have no heme but they transport oxygen. In
each sub unit of hamocynin, there is a pair of copper atom. Haemocyanin, binds and
transports oxygen in mollusca and orthopodophyla species. These are macromolecules
which have generally more than 10037 units and molecular weights in between 25000
to 75000. The pair of copper atoms in haemocyanins bind dioxygen molecule. The
protein is colorless when it does not has oxygen, but when it binds oxygen it is blue,
because Cu(I) is oxidized into Cu(II). Oxyhaemocyanins, although are completely
magnetic, but give antimagnetic reactions on copper centers.

In oxyhaemocyanins the two copper atoms are separated at a distance of 367 Ao, and
are linked with three histine ligands and peroxy group and phenolate of tyrosine (Fig.
2.6).

The analysis of EXAS data of  and  compounds of Megathura crenuleta and flelix
pomatia indicates that two histidine ligands and two x ligands (N or O) are present on
each copper atom at a distance of 3.55 A o. Copper atoms are almost in a square planar
geometry and are linked sharing X and peroxide oxygen. The coordination number of
these (two) copper atoms is low but these are quite suitable to bind dioxygen
molecules. After oxidation of copper (I) into Cu(II), these copper atoms are interlinked
through X atoms. The peroxide linked to these copper is in binuclear state.
 Fig. 2.6: Active positions in oxyhaemocynin

Haemerythin:

Haemerythin is used for transportation and storage of dioxygen in different

nonvertebrate species of sea. Oxygen-less haemerythin is colourless or red and it turns
pink after oxidation. The haemerythis of Golfingia goulie insect has molecular weight
approximately 108000 and is made of eight similar subunits. Each of these sub unit has
two iron atoms linked with an oxygen molecule. Myohaemerythin, present in tissues of
muscles is a mononumeric species. Oxymyohaemerythin is more stable, just as
myoglobin is than haemoglobin.

Oxygen-less haemerythin after reaction with dioxygen oxidises the two Fe(II) centers
into Fe(III) linked with peroxide.

Oxyhaemerythin in solution and by oxidation gives Fe(III), which does not react with
oxygen, although Fe(III) links with anions. Probably these anions bind in place of O2:

Fe(II) - Fe(II) + O2 Fe (III) - Fe(III) + O2-

Deoxy  L-
Fe (III) Met Fe (III) + O2-

[Fe (II) . Fe (III)]8 (Fe (II))4 . (Fe III)4

Oxygen-less haemerythins are diamagnetic and have two high spin Fe(II) centers. The
two peroxide atoms have different atmosphere and the Fe(III) centers different.
 Model synthetic complexes of iron and copper

Although, the most common mode of reaction by which molecular oxygen binds with
transition metal complexes is oxidation, yet there are several instances wherein dioxygen
can function as neutral ligand. Certain metal complexes provide the delicate balance
needed to form adducts with dioxygen without the metal or the ligands being irreversibly
oxidized. Such type of metal complexes which can be effectively utilized in transport
and storage of oxygen are often known as synthetic oxygen carriers. The equation for
adduct formation in which these oxygen carriers take up and release dioxygen reversibly

is written as:
Molecular oxygen can be reduced by one electron process without O-O bond being
broken:
Each of these species can act as a ligand towards transition metals.

To understand all about synthetic oxygen carriers one should consider the following the
electronic structure of oxygen molecule (Figure 1):
Figure 1: Electronic structure of oxygen molecule

Synthetic model complexes of iron

Coordination researchers and chemists for many years were unable to develop synthetic
iron- containing oxygen carriers to mimic the natural occurring hemoglobin and
myoglobin proteins since in all the cases Fe (II) complexes were irreversibly oxidised by

oxygen to produce μ-oxo Fe (III)-O-Fe (III) complexes (as elucidated in the following
equation):

In the 1970s, investigators designed new synthetic approaches to avoid the formation of
μ-oxo Fe complexes in order to successfully fabricate synthetic iron oxygen carriers and
for this purpose they have used following approaches: (i) Steric: introduction of the bulky
groups on the ligands in such a fashion which sterically inhibit the formation of Fe-O-Fe
dimer (ii) Low temperature: use of low temperature slows down the rate of reactions
leading to dimerization. (iii) Surface attachment: attachment of the iron complex on the
rigid surface prevents dimerization process. However, amongst the above methods to
synthesize modified porphyrins, steric hindrance has produced much more elegant work.
Initial attempt was made to test this concept to synthesize tail base complexes (i) in which
an imidazole is covalently attached Fe(II) porphyrins (Figure 2). The studies on such tail
base complexes revealed that it leads to the formation of 1:1 adducts only at low
temperatures. On the other hand, the strapped complexes (ii) and (iii) with a hydrocarbon
chain linked over the face of the porphyrin do not undergo reversible binding with oxygen
because of lack of rigidity of the chain, which can be pushed out of the way. Further, the
use of steric constraints has been very nicely demonstrated by Collman and Baldwin.
Collman et al. reported the “picket fence” porphyrins (iv) and (v) that not only favour
five-coordination but also inhibit above said bimolecular interation. In this model, on one
side of the porphyrin ring there should be great steric hindrance with suitable ligand such
as N -alkyl imidazole while the other side should be left unencumbered to provide
hydrophobic site for oxygen. Further, Baldwin and co-workers demonstrated that
‘capped’ porphyrin (vi) also provides satisfactory steric protection and hence could react
reversibly with oxygen in pyridine.
Figure 2: Different types of to iron porphyrin complex to avoid the formation of oxo complex so that
reversible binding is possible (i) tail base (ii) strapped (iii) Strapped (iv) Picket fence (v) Picket
fence/tailbase (vi) Capped porphyrins

Synthetic model complexes of copper

During the past few decades, the dicopper sites of hemocyanin which play a crucial role
in the biological reactions of oxygen, have drawn the attention of several inorganic
chemists worldwide as they exhibit interesting spectroscopic and magnetic properties.
Therefore, several attempts have been made to develop suitable copper complexes that
would mimic some of the properties of hemocyanin. A major breakthrough in synthetic
modelling chemistry came in 1989 when Kitajima and co-workers synthesized and
characterized a di-CuII peroxo complex {Cu[HB(3,5- iPr2pz3]}2 using sterically
encumbering tris(pyrazolyl)borate ligand (Figure 3). They found that oxygen binding
occurs between two Cu centers in a side-on mode. Besides, studies revealed that this
compound could be generated at reduced temperatures in solution by O2 reaction with 1
or via an acid-base reaction of hydrogen peroxide with dicopper (II) species 2.
 Figure 3: Synthesis of di-CuII peroxo
complex {Cu[HB(3,5-iPr2pz3]}2 using
sterically encumbering
tris(pyrazolyl)borate ligand

Recently, Simmons and Wilson have reported a synthetic copper(I) complex containing
two imidazole donors that are capable of binding dioxygen reversibly in both solid state
as well as in solution. Firstly they synthesized the pentadendate ligand [2,6-diacetyl-1-(2-
imidazole-4- ylethylimino)ethyl)pyridine] abbreviated as “DIEP” via condensation of 2,6
diacetylpyridine with two moles of histamine. Subsequently, [Cu(I)Me(CN)4]ClO4 was
added under nitrogen atmosphere to generate the dark red coloured [Cu(I)(diep)(ClO 4)]
complex in which copper is presumed to be five-coordinated (Figure 4). It was found
that synthesized copper (I) complex absorbed one mole of O2 per 2 moles of copper and
this reaction could be readily reversed by gentle heating and degassing with
nitrogen.