Dr. Shubhra Bikash MaityDepartment of ChemistryCV Raman Global University

Bioinorganic Chemistry

Dioxygen Transport

Structures of Amino Acids

H2N COOH

H

H2N COOH

CH3

H2N COOH H2N COOH H2N COOH

HOH OH SH

H2N COOH H2N COOH H2N COOH H2N COOH

H2N COOHH2N COOH H2N COOH H2N COOH H2N COOH

H2N COOH H2N COOH H2N COOH H2N COOH H2N COOH

S

NH

COOH

OHNH

OH

OOHO

NH2

OO NH2

NHHN

NH3

NH

NH2H2N

Glycine (Gly) G Alanine (Ala) A Serine (Ser) S Threonine, Thr (T) Cysteine (Cys) C

Small Nucleophile

Hydrophobic

Aromatic Acidic

Amide Basic

Phenylalanine (Phe) F Tyrosine (Tyr) Y Tryptophan (Trp) W Aspartic Acid (Asp) D Glutamic Acid (Glu) E

Valine (Val) V Leucine (Leu) L Isoleucine (Ile) I Methionine (Met) M Proline (Pro) P

Asparagine (Asn) N Glutamine (Gln) Q Histidine (His) H Lysine (Lys) K Arginine (Arg) R

The Primary Structure of a Protein

Primary Protein Structure: Sequence of a chain of amino acids

The Secondary Structure of a Proteins

Hydrogen bonds can form betweennearby amino and carbonyl groupson the same polypeptide chain.

Left: an �-helix (alpha-helix), in which the polypeptide’s backbone is coiledRight: a �-pleated sheet (beta-pleated sheet), in which segments of a peptide chain bend 180° and then fold in the same plane

Tertiary & Quaternary Structure of Proteins

Interactions that determinethe tertiary structure ofproteins

TERTIARY STRUCTURES

QUATERNARY STRUCTURES

The Cro protein is a dimer—it consists of twoidentical polypeptide subunits

Hemoglobin is a tetramer—it consists of four polypeptide subunits. two identical α subunits and

two identical β subunits

Summary: Protein Structure

NON-PROTEINSPROTEINS

ELECTRON TRANSPORT

DIOXYGEN MANAGEMENT

METAL MANAGEMENT

PHOTOREDOX

METAL STORAGE & TRANSFER

Iron-sulfur (Fe), Blue copper (Cu),& Cytochromes (Fe)

Hemoglobin (Fe), Myoglobin (Fe), Hemocyanin (Cu), & Hemerythrin (Fe)

Ferritin (Fe), Transferrin (Fe),& Ceruloplasmin (Cu)

MetalloenzymesTRANSPORT & STORAGE

HydrolasesePhosphatases (Mg, Zn, Cu)Aminopeptidases (Mg, Zn)Carboxypeptidases (Zn)

Oxidoreductases

Oxidases (Fe, Cu), Reductases (Fe, Cu, Mo)Superoxide dismutases (Cu, Zn, Mn)

Isomerases and synthetases

Vitamin B12 coenzyme (Co)

Transferases Kinases (Mg)

METALLOBIOMOLECULES

Lyases

Lygases Glutamin synthetase (Mg, Mn)

Enolases (Mg)

Chlorophyll (Mg)

Siderophores (Fe)

Porphyrin and Metalloporphyrin22π electrons but only 18 are delocalized (n= 4 in Hückel’s rule)

Hans Fischer (1881-1945)Nobel Prize in Chemistry (1930)

NH

NH HN

HN NH

N HN

N

Porphyrinogens Porphyrins/Porphinshighly colored

(purple)Colorless

Pyrrole ring

h O2N

N N

N

Mn+

Metalloporphyrins

Mn+

NH

N HN

NA B

CD

1

2 3

4

567

8

2.05

How to draw Porphyrin molecule??

NH

NH HN

HN

Hemoglobin: A Protein that transports O2 to all organs of the body though blood

Oxygen Carrying & Storage Proteins….

Heme

Myoglobin: A Protein thatstores O2

PDB ID: 1GZX PDB ID: 3RGK

Some Properties of Oxygen Carrying Proteins

Property Myoglobin (Mb) Hemoglobin (Hb)

Metal Fe Fe

Oxidation state of metal in deoxyprotein

(II) (II)

Metal:O2 Fe:O2 Fe:O2

Oxidation state of metal in oxy protein

(III) (III)

Color, deoxy state Red-purple Red-purple

Color, oxy state Red Red

Environment around Metal centre Porphyrin ring Porphyrin ring

Molecular weight (Da) 17,100 65,000

Number of subunit(s) 1 (α = 161 residues) 4 (2α + 2: α = 141 residues& = 146 residues)

Deoxy-Hb/Mb Oxy-Hb/Mb

Oxygen Binding Changes the Position of Iron Ion in Hb/Mb

High spin Fe(II) Low spin Fe(III)

Dioxygen and its Reduced Species

Species νO-O (cm-1) dO-O (Å)

O2+ 1905 1.12

O2 1580 1.21

O2- 1097 1.33

O22- 802 1.49

From resonance Raman spectroscopy the O-O stretch in oxy-Mb is measured to be ̴1105 cm-1. The protein is also diamagnetic (d5, FeIII and O2

- couple)

Oxygen Binding to Hb: Statistical Probability

Musical Chair!!

Oxygen Binding to Hb: In RealityIn reality the successive O2 binding constants of Hb gradually increases, i.e.K1 < K2 < K3 < K4 . This is due to cooperative binding of O2 with Hb.

This happens like chain and pulley. Thepulling of the proximal histidine alongwith the activity of Fe getting into theplane of the porphyrin triggers thisactivity

Cooperativity in O2 Binding & Release in Hb

Oxygen binds to one Hb sub-unit

Fe(II) contracts & moves into plane of porphyrin ring

Moves the histidineattached to it

Triggers conformational changes in the globin chain

Translated through H-bond network

Enhances the ability of other three units to bind O2

NB: Due to MONOMERIC in nature Mb does not possess such cooperative interactions.

In a similar way when blood reaches the muscle, only one O2 is released , the others arereleased even more easily due to the cooperative effect in reverse way.

The Phenomenon is called Cooperative Effect

O2 Affinity of Mb & Hb: Hill PlotDue to monomeric in nature & absence of cooperative interaction Mb takes up O2 in

1:1 ratio. The equilibrium expression is therefore expressed as

On the other hand due to tetrameric nature & cooperative interaction oxygenation of Hb may be expressed as

Fraction (f) of Mb & Hb oxygenated could be expressed as

Similarly for Hb -

Which gives the Hill equation for oxygenation of Mb and Hb respectively -

O2 Affinity of Mb & Hb: Hill Plot

O2 Binding Curve: Effect of Cooperativity

Effect of pH on Oxygenation of Hb/Mb

Oxygenation of Hb becomes pH dependent: Bohr Effect

Christian Bohr, father of Niels Bohr discoveredthis effect. An increase in concentration ofprotons and/or carbon dioxide will reduce theoxygen affinity of hemoglobin

The chemical basis for the Bohr effect is due tothe formation of two salt bridges of thequaternary structure. One of the salt bridges isformed by the interaction between Histidine 146and Lysine 40. This connection will help to orientthe histidine residue to also interact in anothersalt bridge formation with the negatively chargedaspartate 94. The second bridge is formed withthe aid of an additional proton on the histidineresidue.

Below a pH of 6, theimidazole ring ofhistidine is mostlyprotonated thusfavoring salt bridgeformation

Role of Hb & Mb in O2 & CO2 transport

Role of Hb & Mb in O2 & CO2 transport

2, 3 biphospho glycerate (BPG)

2,3- Biphosphoglycerate

The organic compound 2, 3 biphospho glycerate (BPG) binds to hemoglobin Aand reduces its O2 affinity by a factor of 26. This at the first instance will makeone wonder why? Interestingly, this increases the oxygen-binding affinity of fetalhemoglobin (Hb-F) relative to that of maternal (Hb-A) hemoglobin. Thisdifference in oxygen affinity allows oxygen to be effectively transferred frommaternal to fetal red cells, the transport of oxygen from mother to fetus.

Hemoglobin A and Hemoglobin F Differences Babies are born with hemoglobin F (Fetal),but after a few months, the body shuts off itssynthesis and starts making hemoglobin A(Adult). That's called the hemoglobin switch.

From the structural point of view, the adult hemoglobin is composed of 4heme groups, 2 alpha chains and 2 beta chains. The fetal hemoglobin isalso composed of 4 heme groups, 2 alpha chains and 2 delta chains. The chain is 72% identical in amino acid sequence with the β chain. Onenoteworthy change is the substitution of a serine residue for Histidine143 in the β chain. In addition, the fetal hemoglobin and adulthemoglobin are found to be different near the 2,3 BPG binding site. The2,3 BPG binds less tightly with the deoxy form of fetal hemoglobin ascompared to the deoxy form of adult hemoglobin.

Hb A chain

Histidine

Hb F d chain Serine

Hemoglobin S (Sickle Cell Anemia)

Sickle-cell anemia is caused by a mutation in the β-globin chain ofhaemoglobin, causing a hydrophilic amino acid glutamic acid to bereplaced with the hydrophobic amino acid valine.

In areas where malaria is a problem, people's chances of survival actually increaseif they carry sickle-cell trait (Carrier). The malaria parasite has a complex life cycleand spends part of it in red blood cells. In a carrier, the presence of the malariaparasite causes the red blood cells with defective haemoglobin to ruptureprematurely, making the plasmodium unable to reproduce. The polymerization ofHb S affects the ability of the parasite to digest Hb.

Role of Distal Histidine: Binding of CO vs O2

Role of Distal Histidine: Binding of CO vs O2

Role of Distal Histidine: Binding of CO vs O2

Role of Distal Histidine: Crystallographic Evidence

Binding Effect of CO vs O2 to Hb and Mb

Role of Protein Chain

Synthetic Hemes (without protein chains)

Balch et al. J. Am. Chem. Soc. 1984, 106, 7779

Model Systems for Hemoglobin and MyoglobinMethods used to inhibit the formation of μ-oxo-diiron(III)hemes: The use of low temperature so that the reactions leading to dimerization are very slow

Using steric constrains in such a way that the dimerization is inhibited: With the use ofsteric constrains a good no of Fe(II) porphyrin have been synthesized which have specialstructural features designed to facilitate reversible binding of oxygen without oxidation.Some of them are represented in the figures below namely- “picket-fence”, “strapped”,“capped”.

At room temperature this “tail-base” complex is capable offorming a μ–oxo–dimer and however it undergoes reversibleoxygenation at -45 °C leading to dimerization is prevented.

A= NH, R = H, CH2CH2COOH, CH2CH2COOMe

“picket-fence” porphyrin withsubstituted imidazole axial ligands

single-strapped, and double-strapped Capped porphyrinates

Rigid surface attachment of the iron complex to a surface (e.g., silica gel) so thatthe dimerization is prevented

Treatment of polystyrene–Fe(II)(TPP)Im complex with O2 in benzene led to oxidation and formation of the μ-oxo dimer:

Therefore, the cross-linked polystyrene ligand was not sufficiently rigid to prevent dimerizationon treatment with oxygen. However, Fe (II)(TPP)Im–silica gel complex reversibly binds O2:

Polystyrene-Fe(II)(TPP)Im complex

Attached to modified silica gel

NB: CoboglobinA coboglobin is a synthetic compound, a metalloprotein chemically similar to hemoglobin ormyoglobin but using the metal cobalt instead of iron (hence the name). Just like hemoglobin andmyoglobin, the coboglobins are able to reversibly bind molecular oxygen (O2) at the metal atom.However they lose this ability much faster than the natural molecules.

Hemocyanin, Hc (Greek: Blue Blood)

O2 carrying protein with Cu centre as active metal site.

Hc functions as O2 transporter and ensures sufficient O2 in the bodytissues of these “SLOW MOVERS”.

The Asymmetric unit of the hemocyanin crystal consists of six protein subunits each of mass ca.75kDa with an overall mass ca. 460kDa.

Found in some arthropods (Shrimps, Crabs) & Mollusks (Octopus,Snails).

Hemocyanin, Hc (Greek: Blue Blood)

Deoxy-form: Colorless

Oxy-form: Blue (LMCT)

Each Cu (I) centre (d10 ) is diamagnetic.

Each Cu centre in a sq. planer environment with empty coordination site for O2 binding.

Cu-Cu distance (internuclear): 3.7 ± 0.3 Å& Cu-N bond distance: 2Å

Intranuclear Cu-Cu distance 3.6 Å

Oxygen coordination with Cu centers distort the histidines bind protein chains.

Strong antiferromagnetic coupling between two Cu (II) centre (d9) at RT essentially make them diamagnetic.

Spectral signature of deoxy-Hc & oxy-Hc

deoxyhemocyanin

oxyhemocyanin

Significance charge transfer occurs between the coordinated peroxo group and the metal

centers.

Resonance Raman for O-O stretching frequency at 803 cm-1

confirms the peroxo linkage Bertini, I.; Gray, H. B.; Lippard, S. J.; Valentine, J. S.; Bioinorganic Chemistry; UniversityScience Books;

Mill valley, California, 1994

X-ray structure of deoxy-Hc and oxy-Hc

PDB ID: 1LLADeoxy-hemocyanin

PDB ID: 1NOLOxy-hemocyanin

Hemocyanin: Model Complex

N N

NN

NNHB

N N

NN

NNCu BHCuO

O

Kitazima et al. J. Am. Chem. Soc. 1992, 114, 1277-1291

Illustration of the relevant steps for O2 binding to deoxy-hemocyanin

CuI

NN

N

CuI

N

N

N

+

O O 2

deoxyHc

oxyHc

Antiferromagnetic(superexchange stabilization)

S/T - ISC

Ferromagnetic (exchange stabilization)

Charge transfer

delocalization - reduced exchange stabilization

Metz, M.; Solomon, E. I.; J. Am. Chem. Soc. 2001, 123, 4938-4950.

O2 carrying protein with binuclear Fe complex as active site.

Hemerythrin, Hr : Di-oxygen Carrier

Have been found in marine invertebrate phyla including thesipunculids, the brachiopods, the priapulids, and some annelids.

Consists of eight identical subunits (Mol. Wt. 107-108 kDa), eachcontaining two Fe(II) atoms in deoxy form.

Hemerythrin, Hr : Di-oxygen Carrier

PDB ID: 1HMO

Spectral features of deoxy-Hr, met Hr & oxy-Hr

Bertini, I.; Gray, H. B.; Lippard, S. J.; Valentine, J. S.; Bioinorganic Chemistry; UniversityScience Books; Mill valley, California, 1994

Considerable charge transfer occurs between the coordinated peroxogroup and the metal center.

Resonance Raman for O-O stretching frequency at 844 cm-1 confirms the peroxo linkage

Azidomet Hemerythrin & Structural Model of Met-Hr

FeIII

NN

N

HN

NH

HN

His73

His101

His77

FeIII

N

N

HN

NH

His54

His25

O

OO

Asp106O

Glu58

O

N3

An inactive form of protein

Contains (μ-oxo) diiron (III) centers

Azide anion( N3-) is coordinate to

Fe, at the site normally occupied by O2 in oxy-Hr.

Antiferromagnetic spin exchange between the two high spin Fe(III) centers.

FeIII

NN

N

FeIII

N

N

O

OO

RO

R

O

NNH

NH

NH

N3 =

(A). Holmes, M.A.; Stenkamp, R. E.; J. Mol. Biol. 1991, 220, 723-737; (B). Armstrong, W. H.; Lippard, S. J. J. Am. Chem. Soc. 1983, 105, 4837-4838; (C). Wieghardt, K.; Pohl, K.; Gebert, W. Angew. Chem., Int. Ed. Engl. 1983, 22, 727.

Structural Models for Hemerythrin

Mizoguchi et al. Inorg. Chem. 2001, 40, 4662-4673

Optical spectra of [Fe2(µ-OH)(µ-Ph4DBA)(DPE)2(OTf)] in EtCN at -78 °C before (- - -) and after (-) addition of dioxygen.

Schematic Summary of the Redox State s of the binucler ironcenter & their Interconversion

Handbook of Metalloproteins, Online © 2006 John Wiley & Sons, Ltd

Metalloproteins Active Site (deoxy)

Color Change(deoxy to oxy)

Molecular Weight

Source

Hemoglobin Fe(II) [in Heme]

Purple to Red 64, 000 Higher form of life

Myoglobin Fe(II) [in Heme]

Purple to Red 17,100 Higher form of life

Hemocyanin Cu(I)….Cu(I) Colorless to Blue

~ 9 x 106 Arthopods, Molluscs

Hemerythrin Fe(II) ….Fe(II) Colorless to Burgundy

108,000 MarineInvertebrates

O2 Carrying Protein in Biology

1. Give brief descriptions of the following: (a) peptide; (b) naturally occurring amino acids; (c)metalloprotein; (d) haem unit

2. Compare the modes of binding of O2 to the metal centres in (a) myoglobin, (b) haemerythrinand (c) haemocyanin. Indicate what supporting experimental evidence is available for thestructures you describe.

3. Give an account of the storage and transport of metalloproteins in mammals. How does theuptake of iron by aerobic microorganisms differ from that in mammals?

4. (a) Briefly describe the mode of binding of O2 to the iron centre in one haem unit ofhaemoglobin. (b) What are ‘picket fence’ porphyrins and why are they used inmodel studies of O2 binding to myoglobin or haemoglobin? (c) The binding of O2 tohaemoglobin exhibits a ‘cooperativity’ effect. What is meant by this statement? (d) Why isthe change from deoxyhaemoglobin to the oxy-form accompanied by a decrease in theobserved magnetic moment?

5. Draw the active site structures of deoxy & oxy form of (a) haemoglobin (b) hemocyanin and (c) hemerythrin?

6. Explain the term “Bohr Effect”?7. Explain the term haematin formation with the possible fate of Fe (II) centre?

Problems