BAHAN KULIAH BIOKIMIA POWER POINT BAGIAN 1 /BIOCHEMISTRY POWER POINT LECTURES PART 1 | Karya Tulis Ilmiah

Protein Function

Hemoglobin and Myoglobin

Because of its red color, the red blood pigment has been of interest since antiquity.

•_{First protein to be crystallized - 1849.}

•_{First protein to have its mass accurately measured.} •_{First protein to be studied by ultracentrifugation.} •First protein to associated with a physiological

condition.

•_{First protein to show that a point mutation can cause} problems.

•_{First proteins to have X-ray structures determined.} •Theories of cooperativity and control explain

The structure of myoglobin and

hemoglobin

Andrew Kendrew and Max Perutz solved the structure of these molecules in 1959 to 1968.

The questions asked are basic. What chemistry is responsible for oxygen binding, cooperativity, BPG effects and what

alterations in activity does single mutations have on structure and function.

Myoglobin: 44 x 44 x 25 Å single subunit 153 amino acid residues

121 residues are in an a helix. Helices are named A, B, C, …F. The heme pocket is surrounded by E and F but not B, C, G, also H is near the heme.

Hemoglobin

Spherical 64 x 55 x 50 Å two fold rotation of

symmetry  and  subunits are similar and are placed

on the vertices of a tetrahedron. There is no D helix in the  chain of hemoglobin. Extensive interactions

between unlike subunits 2-2 or 1-1 interface

has 35 residues while 1-2 and 2-1 have 19

residue contact.

Quaternary structure of deoxy- and oxyhemoglobin

Oxygenation rotates the 11 dimer in relation to 22 dimer about 15°

The conformation of the deoxy state is called the T state The conformation of the oxy state is called the R state

individual subunits have a t or r if in the deoxy or oxy state.

What causes the differences in the

conformation states?

The positive cooperativity of O

₂

binding to Hb

arises from the effect of the ligand-binding

state of one heme on the ligand-binding

affinity of another.

The Fe iron is about 0.6 Å out of the heme

plane in the deoxy state. When oxygen binds

it pulls the iron back into the heme plane.

Binding of the oxygen on one heme is more difficult but its binding causes a shift in the 1-2 contacts

and moves the distal His E7 and Val E11 out of the oxygen’s path to the Fe on the other subunit. This

process increases the affinity of the heme toward oxygen.

The 1-2 contacts have two stable positions.

These contacts, which are joined by different but equivalent sets of hydrogen bonds and act as a

The energy in the formation of the Fe-O2 bond formation drives the T R transition.

Hemoglobins O₂ -binding Cooperativity derives from the T  R Conformational shift.

•_{The Fe of any subunit cannot move into its heme plane}

without the reorientation of its proximal His so as to prevent this residue from bumping into the porphyrin ring.

•_{The proximal His is so tightly packed by its surrounding}

groups that it can not reorient unless this movement is

accompanied by the previously described translation of the F helix across the heme plane.

•The F helix translation is only possible in concert with the quaternary shift that steps the 1C-2FG contact one turn

•The inflexibility of the 1-1 and the 2-2 interfaces requires

that this shift simultaneously occur at both the 1-2 and 2-1

interfaces.

No one subunit or dimer can change its conformation.

The t state with reduced oxygen affinity will be changed to the r state without binding oxygen because the other subunits switch upon oxygen

Hemoglobin function

₂,₂ dimer which are structurally similar to myoglobin

•_{Transports oxygen from lungs to tissues.}

•O₂ diffusion alone is too poor for transport in larger animals.

•Solubility of O₂ is low in plasma i.e. 10-4 M.

•But bound to hemoglobin, [O₂] = 0.01 M or that of air •Two alternative O₂ transporters are;

•Hemocyanin, a Cu containing protein.

Myoglobin facilitates rapidly respiring

muscle tissue

The rate of O2 diffusion from capillaries to tissue is slow because of the solubility of oxygen.

Myoglobin increases the solubility of oxygen.

Myoglobin facilitates oxygen diffusion.

Oxygen storage is also a function because

The Heme group

Each subunit of hemoglobin or myoglobin contains a heme. •Binds one molecule of oxygen

•Heterocyclic porphyrin derivative •Specifically protoporphyrin IX

The iron must be in the Fe(II) form or reduced form. (ferrous oxidation) state.

Loss of electrons oxidation LEO

Gain of electrons reduction GER

The visible absorption

spectra for hemoglobin

The red color arises from the differences between the energy levels of the d orbitals around the Ferrous atom.

There is an energy difference

between them, which determines the size of the wavelength of the maximal absorbance band.

Fe(II) = d6 electron

Binding of oxygen

rearranges the electronic distribution and alters the d orbital energy.

This causes a difference in the absorption spectra.

Bluish for deoxy Hb Redish for Oxy Hb

Measuring the absorption at 578 nM allows an easy

method to determine the

When Fe(II) goes to Fe(III), oxidized, it

produces methemoglobin which is brown

and coordinated with water in the sixth

position. Dried blood and old meat have this

brown color.

Butchers use ascorbic acid to reduce methemoglobin to make the meat look fresh!!

O

₂

binding to myoglobin

2

2

MbO

O

Mb





]

[MbO

]

[Mb][O

Kd

2 2



]

[O

Kd

]

[O

]

[MbO

[Mb]

]

[MbO

Y

2 2 2 2 O₂









Written backwards we can get the dissociation constant

How do you measure the concentration of oxygen?

Use the partial pressure of O₂ or O₂ tension. = pO₂

2 d 2 O

pO

K

pO

Y

2





P50 = the partial oxygen

pressure when Y_O2 = 0.50

2 50 2 O

pO

P

pO

Y

2





What does the value of P₅₀ tell you about the O₂ binding affinity?

P₅₀ value for myoglobin is 2.8 torr

or

1 torr = 1 mm Hg = 0.133 kPa 760 torr = 1 atm of pressure

Mb gives up little O₂ over normal physiological range of oxygen concentrations in the tissue

i.e. 100 torr in arterial blood

30 torr in venous blood

YO₂ = 0.97 to YO₂ = 0.91

What is the P50 value for Hb?

The Hill Equation

E = enzyme, S = ligand, n= small number

ESn

nS

E





This is for binding of 1 or more ligands

O

₂

is considered a ligand

ESn]

[

[E][S]

K

n



1.

n



[E]

[ESn]



n[ESn]

Ys





2.

As we did before, combine

1. + 2. = 3.





K

S]

[

1

]

E

[

K

[E][S]

Ys

_n n





or n n

[S]

K

[S]

Ys





Look similar to Mb + O2 except for the n

Continuing as before:

 

n

50

P

K







 





n

2 n 50 n 2 O

pO

P

pO

Y

2





4.

n = Hill Constant, a non integral parameter relating

Degree of Cooperativity among interacting ligand-binding sites or subunits

The bigger n the more cooperativity (positive value)

If n = 1, non-cooperative

Hill Plot

Rearrange equation 4.

logK

nLog[S]

Ys

-1

Ys

Log

















y = mx + b

Things to remember

Hb subunits independently compete for O₂ for the first oxygen molecule to bind

When the Y_O2 is close to 1 i.e. 3 subunits are occupied by O₂ , O₂ binding to the last site is independent of the other sites

However by extrapolating slopes: the 4th O2 binds to hemoglobin 100 fold greater than the first O2

A G of 11.4 kJ•mol -1 in the binding affinity for oxygen

Contrast Mb O

₂

binding to Hemoglobin

YO2 = 0.95 at 100 torr

but

0.55 at 30 torr

a YO₂ of 0.40

Understand Fig 9-3

Function of the globin

Protoporphyrin binds oxygen to the sixth ligand of Fe(II) out of the plane of the heme. The fifth ligand is a Histidine, F8 on the side across the heme plane.

His F8 binds to the proximal side and the oxygen binds to the distal side.

The heme alone interacts with oxygen such that the Fe(II) becomes oxidized to Fe(III) and no longer

Fe O O Fe

A heme dimer is formed which leads to the

formation of Fe(III)

By introducing steric hindrance on one side of the heme plane interaction can be prevented and oxygen binding can occur.

The globin acts to:

•_{a. Modulate oxygen binding} affinity

The globin surrounds the heme like a hamburger is surrounded by a bun. Only the propionic acid side chains are exposed to the solvent.

The Bohr Effect

Higher pH i.e. lower [H+] promotes tighter binding of

oxygen to hemoglobin

and

Lower pH i.e. higher [H+] permits the easier release of oxygen from hemoglobin

 

 











O

Hb

O

xH

H

O

Hb

_{2 n x 2 2 n 1}

Where n = 0, 1, 2, 3 and x  0.6 A shift in the equilibrium

Origin of the Bohr Effect

The T  R transition causes the changes in the pK’s

of several groups. The N-terminal amino groups are responsible for 20 -30% of the Bohr effect. His146

accounts for about 40% of the Bohr effect salt

bridged with Asp 94. This interaction is lost in the

To help you understand look at the relation between pH and the P₅₀ values for oxygen binding. As the pH increases the P₅₀ value decreases, indicating the

oxygen binding increases. The opposite effect occurs when the pH decreases.

At 20 torr 10% more oxygen is released when the pH drops from 7.4 to 7.2!!

As oxygen is consumed CO₂ is released. Carbonic Anhydrase catalyzes this reaction in red blood cells.

-3 2

2

H

O

H

HCO

About 0.8 mol of CO₂ is made for each O₂ consumed. Without Carbonic Anhydrase bubbles of CO₂ would form. The H+ generated from this reaction is taken up by the

hemoglobin and causes it to release more oxygen. This proton uptake facilitates the transport of CO₂ by

stimulating bicarbonate formation.

R-NH

₂

+ CO

₂



R-NH-COO

-

+ H

+

About 5% of the CO₂ binds to hemoglobin but this accounts for the 50% of the exchanged CO₂ from the blood. As oxygen is bond in the lungs the CO₂