Outline A. Oxygen transport within the body

1. Gases in the respiratory system (figure 1) (Campbell pg. 845).

2. Venal / arterial blood flow

3. Partial pressure (the torr), concentration gradient and gas exchanges.

4. Gases diffuse from higher pp to lower pp.

B. Role of carrier proteins

C. The Heme group

1. Porphyrin ring: four available �claws� (Figure 1a: Porphyrin ring and iron

complex)

2. Iron coordination is six: binds to four porphyrin atom, to a Histidine above the

plane and to an oxygen molecule in the below the plane. (Figure 1b. Heme

Complex) Note: Figure applies to all Heme- iron- oxygen �histidine complexes

in this lesson.

3. Four Heme groups in Hemoglobin (Figure 1c: Heme in Hb)

D. Myoglobin: (Figure 2: Structure of Myoglobin)

1. Delivers increased amounts O2 to working muscles (Figure 3a: Oxygen

dissociation for Hb & Mb)

2. Protects Heme from oxidation (Figure 2a: Heme in Myoglobin).

3. Stores O2 (predominantly in aquatic mammals)

E. Hemoglobin: (Figure 3:Structure of Hemoglobin)

1. Transports O2

2. Dissociates majority of Oxygen to working cells (Figure 3a: Oxygen dissociation

for Hb & Mb)

F. Cooperative binding in Hb

1. O2 binding alters Heme complex (Figure 4: Heme conformational shift)

2. Lowers displacement of Heme group above porphyrin plane

3. Makes other Heme groups available for oxygenation

4. Four Heme complexes bind four oxygen molecules

5. Altered Quaternary structure (Figure 4a: Conformational shift in oxy �

deoxyhemoglobin)

a. α1β1, α 2β 2 remain fixed

b. α 1β2 , α 2 β 1 sliding contacts

6. Taut and Relaxed forms (Figure 5: Relaxed and Taut States of Hb)

G. Dissociation of oxygen

1. Bohr Affect (Figure 6: Affect of pH on Hb Oxygen dissociation)

2. HbO2 + H+ ! HbH+ + O2 ( i.e. working muscles)

3. Presence of H+ (acid) promotes oxygen dissociation

H. CO2 from intercellular region (Figure 7:Carbon Dioxide Transport)

1. Carbonic anhydrase catalyzes formation of carbonic acid in erythrocytes

a. H20 + CO2 ! H2CO3

b. H2CO3 ! HCO3- + H+

c. Hb reacts with H+: carries it and some CO2 to lungs.

d. In lungs: HbH+ + O2 ! HbO2 + H+

e. Further reaction in lungs: H+ + CO2 !. H2CO3 ! H2O + CO2 (exhaled)

I. BPG defined (2-3,Bisbiphosphoglycerate)

1. Structure of BPG binding (Figure 8: Binding of BPG).

2. Allosteric function

3. Presence of 2-3 BisPG affects oxygen dissociation (Figure 9)

a. Figure 10: Affect of BPG on Hb oxygen saturation

Discussion:

Introduction The solubility of oxygen in blood is so low (less than 10-4 M at physiological pH), that

large complex organisms require a substance that will help transport oxygen to the sites

of oxidative respiration. The substance should efficiently (and reversibly) bind sufficient

oxygen to support cellular metabolism, be �willing� to deliver it where needed and

respond to increased demand by delivering more oxygen to working tissue The carrier

protein hemoglobin and its analog myoglobin fulfill these functions within our bodies.

Hemoglobin also assists in the vital functions of carbon dioxide removal and the

buffering of blood pH levels. This lesson will explore these functions of Hemoglobin

(Hb) and the allosteric interactions that facilitate its function as our body�s oxygen carrier

protein.

Outline Notes

A. The introduction on gas transport should be brief, as most students know the

difference between venal and arterial blood. Figure 1 is useful for determining the

relationship between partial pressure and delivery or uptake of a given gas. Use the

figure to explain partial pressure and the direction of gas flow at different point in the

body. The pressure gradient at the tissue levels explains why O2 diffuses of the blood

and into the tissues and CO2 diffuses into the blood, a similar logic explains why the

reverse reactions occur in the lungs.

B. Hemoglobin and Myoglobin are both globular proteins with the capacity to bind

oxygen as both have the heme porphyrin complex. The structure of Hb is such that it

can bind to four oxygen molecules. Mb can bind to only one oxygen but its carrier

function is restricted to the muscle systems.

C. Porphyrins are molecules with the capability to bind to (complex) metals ions. They

are referred to as mono, bi, tri, and tetra dentate ligands: the prefixes refer to the

number of bonds that they form with the metal. Heme is the porphyrin iron

complex within Mb and Hb that binds to the oxygen molecule. Chlorophyll is

familiar molecule that contains a Magnesium / porphyrin complex. Ligands contain

atoms with lone pair of electrons that can serve as Lewis bases and donate electrons

to a Lewis acid: the metal. In the case of the porphyrin ring, four nitrogen atoms

(located in four pyrrole groups) bind to a Fe2+ atom. The iron can coordinate to six

atoms, thus it can bind to two more entities. In the case of these proteins, the iron

binds to the Histidine F8 residue of the Hb, thus leaving one coordination site

available for the oxygen molecule. In addition to its function as a carrier protein, Hb

has two other important functions that will be discussed in detail later in the lesson.

Myoglobin is usually referred to as an oxygen storage protein, however this is not the

main function in human beings. An interesting aside is to discuss Mb�s role in aquatic

animals that dive for long periods of time: an example is the Weddell seal that can

store ~ 25% of its oxygen in its muscles, to aid in hour-long dives

D. Figure 3a is the oxygen dissociation graph for Hb and Mb. It shows how much

oxygen Hb can deliver to cells (from almost 100 torr pp in the lungs to pp of 40 torr

in cells). In contrast Mb does not dissociate as much oxygen for the same change in

pp. However as the cells work and the partial pressure begins to drop below 20 torr,

Mb delivers an enormous amount of oxygen. One text refers to it as the oxygen

�bucket brigade�. Students should discuss the various animals that store oxygen for

different reason. A good discussion of the different roles of Mb in these animals is an

interesting classroom discussion. It should also be noted that the Mb and Hb serve to

protect the Heme iron from oxidation from the Fe2+ to the Fe3+ state. This is one of

the often-overlooked functions of these proteins.

E. Hemoglobin is the main focus of this lesson. Students should note the positions of the

subunits and their relation to each other. This will help in seeing how the

conformation changes once an oxygen molecule is bound to the heme.

F. Hemoglobin�s cooperative binding occurs because the binding of one oxygen

molecule alters the relation of the heme iron atom to the porphyrin plane. Figure 3

shows the heme porphyrin location very well. Figure 4 helps to show how the heme

iron makes a dome with the porphyrin plane (in the T position) and how the binding

of oxygen lessens the dome (in the R position). The numbers for this are impressive:

in the T formation the heme is 0.06nm out of the plane, binding lessens this by

0.039nm to 0.021nm out of plane. The binding pulls the heme iron, the F8 histidine

and the F helix, the FG and EF corner down with it. Figure 4a is good for seeing the

shift; figure 5 shows how the shift exposes the other heme complexes in the R state

for oxygenation.

G. Exercising muscles produce acids ( H+), which binds readily to oxyhemoglobin.

Figure 6 shows how Hb affinity for oxygen decreases as pH drops. This is perfect, as

working muscles need extra oxygen. This relationship is known as the Bohr affect.

(You may mention the relationship between Christian Bohr and Niels Bohr). This

reaction is reversed in the lungs where the reaction with bicarbonate ion helps Hb

release the protons and pick up oxygen. (Equations are in outline)

H. Carbon dioxide produced in the cells affects dissociation, as the byproduct of its

reaction in the blood is protons, which are also picked up by Hb. Carbonic anhydrase

catalyzes the formation of carbonic acid, which breaks down into bicarbonate ion and

protons. The bicarbonate ions and the Hb-proton molecules travel to lungs via the

blood. In the lungs the reactions are reversed, carbonic acid is formed and

decomposes to H20 and CO2, while the Hb picks up O2. (Equations are in outline)

I. The final molecule that assists Hb delivery of oxygen is a true allosteric molecule. 23

BPG. Figure 8 shows how the molecule fits into the pocket of the Hb molecule.

Figure 9 explains how the molecule affects the dissociation of oxygen. Figure ten

shows the graphic affects of the binding of BPG: without it, hemoglobin would not

tend to dissociate oxygen and thus be useless as the body�s carrier protein. This is an

excellent example of an allosteric relationship.

Figure 1: Loading and Unloading of respiratory gases: Campbell Biology 4th Edition page 845

Figure 1a: Porphyrin ring with Iron.

Source: http://chem.ch.huji.ac.il/~eugeniik/mediator2.htm

Figure 1b. Heme Porphyrin Complex

http://departments.oxy.edu/biology/Stillman/bi322/021400/lecture.htm

Figure 1c: Heme in Hemoglobin Source: http://www.medical-definitions.net/hemoglobin.htm

Figure 2: Structure of Myoglobin

Source: http://tooldoc.wncc.nevada.edu/chap1.htm Figure 2: Structure of Myoglobin

Source: http://chem.ch.huji.ac.il/~eugeniik/mediator2.htm

Figure 2a: Heme in Myoglobin

Source: http://departments.oxy.edu/biology/Stillman/bi322/021400/lecture.htm

Figure 3: Structure of Hemoglobin

Source: http://faculty.etsu.edu/currie/images/hemat3.jpg

Figure 3a: Oxygen Dissociation Curve for Hemoglobin & Myoglobin Source: http://www.kcl.ac.uk/ip/christerhogstrand/courses/hb0223/respirat.htm

Figure 4: Heme Conformational Shift

Source: http://departments.oxy.edu/biology/Stillman/bi322/021400/lecture.htm

Figure 4a: Conformational shift in oxy � deoxyhemoglobin

Figure 5: Relaxed and Taut States of Hemoglobin

Source: http://departments.oxy.edu/biology/Stillman/bi322/021400/lecture.htm

Figure 6: Affect of pH on Hb Oxygen dissociation

Source: http://members.aol.com/Cappuccinno21/HWAns/hw10a.html

Figure 7:Carbon Dioxide Transport

Source: http://www.cdli.ca/~dpower/resp/exchange.htm

Figure 8: Binding of BPG

Source: http://departments.oxy.edu/biology/Stillman/bi322/021400/lecture.htm

Figure 9: Affect of BPG on Hb

Source: http://beagle.colorado.edu/courses/3280/lectures/class07-1.html

Figure 10. Affect of BPG on Hb oxygen saturation Source: http://web.macam98.ac.il/~rafid/BIOLOGY/biochem/Hemoglobin/s-18.gif