O2 transport

Question 1

What are the 3 types of oxygen transport?

Answer 1

Haemoglobin (Hb)
Haemerythrin (Hr)
Haemocyanin (Hc)

Question 2

Haemoglobin

Answer 2

65 kDa
[Fe:O2]4 (1 Fe binds to 1 O2)
Tetramer (2 alpha, 2 beta)
No O2 = purple, + O2 = red

Question 3

Haemerythrin

Answer 3

108 kDa
[2Fe:O2]8 (2 Fe bind to 1 O2)
Octamer
No O2 = colourless, + O2 = violet/pink
Marine worms

Question 4

Haemocyanin

Answer 4

400-20000 kDa
[2Cu:O2]many
No O2 = colourless, + O2 = blue

Question 5

Haemoglobin structure

Answer 5

An assembly of 4 globular protein subunits
Each subunit is composed of a protein chain tightly associated with a haem group (= Fe ion bound in the centre of a porphyrin ring)
Fe strongly (covalently) bound to protein via a His residue below the porphyrin ring (“proximal His”)
A 6th (distal) position can reversibly bind O2 via a coordinate covalent bond

Question 6

Geometry of Fe in haemoglobin when no O2 is bound

Answer 6

Square pyramidal

/distorted octahedron when weakly bonded H2O fills 6th site

Question 7

Geometry of Fe in haemoglobin when O2 is bound

Answer 7

Octahedral

Question 8

How does O2 bind to Fe in haemoglobin?

Answer 8

End-on-bent geometry i.e. one O bound to Fe and other O protrudes at an angle

Question 9

Myoglobin

Answer 9

Monomer (similar to one of the Hb subunits)
O2 storage
Distal His ‘hovers’ over distal face of porphyrin ring - not bound to Fe but available to interact with the substrate O2

Question 10

What is the form of O2 in haemoglobin?

Answer 10

Superoxide (O2^-)

Raman spectroscopy gives wavenumber of 1105 cm-1

Question 11

O2 bonding modes

Answer 11

End-on
Side-on
End-on bridging
Side-on bridging
End-on/side-on bridging
End-on/4-fold bridging

Question 12

Deoxo form of haemo/myoglobin

Answer 12

Fe(II)
High spin
Lies 0.4 Å under haem plane - haem is slightly bent

Question 13

Oxo form of haemo/myoglobin

Answer 13

Fe(III)
Low spin
Fits into haem plane perfectly
His is ‘pulled down’ which destabilises O2 coordination, making it reversible

Question 14

3 forms of haemo- and myoglobin

Answer 14

1. Deoxy(Mb/Hb) = functional, no O2 bound
2. Oxy(Mb/Hb) = functional, O2 bound
3. Met(Mb/Hb) = non-functional, oxidised (Fe3+). Spin state depends on nature of 6th ligand

Question 15

Necessary components for reversible O2 binding

Answer 15

Haem
Imidazole (from His)
O2

Question 16

How are oxygen affinities measured?

Answer 16

In terms of the partial pressure of O2 needed for half saturation (i.e. for half of the Fe in a sample to be bound to O2)

Question 17

Low PhalfO2

Answer 17

High O2 affinity

Question 18

High PhalfO2

Answer 18

Low O2 affinity

Question 19

Why does the picket fence porphyrin model have a substantially higher affinity for O2 than Mb?

Answer 19

1. The encapsulating protein in Hb/Mb restricts O2 access, making it more difficult for an O2 molecule to reach the Fe
2. In the deoxy, 5-coordinate state, Fe(II) is displaced below the plane
   When O2 binds, Fe(II) is oxidised to Fe(III), its radius decreases and it can move into the plane of the porphyrin
   This therefore requires the movement of the coordinated His/imidazole - not a problem in the PF model, but movement of this His in Mb/Hb required movement of the whole protein chain
   This large scale rearrangement makes binding of the 6th ligand is less favourable so reduces affinity

Question 20

Change in O2 affinity in haemoglobin

Answer 20

Low O2 affinity at low [O2]
High O2 affinity at high [O2]
This enables O2 to be delivered to where it is needed - i.e. regions with low [O2]

Question 21

Cooperativity in haemoglobin

Answer 21

The 4th O2 binds to Hb 300-400x more strongly than the 1st O2
The movement in the subunit when Fe binds to O2 is transmitted through the rest of the Hb structure via salt bridges/H-bonding
This makes the same movement easier for the other subunits, favouring O2 binding

Question 22

Why is myoglobin required to have a higher affinity than haemoglobin?

Answer 22

Esp in tissues

The function of Mb is O2 storage

Question 23

Why is CO toxic?

Answer 23

Competes with O2 for Hb binding sites
CO is higher in electrochemical series than O2, binds CO 200-300x more strongly than O2
Bound CO also reduces the ability of Hb to release O2 from its remaining sites

Question 24

How are we protected from CO toxicity? How do Hb and Mb reduce their affinity for CO?

Answer 24

1. CO normally binds in an “end-on linear” fashion, but this is statically prevented in Hb by the surrounding protein, leading to a less favourable bent geometry
2. O2 forms a much stronger H bond to the distal His than CO. A H2O—His H-bond must be displaced upon O2/CO binding. In the case of CO binding, this energy is not recovered through forming another strong H bond

Question 25

Haemerythrin structure

Answer 25

(draw)

Raman 844 cm-1 = HOO-

Question 26

Haemocyanin structure

Answer 26

(draw)

Raman 803 cm-1 = O2^2-

Question 27

Two major classes of enzyme for ‘mopping up’ ROS

Answer 27

1. Hydroperoxidases

2. Superoxide dismutases

Question 28

Hydroperoxidases

Answer 28

2 types:

a) Catalases: catalyse disproportionation of peroxides
2H2O2 —> 2H2O + O2
or 2ROOH —> 2ROH + O2

b) Peroxidases: catalyse reduction of peroxides
H2O2 + H2S —> 2H2O + S
ROOH + H2S —> ROH + H2O + S
(S = substrate)

Question 29

Superoxide dismutases

Answer 29

Catalyse the removal of superoxide O2^-

Question 30

Nearly all organic substances can be oxidised thermodynamically, but why is this process generally very slow?

Answer 30

For kinetic reasons

1. Concerted 4 electron reduction of O2 is (nearly) impossible (intermediates are also highly toxic/reactive, scavengers are needed)
2. Kinetic inhibition - O2 is a paramagnetic triplet radical so most reactions are spin-forbidden

Question 31

What are the 3 types of O2 activation in biological systems?

Answer 31

1. Oxidases (reduce O2 —> 2H2O [energy production])
2. Monooxygenases (one O incorporated into substrate, other O reduced to H2O)
3. Dioxygenases (both O incorporated into substrate)

Question 32

Example of a monooxygenase

Answer 32

Cytochrome P450 (= haem protein)
Catalyses R3CH + O2 + 2H+ + 2e- —> R3COH + 2H2O
(as well as alkene epoxidations/N&O dealkylations/ S oxidation

Question 33

Cytochrome P450 mechanism

Answer 33

C-H is activated by a “radical rebound” mechanism
R-H + Fe=O –> [R* + Fe-OH] –> ROH + Fe
Selective for specific C-H bonds through pre-orientation of the substrate via H-bonding

Question 34

Cytochrome P450 cannot oxidise…

Answer 34

…methane

Question 35

Methane monooxygenase (MMO)

Answer 35

Oxidises methane
Simple iron compounds, non-haem/porphyrin based
CH4 + O2 + 2H+ + NADH —> CH3OH + H2O + NAD+