Hemoglobin : a portrait of a soluble protein with 4

1
Hemoglobin a portrait of a soluble protein with
4 stuctureTHE OBJECTIVES

• use hemoglobin/myoglobin as an example of a
  soluble protein molecule to review many important
  principles of protein structure and function
• to introduce you to the concept of allosterism -
  interactions between spatially distinct sites

2

• Evolution from Anaerobic to Aerobic Life
• Timeline
• Universe is 12-20 billion years old
• Earth is 4.6 billion years old
• Life began 3.5 billion years ago (Anaerobic)
• Aerobic Life (Us) began 0.6 billion years ago
• Iron, Oxygen, and Life
• Pre-Biotic little O2, more CH4, H2S, H2
• Iron primarily Fe2
• Fe2 2 x 10-4 M in water
• Only simple transport of Fe2 needed
• Anaerobic Life
• Simple, 1-celled organisms
• Dont use O2 for metabolism
• Blue-green Algae Develop Photosynthesis
• O2 produced as biproduct
• Fe2 oxidized to Fe3
• Fe3 10-7 M in water

3

• 0.6 Billion Years Ago, O2 reaches 1
• Aerobic Life Evolves
• Use O2 in Metabolism
• 18 times as much energy from glucose in the
  presence of O2 as without it
• O2 1.2 x 10-3 M in water
• O2 transport molecules must evolve
• Oxygen Carrying Molecules
• Hemorythrin
• O2 transport protein in certain sea worms
• Uses a diiron binding site
• Hemocyanin
• O2 transport protein in mollusks and arthropods
• Uses a dicopper binding site

4
Myoglobin and Hemoglobin

• Myoglobin and Hemoglobin are oxygen carrying
  molecules that overcome the problem that
  vertebrates have with the low solubility of
  oxygen in water

O2
O2
O2
Hemoglobin serves as the carrier of oxygen in
blood AND also aids in the transport of carbon
dioxide and H Myoglobin provides muscle tissue
with an oxygen reserve AND facilitates
oxygen movement in muscle
5
Oxygen binds to the Heme prosthetic group
top view
side view
6

• Myoglobin
• Heme Prosthetic Group
• Prosthetic Group non-polypeptide unit of a
  protein that can function without the protein
• Apoprotein protein without its P.G.
• Many proteins require a P.G. for activity
• Protoporphyrin IX Fe is the Heme P.G.
• Many porphyrines exist in organisms
• Naturally occurring macrocyclic ligand
• Ligand organic molecule which binds a metal ion
  by donating 2 e- from a donor atom (N)

7
Topological and Rigidity Effects
Increasing Topological Contraint and Complex
Stability
Increasing Rigidity and Complex Stability
Porphyrins are Topologically complex And Rigid
8

• Fe in the porphyrine makes it a Heme
• When Fe3, this is the Ferrimyoglobin state. It
  can only bind water, not O2.
• The Fe2 species, Ferromyoglobin, binds and
  releases O2.
• Fe2 has 6 d electrons
• When 5-coordinate, Fe2 is high spin
• High spin ions are larger than low spin
• Fe2 is slightly out of plane (0.3Å)
• When 6-coordinate, Fe2 is low spin
• Spin state and size changes when O2 binds to Fe
  heme
• Iron atom nearly in plane of the ring

9
The heme environmment is crucial for its funciton

• the heme is embedded in a non polar crevice
  (white cpk) with its polar side chains on the
  surface of the molecule
• a PROXIMAL His provides the 5th coordination
  position for the Fe. A DISTAL His provides
  essential STERIC constraints

DISTAL
PROXIMAL
heme
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• Myoglobin Structure
• One of the first proteins characterized by
• X-Ray Crystallography (Kendrew, 1959)
• Sperm whale muscle tissue source
• Small, stable protein grows good crystals
• Structural Features
• Compact Globular 153 A.A. protein
• 75 a-helical conformation
• 8 helical regions named AH
• 5 nonhelical regions named ABGH
• Interior is mostly nonpolar residues
• Leucine, Valine, Phenylalanine
• 2 internal Histidines at binding site
• Exterior has mix of polar/nonpolar A.A.s

Mammalian Myoglobin
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• Heme Binding Site Before O2 Binds
• Heme sits in a crevice with polar COO- groups at
  the surface
• F8 Proximal Histidine directly bound to Fe
• E7 Distal Histidine is near opposite face of
  Fe, but not bound to it
• Fe is about 0.3Å out of the plane (77pm radius
  for h.s. Fe2)

12

• Heme Binding Site After O2 Binds
• O2 binds at distal side of Heme
• Fe2 goes low spin (69 pm radius) and moves into
  porphyrin plane
• Distal Histidine NH..OO H-Bond stabilizes the
  bonded O2
• Bulk of the protein prevents thermodynamically
  favored dimerization
• Synthetic O2 carriers must overcome this

Cyclidene Synthetic O2 Carriers (D.H. Busch)
13
The heme environment is crucial for its function

• the reactivity of the heme group is different in
  the presence or absence of the polypeptide

eg. CO binds 25,000 times as strongly as O2 in
the isolated heme, but only 200 times as
strongly as O2 in myoglobin or hemoglobin
DISTAL
PROXIMAL
O2
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• CO Binding in Myoglobin and Hemoglobin
• CO is a poison because it displaces O2
• CO prefers linear coordination, O2 bent
• Distal His forces bent coordination of CO
• Lets O2 compete with CO
• CO produced in body takes 1 Hb
• Without Distal His, CO gt 99 Hb

ISOLATED HEME
1
1
N
Fe
O
O
ISOLATED HEME
25,000
N
Fe
C
O
HEME WITH POLYPEPTIDE ENVIRONMENT
200
N
Fe
C
O