Hemoglobin : a portrait of a
soluble protein with 4° stucture
THE 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
• 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
• Don’t use O2 for metabolism
» Blue-green Algae Develop Photosynthesis
• O2 produced as biproduct
• Fe2+ oxidized to Fe3+
• [Fe3+] = 10-7 M in water
• Molecules Evolve to Destroy O2 (catalase)
• Molecules Evolve to Solubilize and
Transport Iron
– Reduce Fe3+ to Fe2+
– Keep iron from oxidizing back to Fe3+
» 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
H
H
NHis
NHis
NHis
O
Fe
Fe
O
NHis
O
O
O
NHis
O2
NHis
NHis
NHis
O
O
O
Fe
Fe
O
O
O
O
» Hemocyanin
• O2 transport protein in mollusks and
arthropods
• Uses a dicopper binding site
• Gives them blue blood
NHis
NHis
NHis
Cu
NHis
NHis
Cu
NHis
NHis
O2
NHis
NHis
Cu
O
O
NHis
NHis
Cu
NHis
NHis
NHis
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 O
2
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
Oxygen binds to the Heme
prosthetic group
top view
side view
• 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.
-
-
COO-
OOC
NH
N
N
HN
Fe2+
COO-
OOC
N
N
Fe
N
N
• Many “porphyrines” exist in organisms
• Naturally occurring macrocyclic ligand
– Ligand = organic molecule which
binds a metal ion by donating 2 efrom a donor atom (N:)
– Macrocycle = ligand with donor atoms
arranged in a ring
– Strongly binds Fe because of rigid
macrocyclic structure
Topological and Rigidity
Effects
NH2
NH
NH3
NH
HN
NH
HN HN
NH
HN
NH
HN HN
HN
NH2
NH2 H2N
Increasing Topological Contraint and Complex Stability
NH2
H2N
N
N
N
Increasing Rigidity and Complex Stability
-
COO-
OOC
NH
N
N
HN
Porphyrins are:
Topologically complex
And Rigid
N
» 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)
N
N
N
2+
Fe
N
N
– 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
O
O
N
2+
Fe
N
N
N
N
• Heme is only bound to the protein by a
single N(His) coordinate bond at the axial
site of Fe2+ (Proximal Histidine)
• Noncovalent binding (hydrophobic)
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
– 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 A…H
– 5 nonhelical regions named AB…GH
• 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
» Heme Binding Site Before O2 Binds
• Heme sits in a crevice with polar –COOgroups 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+)
» 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 N—H…..O—O H-Bond
stabilizes the bonded O2
O
N
O
Fe2+
N
N
• Bulk of the protein prevents
thermodynamically favored dimerization
N
N
N
N
Fe
N
N
N
N
O
N
Fe
N
N
N
Irreversible = DEAD
• Synthetic O2 carriers must overcome this
Cyclidene Synthetic
O2 Carriers (D.H. Busch)
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
O2
PROXIMAL
»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 > 99% Hb
ISOLATED HEME
N
Fe
1
O
1
O
ISOLATED HEME
N
Fe
C
O
25,000
HEME WITH POLYPEPTIDE ENVIRONMENT
N
Fe
200
C
O