• 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

Hemoglobin : a portrait of a soluble protein with 4° stucture

THE OBJECTIVES

Hemoglobin : a portrait of a soluble protein with 4° stucture

THE OBJECTIVES

• 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

» Hemocyanin

• O2 transport protein in mollusks and arthropods

• Uses a dicopper binding site

• Gives them blue blood

O

Fe

NHis

NHis

OO

NHisFe

NHisO

O

NHis

H

O

Fe

NHis

NHis

OO

NHisFe

NHis

O

OO

NHis

HO

O2

Cu

NHis

NHis

NHis Cu

NHis

NHis

O

O

NHisO2Cu

NHis

NHis

NHis Cu

NHis

NHis

NHis

Myoglobin and Hemoglobin 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 ofoxygen in blood AND also aids in thetransport of carbon dioxide and H+

Myoglobin provides muscle tissue withan oxygen reserve AND facilitates oxygenmovement in muscle

Oxygen binds to the Heme prosthetic group

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.

• 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:)

–Macrocycle = ligand with donor atoms arranged in a ring

–Strongly binds Fe because of rigid macrocyclic structure

N HN

NH N

COO--OOC

Fe2+

N N

N N

COO--OOC

Fe

NH

NH HN

HN

NH3

NH2

NH HN

H2NNH

NH HN

HN

HN

HNNH2

NH2

Topological and Rigidity Effects

H2N NH2 NN N N

Increasing Rigidity and Complex Stability

Increasing Topological Contraint and Complex Stability

N HN

NH N

COO--OOC

Porphyrins are:Topologically complexAnd Rigid

» 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

• 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)

N N

NNN

Fe2+

OO

Fe2+

N

N N

NN

The heme environmment is crucial for its funciton

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% -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.’sMammalian Myoglobin

» 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+)

» 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

• Bulk of the protein prevents thermodynamically favored dimerization

• Synthetic O2 carriers must overcome this

N N

NNN

Fe2+

OO

FeN

N

NN

N

O Fe N

N

N N

N

Irreversible = DEAD

Cyclidene SyntheticO2 Carriers (D.H. Busch)

The heme environment is crucial for its function

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

11

25,000

N Fe OO

N Fe C O

200N Fe C

O

ISOLATED HEME

ISOLATED HEME

HEME WITH POLYPEPTIDE ENVIRONMENT

»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