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

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 • Don’t use O2 for metabolism • Blue-green Algae Develop Photosynthesis • O2 produced as byproduct • 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+

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 • Hemerythrin • 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

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 “porphyrins” 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

7. Topological and Rigidity Effects Increasing Topological Constraint and Complex Stability Increasing Rigidity and Complex Stability Porphyrins are: Topologically complex And Rigid

8. Fe in the porphyrin 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)

9. The heme environment is crucial for its function • 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

10. 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

11. 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 N—H…..O—O 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

14. 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 1 1 N Fe O O ISOLATED HEME 25,000 N Fe C O HEME WITH POLYPEPTIDE ENVIRONMENT 200 N Fe C O