1. 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

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 O 2, more CH 4, H 2 S, H 2 Iron primarily Fe 2+ [Fe 2+ ] = 2 x 10 -4 M in water Only simple transport of Fe 2+ needed »Anaerobic Life Simple, 1-celled organisms Don’t use O 2 for metabolism »Blue-green Algae Develop Photosynthesis O 2 produced as biproduct Fe 2+ oxidized to Fe 3+ [Fe 3+ ] = 10 -7 M in water Molecules Evolve to Destroy O 2 (catalase) Molecules Evolve to Solubilize and Transport Iron –Reduce Fe 3+ to Fe 2+ –Keep iron from oxidizing back to Fe 3+

3. »0.6 Billion Years Ago, [O 2 ] reaches 1% Aerobic Life Evolves Use O 2 in Metabolism 18 times as much energy from glucose in the presence of O 2 as without it [O 2 ] = 1.2 x 10 -3 M in water O 2 transport molecules must evolve –Oxygen Carrying Molecules »Hemorythrin O 2 transport protein in certain sea worms Uses a diiron binding site »Hemocyanin O 2 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 O2O2 O2O2 O2O2 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:) –Macrocycle = ligand with donor atoms arranged in a ring –Strongly binds Fe because of rigid macrocyclic structure

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

8. »Fe in the porphyrine makes it a Heme When Fe 3+, this is the Ferrimyoglobin state. It can only bind water, not O 2. The Fe 2+ species, Ferromyoglobin, binds and releases O 2. –Fe 2+ has 6 d electrons –When 5-coordinate, Fe 2+ is high spin –High spin ions are larger than low spin –Fe 2+ is slightly out of plane (0.3Å) –When 6-coordinate, Fe 2+ is low spin –Spin state and size changes when O 2 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 Fe 2+ (Proximal Histidine) Noncovalent binding (hydrophobic)

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

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%  -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 O 2 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. Fe 2+ )

12. »Heme Binding Site After O 2 Binds O 2 binds at distal side of Heme Fe 2+ goes low spin (69 pm radius) and moves into porphyrin plane Distal Histidine N—H…..O—O H-Bond stabilizes the bonded O 2 Bulk of the protein prevents thermodynamically favored dimerization Synthetic O 2 carriers must overcome this Cyclidene Synthetic O 2 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 O 2 in the isolated heme, but only 200 times as strongly as O 2 in myoglobin or hemoglobin DISTAL O2O2 PROXIMAL

14. 1 1 25,000 NFeO O N CO 200 NFeC O ISOLATED HEME HEME WITH POLYPEPTIDE ENVIRONMENT »CO Binding in Myoglobin and Hemoglobin CO is a poison because it displaces O 2 CO prefers linear coordination, O 2 bent Distal His forces bent coordination of CO Lets O 2 compete with CO CO produced in body takes 1% Hb Without Distal His, CO > 99% Hb