Protein Function: Structure and Function of Myoglobin and Hemoglobin

O2 Transport

Stryer Biochemistry Chapter 7 – Part 1 Dr. Ray

Movement of O2 in Globins

Mb O2 CO2 O2

Diagram shows diffusion directions for + H , CO2, and O2 between the blood and the muscle cells during exercise. The resulting concentration changes affect What structural features of the active site the blood buffer equilibria (top right). would be necessary for the function of globins (reversible O2 binding)? • Hemoglobin (Hb) is an oxygen transport protein: it efficiently carries O2 from the lungs to the tissues. + Hb also contributes to transport of CO2 and H back to the lungs. • Myoglobin (Mb) is a oxygen storage protein in muscle cells. This O2 is used during high levels of activity (exercise). 2 Why does O2 move from one protein (Hb) to the other (Mb)? Comparison of Hb and Mb Structure and Function

Globins are oxygen (O2) binding and storage proteins.

Myoglobin (Mb) • present in peripheral tissues • picks up O2 at capillaries and stores it in muscle cells for use when needed for respiration! • single polypeptide protein, can bind one O2 per protein (full 3D structure: 3o structure)

Hemoglobin (Hb) • found in erythrocytes (red blood cells)

• binds O2 at high O2 partial pressure (pO2) in lungs and transports O2 through circulatory system (blood stream) to capillaries where Hb releases a substantial amount of its bound O2, in the low O2 pressure environment of the peripheral tissues o • 4 subunit protein, so full 3D structure involves 4 structure • Hb is an a2b2 tetramer, that can bind four molecules of O2 per protein

How and why is O2 transferred from Hb to Mb at the tissues? Myoglobin: 3D structure • Describe the levels of protein structure present in Mb: 1o = 2o =

3o =

4o = Hemoglobin: Regulation of Activity via Allosteric Control The precise regulation of protein activity is necessary to allow proteins to: • function properly, at the right time and in the right place • alter their function in different environments Many proteins have more than one active state (3D shape, conformation) with somewhat different biochemical functions: T-state & R-state

• Hemoglobin has two states, one when O2 ligand is bound in the active site (oxyHb), and another slightly different 3D conformation when the protein is ligand-free (deoxyHb).

• In Hb, conversion between the two states is regulated over long distances4 within the protein (allostery), in response to O2 binding. O2 and CO2 Gas Exchange Mechanism in the Blood 1. Blood rich in carbon dioxide is pumped from the heart into the lungs through the pulmonary arteries.

2. In the lungs, CO2 in the blood is exchanged for O2. 3. The O2-rich blood is carried back to the heart through the pulmonary veins.

4. This O2-rich blood is then pumped from the heart to the many tissues & organs of the body, through the systemic arteries. 5. In the tissues, the arteries narrow to tiny capillaries, where O2 in the blood is exchanged for CO2. 6. The capillaries widen into the systemic veins, which carry the CO2-rich blood back to the heart. Arteries are blood vessels carrying Hemoglobin's biological function is blood away from the heart; veins regulated by changes in the overall carry blood to the heart. protein 3D structure. 5 http://www.chemistry.wustl.edu/~edudev/LabTutorials/Hemoglobin/MetalComplexinBlood.html#HelixMovie The roles of Hemoglobin and Myoglobin in: transport of O from the lungs to respiring tissues: 2 O2 (g)  Hb-O2  Mb-O2  muscle (respiration)

understand O2 transfer steps!

different There are two functional [Hb] – deoxy Hb T-state states forms of globins: [HbO2] – oxy Hb R-state These differ by the absence or presence of bound oxygen and the two forms have slightly different 3D structures ! Ref: Voet, Voet, Pratt Biochemistry  Know this flow chart well Metal Complexes in the Body http://www.chemistry.wustl.edu/%7Ecourses/genchem/ Tutorials/Hemoglobin/151_T3_hemoglobin.htm • The ability of metal ions to coordinate with (bind) and then release ligands in some processes, and to oxidize and reduce bound ligands in other processes makes them ideal for use in biological systems. Iron complexes are used in the transport of oxygen in the blood and tissues. • Metal-ion complexes consist of a metal ion that is bonded via "coordinate-covalent bonds“ to a small number of anions or neutral molecules called ligands. • A coordinate-covalent bond (represented by a green arrow in diagram) forms when both of shared electrons come from the same atom, called the donor atom (blue). •An anion or molecule containing the donor atom is known as a ligand. The ligand can be a monodentate ligand (a ligand that contains only one electron-pair-donor atom, shown in light blue in top figure) or it can be a bidentate ligand (a ligand that contains two donor atoms simultaneously coordinated to the metal ion, shown in yellow in bottom figure). Porphyrin • Porphyrins that coordinate to iron in hemes are polydentate ligands. 7 3D structure of Sperm Whale Myoglobin (Mb) • aquatic mammals (seals and whales) have ten times higher concentrations of muscle Mb than terrestrial animals O2 • 153 residue, 16.9 kD • Has 8 a-helices, A – H

• active site heme is a prosthetic group , a specific non-polypeptide cofactor critical for biological activity. • endogenous axial ligand is donated by His 93 (His F8)  His binds to Fe • Hemoglobin (Hb) is a tetramer with four myoglobin-like subunits • A protein (or enzyme) without its prosthetic group is called an apoprotein or apoenzyme . Apoproteins (apoenzymes) do NOT have biological activity. • A protein (or enzyme) containing its prosthetic group is called a holoprotein or holoenzyme. Holoproteins (& holoenzymes) DO have biological activity. 8 Iron-porphyrin (heme) cofactor in active site of Globins

• O2 binds to the Fe-porphyrin prosthetic group in globins • The Fe is coordinated to 4 N donor atoms of porphyrin ring, from four pyrrole rings • Protoporphyrin IX (PPIX) is a tetrapyrrole, a conjugated planar aromatic macrocycle, with: 4 equivalent Fe-N bonds 2 carboxylate, 2 vinyl, and 4 methyl substituents • Iron Protoporphyrin IX, forms four coordinate covalent bonds (L:  M) with the porphyrin ring nitrogens. In such bonds, the nitrogen lone-pair provides both electrons for the coordinate-covalent Fe-N bond. • Iron (Fe) can form up to 6 coordinate covalent bonds! Why? It is a d-block transition metal with an expanded octet. 9 Structure of Active Site Heme Two typical coordination numbers, geometries and metal atom positions are Distal axial O2 ligand possible for complexes of iron porphyrins. Central Fe can be either: • 5-coordinate (5c) square pyramidal as in deoxyMb (in absence of O2), with the 5th coordination position of Fe2+ occupied by the proximal His, and 6th coordination site empty.

• 6-coordinate (6c) octahedral when O2 is bound on the distal side, in oxyMb form.

Proximal axial His ligand Side chain = Imidazole ring 6-coordinate 5-coordinate octahedral square pyramidal Equatorial ligand is metal in-plane metal out-of-plane the porphyrin ring 10 Active Site Structure of Myoglobin • 5-coordinate, with proximal Histidine out-of-plane • Fe has open coordination site, O 2 so can bind O2 • other Nitrogen of Proximal His forms two H-bonds

.

. N

N H His H-bond acceptors are Ser sidechain –OH and Leu backbone C=O Rotate view11 & move back for next slide 3-Dimensional Structure of Myoglobin Heme shown in space filling mode 2o and 3o structure: O2 • H-bonds that stabilize a-helix shown in 3D (F-helix) and in 1o sequence (n  n+4 ) • hydrophilic (polar) side- chains on protein exterior • hydrophobic (nonpolar) side- chains in protein interior • heme edge is solvent exposed MOE 12 Axial Ligands in Active Site of Globins • Edge on view of iron-porphyrin ring:

distal axial ligand X

N Fe N Deoxy porphyrin Myoglobin  N

 proximal axial ligand N H Histidine The identity of the axial ligands (above and below the heme plane) varies among different heme proteins: • Proximal Ligand = is endogenous protein ligand (His, Tyr, Met - amino acid side chain) • Distal Ligand (X) = can be empty, or be an endogenous or exogenous ligand (H2O, O2 , H2O2 - oxygen containing small molecule, substrate) The iron (Fe2+) of the heme group of deoxyMb and deoxyHb lies slightly outside the plane of the porphyrin, by 0.4 Å, so porphyrin is domed. Oxygen Binding to Myoglobin

What is the role of the protein in O2 binding, transport and storage?

• O2 only binds to reduced 2+ Fe hemes (it does not bind to oxidized Fe3+ hemes)

• For isolated hemes (Fe-porphyrins in a testube ), O2 binding is irreversible 2+ 3+ as it oxidizes Fe  Fe • The protein prevents this oxidation and allows reversible binding of O2

Mb + O2 ⇆ Mb-O2

• Protein stabilizes bound O2 by non-covalent interactions with specific active site residues in distal pocket 14 O2 binding to Myoglobin in Distal Pocket

1) Is O2 polar or nonpolar? Why? His E7 2) Will O2 have a high or low solubility in blood (water)? Phe CD1

Interaction with active site residues in Val E11 the protein, stabilizes bound O2 : • upper drawing – steric interactions : space-filling representation shows non-polar O2 in van der Waals contact with His F8 distal Val E11 and close to distal Phe CD1 (both have nonpolar sidechains) His E7 • lower drawing – electrostatic interactions: skeletal model shows H-bond between distal His E7 and lone-pair on terminal O atom of bound O2

• solubility of O gas in blood is only ~10–4 M, 2 15 Mb increases the solubility of O2 in muscles O2 binding to Myoglobin in Distal Pocket • After molecular oxygen binds, there is partial 2+ transfer of an electron from Fe to O2, shifting 3+ - towards a species with Fe bound to O2 . This internal electron shift is reversible, so oxygen binds 2+ to and leaves Fe Hb as molecular oxygen (O2). - • Superoxide anion (O2 ) is a reactive oxygen species that can damage biomolecules if released from Mb. Interaction with protein residues - prevents release of O2 . Myoglobin prevents release of this reactive species, by stabilizing bound

O2 using favorable steric and electrostatic interactions with active site residues. • The H-bond between the distal His E7 and the terminal atom of the bound oxygen fine tunes the reactivity of the 16 heme, favoring reversible O2 binding. Heme Group in Active Site of Hemoglobin

• Hemoglobin is composed of 4 subunits, with a heme bound to each subunit. • A His residue forms a coordinate covalent What happens to bond with the central Fe on one side of the the structure of the heme plane (proximal side) , allowing O2 to bind on the other side (distal side). Fe-porphyrin when it binds O2? Hemoglobin's biological function is regulated by changes in the overall 3D protein structure. Effect of Binding O2 Ligand to Hemoglobin 1) Does the proximal His move? 2) Does the rest of the protein move?

T-state R-state

http://www.chemistry.wustl.edu/~edudev/LabTutorials/ Hemoglobin/changemovie.html

This movie shows how the amino acid residues near the heme group in hemoglobin shift as the heme group converts between the nonplanar (domed) and the planar conformation by binding and releasing a molecule of O2. 18 Effect of Binding O2 Ligand 1) What happens to the structure of the Fe-porphyrin when it binds O2? • O2 binding changes the position of iron • Proximal His moves towards the Fe • The rest of the protein moves in Deoxygenated heme group and attached response to O2 binding histidine residue electron clouds push one another apart, and the iron atom in the center is drawn out of the plane. oxyHb has Fe in porphyrin plane (planar) deoxyHb has Fe out-of-plane, (porphyrin = is domed)

Why does iron moves into the plane of the heme on oxygenation? • Binding of O2 changes the electron configuration within the Fe • Thus the Fe becomes smaller in size when it is 6-coordinate • The Fe is pulled into the porphyrin plane 19 Globin (Mb, Hb) Structure and Ligand Binding What happens to the 3D structure of Hemoglobin when it binds molecular oxygen, O2 ?

• Proteins that bind small molecules (ligands) can have one state (3D shape) when the ligand is bound in the active site, and another slightly different 3D shape when the protein is ligand-free (no ligand bound). • Myoglobin (muscle tissue) has three species (different 3D states) in an organism. Hemoglobin (Hb) in blood has similar colors and species. • Each species has a different color, because it has a slightly different electronic (and magnetic) state for the Fe-heme complex:

Coordination Ligation Oxidation Name of Protein Number State State Species Color 5-coordinate Unligated Fe(II) deoxy Hb dark purple in 6-coordinate Ligated Fe(II)-O oxy Hb bright red in 2 5-coordinate Unligated Fe(III) met Hb brown in Oxidized 3D protein shape