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Dioxygen: Uptake, Transport & Storage

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Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage: Hemocyanin/Hemerythrin Hemoglobin/Myoglobin References: M. F. Perutz et al. Acc. Chem. Res. (1987) 20, 309—321. J. M. Riefkind Adv. Inorg. Biochem. (1988) 7, 155—241. M. F. Perutz Annu. Rev. Physiol. (1990) 52, 1—25. M. F. Perutz Ann. Rev. Biophysics and Biomolecular Structure 1998 R. E. Dickerson, I. Geis Hemoglobin 1983 D. Voet & J. G. Voet Biochemistry 2nd ed. 1995 L. Stryer Biochemistry 4th ed. 1997 050902 Dioxygen: Uptake, Transport & Storage Why do we have to bind O2 in first place? Why not molecular diffusion? Despite the huge body of synthetic work, NO model system At 100-mm O2 pressure found in the lungs, 0.3 mL of O2 can dissolve per comes even close to the biological parent! 100 mL plasma…far too little to keep you going. One would have to breath pure O2 at 3 atm pressure for the solubility to rise It’s not only about reversible dioxygen binding! to an acceptable 7 mL O2/100 mL plasma…and that’s just enough to enable you to read this. In contrast, 15g of Hemoglobin (found in 100 mL of blood plasma) are capable of binding 20 mL of gaseous O2..and that’s also 1. The transport molecule must have a high affinity for O2 in the presence of plentiful supply at the lungs and a lowered affinity in why giant man-eating killer ants will only exist in Hollywood! the O2—poorer environment of the muscles…where it is needed! 2. The storage molecule must have a higher affinity for dioxygen What it needs to do “the job”: than the carrier has at low dioxygen concentrations. Every mL of blood has approx. 5 billion red blood cells (erythrocytes) 3. The carrier should not only bind to O , but to carbon dioxide as 2 …each erythrocyte is packed with 280 million molecules of hemoglobin. well to transport CO2 back to the lungs where it can be ejected as a waste product. 4. The carrier should release its dioxygen more readily to working Dickerson & Geis: “If an erythrocyte were enlarged 300 million times, muscles than to resting tissue. it would be the size, and roughly the shape, of the Rose Bowl, piled with 280 million large grapefruits”. Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage The Dioxygen-Carrying Team: Hemoglobin/Myoglobin 1 Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage Myoglobin/Hemoglobin Comparison: Size: Myoglobin: Only 1 heme and 1 polypeptide chain of 153 amino acids. MW = 17,800 Hemoglobin: 4 heme units and 4 polypeptide chains; 2 α chains with 141amino acids, and 2 β chains with 146 amino acids. MW = 64,500 7.5 microns taken from: Dennis Kunkel Microscopy, Inc. Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage Myoglobin/Hemoglobin: The first three-dimensional structures Myoglobin: The first three-dimensional structure analysis of any protein: Nobel Prize 1962 of any protein. Kendrew at al. Nature (1960), 185, 422. John C.Kendrew (born 1917, died 1998) Max Peruz (born 1914, died 2002) The richest source of myoglobin are the muscles of aquatic diving mammals: seals, whales, and porpoises. It is no surprise that the first x- ray crystal structure of myoglobin was obtained from the protein isolated from sperm whale. Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage A Three-Dimensional Fourier Synthesis at 2 Å Resolution. A Three-Dimensional Fourier Synthesis at 2 Å Resolution. Observed (left) and computed (middle) electron density distribution in the plane of the heme. Photograph of a set of sections normal to the plane of the heme group (right). taken from: Kendrew at al. Nature (1960), 185, 422. …requires an awful lot of imagination! 2 …a note on the side: Science & Art Geis (1908-1997) was one of the greatest scientific artists of the 20th century. His innovations, particularly in depicting the structures of biological macromolecules such as DNA, earned him an international reputation. Many of his illustrations appeared in Scientific American, including a painting of the first protein crystal structure, of myoglobin, published in 1961. ©2000 Howard Hughes Medical Institute Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage Myoglobin: Shapes & Perspectives: an oblate spheroid: 44 x 44 x 25 Å Front view: Side view: “Little can be said as yet about the relation between structure and function. • a box for the heme group, • helices range in length from 7 The haem groups are much too far apart for the combination with oxygen of • built up from 8 connected, (C,D) to 26 (H) residues. any one of them to affect the oxygen affinity of its neighbours directly. • right-handed pieces of α helix. • hydrophilic groups outside. Whatever interaction between the haem groups exists must be of subtle and • hydrophobic groups inside. indirect kind that we cannot yet guess.” M. F. Perutz, Nature 1960, 185, 416. Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage Size: Hemoglobin: Structures of Deoxy- (left) and Oxyhemoglobin (right) M. F. Perutz at al. Nature (1960), 185, 416. 2 α and 2 β polypeptide chains. Each of the four chains is folded in much the same way as seen for Myoglobin. The α1β1 and α2β2 interactions (35 residues) are hydrophobic and stronger than α1α2 and β1β2. They are also stronger than the α1β2 (19 residues). 3 Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage Hemoglobin: Symmetry & Perspective: The quaternary structural changes preserve Hb’s two-fold symmetry and takes place entirely across the α1β2 and α2β1 interface: Side view: o Nearly 2,2,2 symmetry (pseudo D ) Oxygenation rotates the α1β1 dimer ~ 15 with respect to the α2β2 dimer (page 33), 2 so that some atoms at the α β interface shift by as much as 6 Å relative to each roughly spherical: 64 x 55 x 50 Å 1 2 other. Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage Myoglobin/Hemoglobin: A comparison Myoglobin/Hemoglobin: A comparison of the amino acid sequences. The similarity of their conformations is evident… Myoglobin Hemoglobin (β-chain) …yet unexpected, because their amino acid sequences are rather different! ² Different amino acid sequences can specify very similar 3-D structures. the three chains are identical at only 24 of 141 position. Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage The interior consists almost entirely of non-polar residues such as Highly conserved amino acid residues in hemoglobins: leucine (Leu), valine (Val), phenylalanine (Phe), and methionene. Position Amino Acid Role F8 Histidine Proximal heme-linked His E7 Histidine Distal His near the heme CD1 Phenylalanine Heme contact F4 Leucine Heme contact B6 Glycine Allows close approach of the B and E helices C2 Proline Helix termination HC2 Tyrosine Cross-links the H and F helices C4 Threonine ? H10 Lysine ? Additionally, highly non-polar interior and polar character of the exterior The only polar residues are of the molecule is conserved. two histidines with their critical function at the binding site. 4 Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage The Role of the Proximal (F8) & Distal (E7) Histidine: The Role of the Proximal (F8) & Distal (E7) Histidine: The imidazole ring of the distal His E7 (pK = 5.5 ) acts a a proton trap, thereby protecting* the iron from H+. Linearly coordinated A bent Fe—CO The dioxygen ligand CO binds ~ 25,000 geometry weakens binds to the iron-heme more tightly to Fe the ligand interaction with an Fe-O-O angle than dioxygen does. significantly. CO binds of ~ 115 – 159o. only 200x stronger. Discriminate in favor of O and against CO. How? *) heme iron oxidation is catalyzed by H+ that are reduced by the heme Fe 2 z− and that in turn reduce O2 to O2 Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage The Electronic Structure of Hemoglobin/Myoglobin: The Role of the Proximal (F8) α-subunit Observation: Diamagnetic Spin Ground State for the Oxy-Form! & Distal (E7) Histidine: Fe-O-O difference between α and β-chain in Hb is not significant (within exp. error); but very different from o Fe-O-O of 115 in Mb—O2 overlay no H-bond to His E7 β-subunit Solution: Antiferromagnetic spin-spin coupling. Dioxygen: Uptake, Transport & Storage Status Quo Hb binds dioxygen and transports O2 to the tissue. Two strikingly similar proteins, hemoglobin (Hb) & myoglobin (Mb), How does Mb manages to get O transferred from Hb? that immobilize and protect the active site, a heme—iron unit, 2 which reversibly bind dioxygen. Hemoglobin is a much more But how does the system address the following important issues: intricate and sentient molecule than Myoglobin is! Hb binds dioxygen and transports O2 to the tissue. + Hb transports H and CO2 How does Mb manages to get O transferred from 2 in addition to O2 Hb—O2 + Mb to Mb—O2 + Hb? O2 binding properties in Hb + are regulated by interactions Active tissue produces CO2 and H . Who takes care of the waste products? between separate, non- adjacent sites. Maternal blood needs to transfer O to fetal blood. 2 Hemoglobin is an How do fetal red blood cells manage to receive O2? allosteric protein; whereas Myoglobin is not! 5 Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage Dioxygen binds cooperatively to hemoglobin! The binding of O2 to hemoglobin enhances the binding of additional O2 to the same hemoglobin (take advantage of high concentrations of O2 in the lungs; sigmoidal curve ). Binding of O2 to Myoglobin is not cooperative; hyperbolic cure). Affinity of hemoglobin for O2 is pH dependent! + H and CO2 promote the release of bound dioxygen (for instance in active tissues such as in muscles). Reciprocally, higher concentrations of O2 promote the release of CO2 (e.g.
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