Transition Metals As Spectroscopic Probes for Structure and Reactivity
1. Transition Metals as Spectroscopic Probes for Structure and Reactivity Brandon Alexander CH414 Inorganic Chemistry II
2. “Spectroscopically Silent” Metals Physiochemical Methods-do well at looking at the environment of metal centers of proteins, enzymes…and other structures. NMR/MS Some metal Ions are not open to such investigation.
3. Common Metal Ions in Biology Na K Mg Ca Zn Difficult to study-Inability to apply spectroscopic methods “Spectroscopically Silent” Metals + + 2+ 2+ 2+
10. Shift Reagents & Relaxation Probes Shift Reagent- Paramagnetic shift reagents have the ability to induce chemical shifts and thus simplify complex NMR spectra. Magnetic Resonance Solutions, Inc--NMR (nuclear magnetic resonance) shift reagents- http://www.macrocyclics.com/topics/NMR-shift-reagents.html
11. Shift Reagents & Relaxation Probes Relaxation - The name spin-lattice relaxation refers to the time it takes for the spins to give the energy they obtained from the RF pulse back to the surrounding lattice, thereby restoring their equilibrium state.
13. When you have a magnetic moment directed at some finite angle with respect to the magnetic field direction, the field will exert a torque on the magnetic moment. This causes it to precess about the magnetic field direction. This is analogous to the precession of a spinning top around the gravity field. The torque can be expressed as the rate of change of the nuclear spin angular momentum “I” equated to the expression for the magnetic torque on the magnetic moment Georgia State Univ. Dept. of Physics and Astronomy. Hyperphysics. “Larmorprecession”
14. which when put in derivative form gives a precession angular velocity Georgia State Univ. Dept. of Physics and Astronomy. Hyperphysics. “Larmorprecession”
15. The Spinning Top in a Gravitational Field Analogy Relaxation Animation MRI - Relaxation BIGS-Germany Georgia State Univ. Dept. of Physics and Astronomy. Hyperphysics. “Larmorprecession”
16. Zinc(II) Environment-Cobalt(II) Probe Zinc metalloprotiens-bind Zn in tetrahedral environments (d10) Remove metal from protein (apoprotein) Removed by “dialysis against a ligand that has a high affinity for Zn “ EDTA or 1,10-phenanthroline 2+ 2+ Cowan, J.A. InorganicBiochemistry 2e
17. Metallo-β-Lactamase “…these proteins bind two zinc ions per molecule as cofactor.” Carfi A, et.al "The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold". EMBO J. 14 (20): 4914–21 Photo: Wikipedia-Public Domain
18. Metallo-β-Lactamase 2.1. Preparation of enzyme species “The preparation of the native and metal-free (apo-) β-lactamase from A. hydrophila AE036 … The Co2+- and Cd2+-substituted forms of the enzyme were prepared by direct addition of metal ions to the apoenzyme in either metal-free 15 mM sodium cacodylate, pH 6.5, or 50 mM HEPES, pH 7.5. The Cu2+-species were produced in 50 mM TES, pH 7.5. The Cu2+-Zn2+-hybrid was obtained by addition of one equivalent of Zn2+ to the Cu22+-enzyme at pH 7.5.” Valladares, M.H., et.al “Kinetic and spectroscopic characterization of native and metal substituted β-lactamases from A. Hydrophila AE306” FEBS Letters, 477, I3, 285
19. Cobalt Analogues Co (2+) d6 NMR— Co-59- standard Natural Abundance Spin 7/2
20.
21. Ribozymes ribonucleic acid enzyme Catalyze the hydrolysis of their own phosphodiester bonds or phosphodiester bonds in other RNA Photo: Wikipedia-Public Domain
24. Magnesium Locations in Ribozymes Coordinate Mg2+ sites to Phosphates in Hammerhead Ribozyme Photo: Zhuang , Z., Kahn, K Department of Chemistry and Biochemistry, UC Santa Barbara. 2003-2004
25. “Possible” Role of Mg2+ In-line transition-state for the hammerhead ribozyme reaction. A general base (B, red) abstracts a proton from the 2'-O, and a general acid (A, blue), supplies a proton to the 5'-O leaving group as negative charge accumulates. The bonds breaking and forming (dotted lines) must be in the axial positions and reside approximately 180° apart, as shown. The reaction product is a 2',3'-cyclic phosphate. Metal can play the role as “general” acid or “general” base
26. Reversibility Westhof, E, A tale in molecular recognition: the hammerhead ribozyme. J. Molecular Recognition, 20:1-3 (2006).
27.
28.
29. “These results demonstrate that 31P NMR representsan important method for both identification and characterization of metal-binding sites in nucleic acids.” Hansen, M., et. al., “Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme” RNA. 1999, (8), 1099–1104. Substituting Mg for Mn
30. Hansen, M., et. al., “Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme” RNA. 1999, (8), 1099–1104.
31.
32. Why the divalent metal is needed—Looking A9 & A13 in HH16 The Phosphate for A9 Lies above the base The Phosphate for A13 lies below the base Why is this? Sigel. Manganese and Its Role in Biological Systems. 37, 1999, 166.
33. Other possibilities The Ribozyme has a “tetraloop” that reverses the directionality of the phosphodiester backbone. Secondary Structure—Metal could help with the folding Sigel. Manganese and Its Role in Biological Systems. 37, 1999, 166.
34. Calcium (II)—Using the Lanthanides Ca 2+ is a hard metal Prefers oxygen ligands “In every calcium binding protein; the ligand environment is made up from O-donors” O donors Glu, Asp, Thr, Ser, backbone carbonyls, H2O Cowan, J.A. InorganicBiochemistry 2e
36. Uses of the Lanthanides Heavy atom derivatives in X-Ray Diffraction Luminescent probe NMR—Shift and Relaxation Reagents
37. Lanthanide Luminescence Eu3+ and Tb3+ Luminesce strongly at room temperature when complexed to ligands Long excited state life times 100-3000μs Cowan, J.A. InorganicBiochemistry 2e
38. Lanthanide Luminescence Similar ionic radii Coordination number of Ln3+ (7-9) close to Ca2+ (6-8) Ln3+ binds ligands~ 105 times more strongly than Ca2+ Department of Chemistry. Oxford.”Lanthenides, Electronic Spectroscopy”
39. Lanthanide Luminescence Used in determining the number of water molecules bonded to an active-site metal ion. The coupling of the 4forbitals to inner sphere ligands is weak but it is strong enough to monitor the emmision life time in water. Different Decay rates Cowan, J.A. InorganicBiochemistry 2e
40. Thermolysin—binding sites Catalyzes the hydrolysis of peptide bonds containing hydrophobic amino acids “…requires one zinc ion for enzyme activity and four calcium ions for structural stability” P. 377-text 3TMN—Ligand Explorer Tajima, M., et. al., Eur J Biochem. 1976. 64(1):243-7.
43. Uses of Lanthenides “…Eu-activated luminescent materials and molecular compounds have been synthesized and tested. Their applications range from electroluminescent devices to security inks, plastic films for agriculture, analytical probes, as well as bioprobesfor immunoassays or for imaging live cells in the context of cancer diagnostics” Shavaleev, N.M., et.al.,” Highly Luminescent Homoleptic Europium Chelates” Inorg. Chem. Comm. 2009, 48, 5611-5613
45. Summary Metal ion centers can be studied by synthesizing a metal derivative of the “spectroscopically silent” Common Replacements Co for Zn Mn for Mg Ln for Ca (Ln = lanthenide)
46. References Cowan, J.A. InorganicBiochemistry2e. Wiley-VCH. 1997 Magnetic Resonance Solutions, Inc--NMR (nuclear magnetic resonance) shift reagents- http://www.macrocyclics.com/topics/NMR-shift-reagents.html MRI - Relaxation Bluck Industrial Graphic Service-Germany http://www.bigs.de/BLH/en/index.php?option=com_content&view=category&layout=blog&id=104&Itemid=272 Carfi A, et.al "The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold". EMBO J. 14 (20): 4914–21 Valladares, M.H., et.al “Kinetic and spectroscopic characterization of native and metal substituted β-lactamases from A. Hydrophila AE306” FEBS Letters, 477, I3, 285 Mason, S. CH 214, Lecture, Spring 2009—Chapter 20-21 Housecroft & Sharpe Inorganic Chemistry. 3e, 2008 Westhof, E, A tale in molecular recognition: the hammerhead ribozyme. J. Molecular Recognition, 20:1-3 (2006). Hansen, M., et. al., “Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme” RNA. 1999, 5, (8), 1099–1104.
47. References Sigel. Manganese and Its Role in Biological Systems. 37, 1999, 166. S.J. Heyes, Department of Chemistry. Oxford.”Lanthenides, Electronic Spectroscopy” http://www.chem.ox.ac.uk/icl/heyes/lanthact/L9.html. 1997-1998 Tajima, M., et. al., “Role of Calcium Ions in the ThermostabilityofThermolysin and Bacillus subtilis var. amylosacchariticus Neutral Protease.” Eur J Biochem. 1976. 64(1):243-7 Shavaleev, N.M., et.al.,” Highly Luminescent Homoleptic Europium Chelates” Inorg. Chem. Comm. 2009, 48, 5611-5613
