Let s continue our discussion on the interaction between Fe(III) and 6,7-dihydroxynaphthalene-2- sulfonate.

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1 Chemistry 5995(133)-8990(013) Bioinorganic Chemistry: The Good, the Bad, and the Potential of Metals Assignment 2- Aqueous Speciation, Magnetism, Redox, UV-Vis Spectroscopy, and Pymol Let s continue our discussion on the interaction between Fe(III) and 6,7-dihydroxynaphthalene-2- sulfonate. The following product is obtained in the solid-state from the interaction of the metal with three mole equivalents of the ligand. In aqueous solution, the interaction is more complex resulting in a different speciation especially because of competition from proton binding. 1. Use the aqueous speciation program and the Fe(III) naphthalenediolate speciation data file (see website) to illustrate the point made in class that metals lower the pka of ligands. 2. In biological fluids, metal aqueous speciation would be even more complex because of the presence of other metal chelators. Let s examine this on a very simple level by incorporating the influence that hydrolysis would have on Fe(III) naphthalenediolate speciation. To the speciation data file add the Fe(III) hydrolysis constants presented in class. Working with an Fe(III) concentration of 20 μm (the amount present in blood), discuss what happens to the Fe(III) speciation at the different 6,7-dihydroxynaphthalene-2-sulfonate concentrations below. Recall that the software assumes the concentration of water to be 1 M. (Note: This is an excellent time for you to review units and conversions especially to prepare for your exams.) [6,7-dihydroxynaphthalene-2-sulfonate] (μm)

2 3. Determine the log β values for the following reactions. Please use simple notations. (Note: You must refresh yourself on how to work with log values.) a. After determining the log β value, calculate the ph specific value for ph 7.4. Is this reaction thermodynamically favorable at this ph? b. To solve this problem, you will need to use the pka (or pkw) of water (provided). After determining the log β value, calculate the ph specific value for ph 7.4. Is this reaction thermodynamically favorable at this ph? 4. The magnetic moment of two Mn complexes were measured Complex μ (BM) at 300 K [Mn(H2O)6] 4.90 [Mn(NH3)6] 2.83 The Mn in the two complexes has the same oxidation states. Explain the magnetic moment data using orbital splitting diagrams.

3 5. Ligand binding to metal ions can change the reduction potential of the metal ion. a. Look at the reduction potentials below and explain what effect does binding of ethylenediamine (en) have on the reduction potential of Cu 2+? E o (V) vs NHE Cu 2+ + e - Cu [Cu(en)2] 2+ + e - [Cu(en)2] b. Looking at the Table 19-2 handout, which of the redox agents will NOT be able to reduce ligand-free Cu 2+? c. Which of the redox agents will be able to oxidize [Cu(en)2] +? d. Is the following electrochemical reaction thermodynamically favorable? Show your math. NAD + as the oxidizing agent and [Cu(en)2] + as the reducing agent. 6. Is ΔO related to the intensity of a d-d electron transition in an octahedral complex? Explain.

4 PYMOL To begin: 1. Download pymol by going to Complete the application. Download the latest version of Pymol. 2. Enter the Protein Data Bank (PDB): Go to the website 3. Search under Everything: 1N84 Crystal structure 1N84 is that of Fe(III) bound to the recombinantly expressed N-lobe human serum transferrin. This is a single-lobed version of the protein. The Fe(III) in this structure is bound in a traditional form where the metal is coordinated to the Asp63, Tyr95, Tyr188, and His249 residues and the synergistic anion carbonate in a closedconformation. 4. Download Files > PDB File (text) 5. Open the file (you may want to use an external mouse) a. Two boxes will open. The white box (the command box) is a text box of sorts that allows you to type in certain commands that will be performed on the protein crystal structure. The black box (the viewer) allows you to visualize the protein. b. To begin, use your mouse or keypad to explore how you can move the crystal structure in the viewer for better visualization. The left button allows you to move the protein around. The right button allows you to zoom in and out. 6. Key Descriptions in the PyMOL Viewer window

5 You will learn some basic key commands for running pymol. For more extensive instructions, refer to the pdf file that is attached on the course website. This will be helpful for your final projects. On the right side of the PyMOL Viewer you should see two tabs, one labeled all and the other labeled 1N84. These tabs both control the look of the protein. As described below, you can select certain residues or parts of the protein that you are interested in observing/studying. When you select these items, a new tab will appear with the label (sele) For a selected item, the following sub-tabs will be present, which will allow you to manipulate the item as you wish. A Action: It has navigation tools, analysis tools and object manipulation S Show: Gives you different ways to represent the protein H Hide: Same as show but allows you to hide instead of show things. L Label: Any atom, amino acid, chain that you want to identify C Colors: Change the colors of the protein by atoms, chains, amino acids, etc. 7. You will be able to view different parts of the protein crystal structure. a. polymer: refers to the protein itself b. organic: refers to nonprotein organic components c. inorganic: refers to nonprotein inorganic components d. solvent: refers to solvent molecules You have 2 options as to how you can play with the visualization of the protein: I. Hide all of the crystal structure and select component by component the areas you are interested in. II. Work with the entire protein and highlight by color certain regions of it. We will focus on example I (hiding all). First go to Display in the menu bar of the white box and click on Sequence to see the protein sequence listed from the N-terminus to the C-terminus in the top part of the visualizer window. You will also see all organic and inorganic components listed right after the protein sequence.

6 Take a moment to scroll across the sequence. You are free to click on any residue. A (sele) tab will open and will control that particular residue. If you click on another residue, this tab will control both residues. In the white command box: NOTE: PyMOL is a very unforgiving software and there is no general undo button and so I suggest that you save each step along the way with a unique name. If you do not and you make a mistake, you will have to start your commands from the beginning and you may even have to reopen the original document. Type in the following commands in the order listed below: a) PyMOL > hide all The crystal structure that was originally present will disappear. b) PyMOL > show sphere, metal This command will allow you to visualize the Fe(III) ion in the crystal structure. c) PyMOL > show stick, organic This command will allow you to visualize the nonprotein organic components of the structures. In this structure, you should see a carbonate anion near the Fe(III). For some reason, the carbonate molecules looks like two carbonates molecules fused, which is an unfortunate glitch. In typical transferrin structures, you would normally see sugar molecules bound to the protein because it is a glycoprotein. d) PyMOL > show stick, inorganic This command will allow you to visualize the nonprotein inorganic component of the structures. You will see that the Fe(III)has bonds to the carbonate. There should only be two but it may look like three because of the unfortunate glitch. In some structures, you may find inorganic anions bound to the protein. e) PyMOL > select binding_site, byres organic expand 6

7 (Show > lines) This command will allow you to visualize the protein residues that are part of the primary and to an extent, the secondary Fe(III) coordination spheres. You can change how much of the secondary sphere that you can see by varying the number in the above command. By clicking on show, you can change the display to sticks. f) To identify the amino acids involves in the active site: Click on L (next to binding site) > residues To remove the labels, click on L > clear i) For better visualization, let s change the background to white. Click Display > Background > White You will see that your protein residues will change in their depth of color depending on their positioning. To make the residues have a consistent color depth, Click Display > Depth Cue j) Let s simplify the image. Change the size of the Fe(III) by right clicking on the metal in the structure. Select object>preset > ball and stick Then click on the select button to deselect the Fe(III) and now reconfigure the binding site so that you can properly see the primary coordination sphere. The image does not have to look perfect. In your spare time, you can learn how to master this program better. k) PyMOL > ray This step gives a nice polish to the image. l) Save your image: File > Save image as > PNG > Write the name for your file > Save

8 m) Insert this image as a picture into your word document. Specific question to address: How can we use the crystal structures of transferrin to compare the difference in protein conformation when the protein is metal bound and unbound? Let s use the residues Lys206 (K206) and Lys296 (K296) in these structures to address this question You will need to use the following three pdb files: 1N84 (the file that you were just using), 2HAV, and 3QYT. Start by opening up a crystal structure and hiding the protein structure. Do this for all three structures one at a time. a. Display sequence and click on the metal binding residues. D63, Y95, Y188, and H249. Click the sub-tab S > Stick b. Click on K206 and K296 Click on the tab corresponding to the lysines. Click on the sub-tab S > Stick Click the sub-tab C > by element >CHNOS (where C is anything but green) c. Now measure the distance between two nitrogens on the R groups of the K206 and K296. To measure the bond distances of any atom or residue: Wizard (in the panel) > Measurement > Select atom 1 > Select atom 2 > Done What are the distances in the three structures? Why are the lengths different for all three structures? Use the assigned readings to answer this question and also descriptions of the crystal structures from the protein database website. Extra credit: Use the command below to examine potential structural differences in two different metal bound human serum transferrin crystal structures. You do not have to stick with Fe(III) bound forms. Find polar contacts of a residue or atom with other residues: Select atom > Action > Find > Polar Contacts > to other atom in object