CHE 472 University of Buffalo Fe S Protein Strucutre Examination Paper

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This homework is better for those who have used RCSB Data Bank. The attachment explains in detail on what to do. It consists of two parts (I and II), you can answer each question under each part. No need to rewrite the question. For example:

Part I

A) "Your answer".

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CHE 472 Assignment (20 pts.) This exercise will use the RCSB Protein Databank (pdb) to view two common Fe-S proteins that are important in electron transport reactions. It will also examine the nature of the FeS clusters found in these proteins. Viewing Protein Structures: Enter the PDB ID in the search box at the RCSB Data Bank: http://www.rcsb.org/pdb. You can view the protein structures using the default NGL viewer by selecting “3D ViewStructure”. You can also view the structure using JSmol viewer by selecting JSmol from the drop-down list under “Select Different Viewer”. Note: JSmol generally has more tools (measuring, coloring, labeling, etc.) and will be required for some of the questions here, but you may find the NGL viewer useful in getting started and familiarizing yourself with the overall structure of the protein. A tutorial on using the features in JSmol can be found at: https://www.andrew.cmu.edu/user/rule/jsmol/jsmol_tutorial.html Viewing the Fe-S Clusters. Generally, any molecules that are not amino acid constituents within a protein are referred to as “Ligands” in the structures downloaded from the Protein Data Bank. Therefore, the FeS clusters in these proteins are called ligands. To view the Fe-S clusters there are 2 options. 1) To use the NGL viewer: select the structure summary tab and scroll down to the table labeled Small Molecules. There you will find a list of all non-amino acid constituents in the protein (in this case the Fe-S cluster and perhaps some solvent molecules). Select the link called “Ligand Interaction”. This will open up a structure of just the ligand and surrounding environment. Once again, the NGL viewer has fewer features than the JSmol viewer but is useful in getting oriented and addressing some of the questions. 2) To use the JSmol viewer: while looking at the structure of the protein in JSmol, scroll down the page to the Table labeled “Ligands” and select the link listed under “View Interactions” for the ligand you are interested in examining. Note- to measure distances and angles for the Fe-S clusters you must use the JSmol viewer. See the JSmol tutorial listed above to learn how to do this. Part I: I) Ferredoxin is a small Fe-S protein found in plants and algae that is involved in photosynthetic and other electron-transport processes. Download the structure for the Ferredoxin from the cyanobacteria Anabaena (PDB ID= 1FXA) at the RSCB Protein databank. A) Examine the three-dimensional structure of Ferredoxin by selecting “3D View- Structure” and selecting the JSmol Viewer. 1) What secondary structure predominates in the protein? 2) The structure shown represents a unique domain, which is found in many Fe-S proteins. This domain is referred to as an 2Fe-2S ferredoxin type domain or a betagrasp domain. Do a web search to determine what secondary elements are present in this type of domain and how they are arranged with respect to each other. Why is the domain often referred to as a beta-grasp? B) Ferredoxin contains a single iron-sulfur cluster. Identify the atoms in the cluster. How many iron atoms and inorganic sulfur atoms are found in the cluster? C) Where is this cluster with respect to the beta sheets in the subunit? Is it buried in the structure or exposed to solvent? (Hint: turn on the space filling scheme and select water accessible surface for atoms.) D) Select backbone as the scheme. Are both the iron atoms buried to the same extent? Which is closer to the surface and thus would have a higher reduction potential? Note: Ferredoxin has a reduction potential of -0.43 V and is a very strong reducing agent. In green plants and photosynthetic bacteria, it transfers electrons to FNR which can in turn reduce NADP+ (2 e- and 1 H+) to NADPH. The reduction potential of NADPH is -0.32 V. E) Go to the summary tab for 1FXA and select the Fe-S cluster from in the list of Small Molecules F) Examine the [2Fe-2S] cluster in Chain A. What amino acids (name and sequence numbers) is each Fe in the cluster coordinated to? (Hint turn on metal interactions in the NGL viewer to have a look.) How are these amino acids interacting with the Fe ions? G) A schematic for the [2Fe-2S] cluster from Ferredoxin is shown below: 1) using the Measure distances option in the JSmol Viewer, measure and record all the Fe-S distances in the cluster. How do they compare to the distances between the Fe ion and the coordinated amino acids? 2) Using the angle measure option under “measures”, measure the bond angles around each of the Fe ions. Supply those angles on the diagram above. 3) What is the approximate coordination geometry for each of the Fe ions? 4) Four of the eight atoms shown above fall in a plane. Circle the planar portion of the structure. H) The [2Fe-2S] cluster is sometimes notated as Fe2S2(Scys)4 to explicitly show the involvement of the Cys S. Assuming that each inorganic (bridging) S carries a formal charge of -2 and that each cysteine sulfur carries a formal charge of -1, calculate the formal charge in the fully oxidized complex given that both Fe’s in the oxidized complex are Fe+3. 1) The reduced form of the [2Fe-2S] cluster has one Fe+2 and one Fe+3 ion. What is the formal charge on the entire complex in the reduced form? 2) When calculating the formal charge on the cluster itself, it is possible to ignore the charge from the Cys S (supplied by the protein) and simply calculate the charge on the cluster using the Fe and inorganic S. The resultant cluster is notated [2Fe-2S]+n where n is the formal charge on the cluster. Using this notation designate the charge on the oxidized and reduced clusters. Part II High-potential Iron Protein (HIPIP) contains an iron-sulfur cluster that differs from the one found in Ferredoxin. This type of iron-sulfur cluster is found in a variety of proteins. As a class, the HIPIP proteins undergo a reversible one-electron transfer reaction at a characteristically high reduction potential (between +0.05 and +0.45 V). A) Examine the structure of the HIPIP from the purple photosynthetic bacterium, A.vinosum (PDB ID= 1HIP). Describe the secondary level of structure in the protein. B) Where is the iron-sulfur cluster located in the protein? Is it buried in the structure or exposed to solvent? Can a water molecule approach the metals? C) Examine the [4Fe-4S] cluster from the HIPIP protein (use the Ligand Viewer to do this). 1) How are the Fe ions attached to the protein? 2) A [4Fe-4S] cluster is often called a cubane given its similarities to a cube. Measure the angles at which each Fe and sulfur atom is bound to the cube. Is the Fe-S cluster shown a perfect cube? Why or why not? 3) Examine the coordination geometry of each Fe ion. What is it? 4) Two of the Fe’s in the cluster are coordinated by a Cys-X-Y-Cys motif. What are the amino acids designated by X and Y? D) Calculate the formal charge on the oxidized form of the [4Fe-4S] cluster given that three of the Fe ions are Fe+3 and one is Fe+2. E) In some proteins, the cube may actually lose an iron atom (becoming a [3Fe-4S] cluster) and later regain it as part of a regulatory function. The variation of the reduction potential of the metal clusters in high-potential iron proteins may be due to the chemical and physical environments of the protein, for instance: 1) exposure of the cluster to solvent, or lack thereof 2) presence of aromatic residues near the cluster 3) flexibility of the protein 4) hydrogen-bonding pattern to the oxidation-reduction center A hydrophobic environment favors the oxidized form of the cluster, with its lower formal charge, and in part accounts for the exceptionally high positive reduction potential in the HIPIP’s. Also, there are fewer instances of hydrogen bonds to the cluster in these proteins, again favoring oxidation and adding to the high positive reduction potential. Using the tools available for examining the Fe-S cluster : 1) Determine which amino acids are nearest to (but not coordinated to) the cluster. Are these hydrophobic or hydrophilic amino acids? 2) Are there any hydrogen bonding interactions between the protein and the cluster? If so, describe them.
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Running Head: EXAMINING FE-S PROTEIN STRUCTURE

Examining Fe-S Protein Structure

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EXAMINING FE-S PROTEIN STRUCTURE

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Examining Fe-S Protein Structure
Part 1
Examining Fe-S Protein Structure
(A.)
1. The secondary structure that predominates in the protein is cysteine (α helix). This
secondary structure is formed as a result of joining molecules to form a water solvent amino
acid. α helix forms residues whose structure is essential is maintaining the rigidity of the protein.
2 The secondary elements that were present in the beta-grasp domain is known as ferredoxin
which is written as Fe2S2. Its primary function in real-life photosynthesis is to transfer electrons
to reductase enzyme which in turn make processes like assimilation possible. On top of that, the
ability of ferredoxin to oxidize or reduce is important in regulation of CO2.
(B.)
This domain is usually referred to as beta-grasp because the two active site consists of two disulphide which are bridged with high spin tetrahedral Iron three ions. Each of the atoms is
coordinated with two non-labile Sulphur atoms of the group terminal cystine. It can be
represented diagrammatically as shown below:
(C.)
With respect to the beta sheets in the subunit, the [2Fe-2S] cluster is located as a scaffolding
in the protein. Its environment is in such a way that it forms a pocket integrated within the
structure. This hydrophobic structure does not expose it to solvents.
(D.)

EXAMINING FE-S PROTEIN STRUCTURE

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There exists a distinctive variation of how the two iron atoms are engrained in ...


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