Iron-Sulfur Cluster Proteins

Understanding how reduction potentials are modulated for specific functions in complex biological systems is a fundamental problem in biological chemistry.  Of particular interest are the electron transport iron sulfur (FeS) proteins, whose structures range from 2Fe-2S diamonds, intermediate 3Fe-4S butterfly-shaped clusters, to 4Fe-4S cluster cubes (Figure 1a-c).  How the iron sulfur clusters can have a range of reduction potentials is a question that faces biological chemists today.

          The factors that govern the reduction potential of iron sulfur clusters have been postulated to include the number of (NH or OH to S) hydrogen bonds to the cluster, the proximity of charged residues, and the exposure to solvent (1,2).  These factors have been implicated based on site-directed mutants, un-natural backbone substitutions, and theoretical calculations (2-6).  In the past, mutations to important residues have produced altered reduction potentials. However, with few exceptions (c.f. 3,7-9), the structures of the mutant proteins have not been determined.  Since mutations can result in unknown structural changes, the conformations need to be detected with crystal or solution structures.  The proposed research will use the 2Fe-2S cluster Rieske protein as a model for FeS proteins, in which proteins with altered reduction potentials will be produced through a rational approach, and both the biochemical and structural changes evaluated.



           Rieske and Rieske-type proteins contain a unique 2Fe-2S cluster that is ligated by 2 cysteines and 2 histidines (Figure 2).  Rieske proteins are part of the electron transport systems, the bc complexes of the respiratory chains and the b6f photosynthetic complex (10, 11).  These proteins have high, positive reduction potentials that are pH dependent.   Rieske-type proteins are part of dioxygenase or other detoxification systems and usually have low or negative reduction potentials that are not pH sensitive in the physiological range.

           The Rieske and Rieske-type proteins share a common fold, although they have little sequence similarity, save for the residues that provide ligands to the cluster (10,12).  The proteins have large and small subunits; the small subunit contains the 2Fe-2S cluster. The large domain is a β- barrel or β-sandwich made up of 8-10 β-strands linked by flexible loops (Figure 2A).

The reduction potentials of the structurally characterized members of the Rieske family range from ~ -150 mV to ~ +475 mV.  The Rieske proteins from SoxF, spinach, bovine, and Thermus thermophilus (Tt) all function within respiratory chains, the arsenite oxidase Rieske-type protein is part of the arsenic detoxification system of bacteria, and biphenyl dioxygenase is also a detoxification protein that breaks down aromatic compounds.

          The structural differences between the Rieske and Rieske-type proteins are in the presence of a disulfide bond in the Rieske protein and the number of hydrogen bonds between NH or OH groups and the S or Sγ of the 2Fe-2S clusters (Figure 3 and  (12,15,18-22)).  Rieske proteins have 5 to 10 hydrogen bonds where Rieske-type proteins have less than 5.  More hydrogen bonds are thought to allow the protein to dissipate the extra negative charge upon reduction, thus allowing the reduction potential to be raised.  Single site-directed mutants have been shown to alter the reduction potential of Rieske proteins by removing hydrogen bonds to the S of the iron sulfur cluster or the Sγ atom of a ligating cysteine. (23-25).

The use of Rieske and Rieske-type proteins to study the factors governing reduction potentials provides a unique system, since there is a naturally occurring range of reduction potentials in a common protein fold (Figure 3).  The Rieske protein from Thermus thermophilus and biphenyl dioxygenase Rieske-type protein will be subjected to mutagenesis.  Residues that are proposed to affect the reduction potential, including those that form hydrogen bonds to the cluster, the disulfide bonded residues, and the charged and hydrophobic residues will be systematically altered.  By mutating residues within the Rieske protein from T. thermophilus with iso-positional amino acids from biphenyl dioxygenase, the reduction potential of the Rieske protein can be potentially lowered and the pH dependence lost.  In a parallel experiment, residues from the Rieske-type protein from biphenyl dioxygenase will be replaced with corresponding residues from T. thermophilus to see if the reduction potential can be raised and pH dependence created.  Since both the Tt Rieske and biphenyl dioxygenase Rieske-type proteins have been crystallized previously, each mutant will also be structurally analyzed using X-Ray crystallography to ascertain any changes that might accompany the mutations and altered reduction potentials, providing a full picture of the effect of these mutations on the protein.  These studies are also precursors to future directions where reduction potential can be a designed feature of a protein that will result in bacteria for use in bioremediation.



 (1)        Klingen, A. R., and Ullmann, G. M. (2004) "Negatively Charged Residues and Hydrogen Bonds Tune the Ligand Histidine pKa Values of Rieske Iron-Sulfur Proteins". Biochemistry 43, 12383-12389.

(2)        Ullmann, G. M., Noodleman, L., and Case, D. A. (2002) "Density functional calculation of pKa values and redox potentials in the bovine Rieske iron-sulfur protein". J. Biol. Inorg. Chem. 7, 632-9.

(3)        Chen, K., Bonagura, C. A., Tilley, G. J., McEvoy, J. P., Jung, Y.-S., Armstrong, F. A., Stout, C. D., and Burgess, B. K. (2002) "Crystal Structures of FdI variants exhibiting large changes in [Fe-S] reduction potential". Nat. Struct. Biol. 9, 188-192.

(4)        Lin, I.-J., Gebel, E. B., Machonkin, T. E., Westler, W. M., and Markley, J. L. (2003) "Correlation between hydrogen bond lengths and reduction potentials in Clostridium pasteurianum Rubredoxin". J. Am. Chem. Soc. 125, 1464-1465.

(5)        Low, D. W., and Hill, M. G. (2000) "Backbone-engineered High Potential Iron Proteins: Effects of Active-Site Hydrogen Bonding on Reduction Potential". J. Am. Chem. Soc. 122, 11039-11040.

(6)        Soriano, A., Li, D., Bian, S., Agarwal, A., and Cowan, J. A. (1996) "Factors Influencing Redox Thermodynamics and Electron Self-Exchange for the [Fe4-S4] Cluster in Chromatium vinosum High Potential Iron Protein: The Role of Core Aromatic Residues in Defining Cluster Redox Chemistry". Biochemistry 35, 12479-12486.

(7)        Binda, C., Coda, A., Aliverti, A., Zanetti, G., and Mattevi, A. (1998) "Structure of the Mutant E92K of [2Fe-2S] Ferredoxin I from Spinacia oleracea at 1.7 Resolution". Acta. Cryst. D54, 1353-1358.

(8)        Camba, R., Jung, Y.-S., Hunsicker-Wang, L. M., Burgess, B. K., Stout, C. D., Hirst, J., and Armstrong, F. A. (2003) "The Role of the Proximal Proline in Reactions of the [3Fe-4S] cluster in Azotobacter vinelandii Ferredoxin I". Biochemistry 42, 10589-10599.

(9)        Hurley, J. K., Weber-Main, A. M., Hodges, A. E., Stankovich, M. T., Benning, M. M., Holden, H. M., Cheng, H., Xia, B., Markley, J. L., Genzor, C., Gomez-Moreno, C., Hafezi, R., and Tollin, G. (1997) "Iron-Sulfur Cysteine-to-Serine Mutants of Anabaena [2Fe-2S] Ferredoxin Exhibit Unexpected Redox Properties and Are Competent in Electron Transfer to Ferredoxin:NADP+ Reductase". Biochemistry 36, 15109-15117.

(10)      Link, T. A. (1999) "The structures of Rieske and Rieske-type proteins". Adv. Inorg. Chem. 47, 83-157.

(11)      Berry, E. A., Guergova-Kuras, M., Huang, L. S., and Crofts, A. R. (2000) "Structure and function of cytochrome bc complexes". Ann. Rev. Biochem. 69, 1005-1075.

(12)      Hunsicker-Wang, L. M., Heine, A., Chen, Y., Luna, E. P., Todaro, T., Zhang, Y.-M., Williams, P. A., McRee, D. E., Hirst, J., Stout, C. D., and Fee, J. A. (2003) "High resolution structure of the soluble, respiratory-type Rieske protein from Thermus thermophilus: Analysis and comparison". Biochemistry 42, 7303 - 7317.

(13)      Schmidt, C. L. (2004) "Rieske Iron-Sulfur Proteins From Extremophilic Organisms". J. Bioenerg. Biomem. 36, 107-113.

(14)      Carrell, C. J., Zhang, H., Cramer, W. A., and Smith, J. L. (1997) "Biological identity and diversity in photosynthesis and respiration: structure of the lumen-side domain of the chloroplast Rieske protein". Structure 5, 1613-25.

(15)      Iwata, S., Saynovits, M., Link, T. A., and Michel, H. (1996) "Structure of a water soluble fragment of the 'Rieske' iron-sulfur protein of the bovine heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5 resolution". Structure 4, 567-79.

(16)      Zu, Y., Fee, J. A., and Hirst, J. (2001) "Complete thermodynamic characterization of reduction and protonation of the bc1-type Rieske [2Fe-2S] center of Thermus thermophilus". J. Am. Chem. Soc. 123, 9906-7.

(17)      Hoke, K. R., Cobb, N., Armstrong, F. A., and Hille, R. (2003) "Electrochemical Studies of Arsenite Oxidase: An Unusual Example of a Highly Cooperative Two-Electron Molybdenum Center". Biochemistry 43, 1667-1674.

(18)      Colbert, C. L., Couture, M. M., Eltis, L. D., and Bolin, J. T. (2000) "A cluster exposed: structure of the Rieske ferredoxin from biphenyl dioxygenase and the redox properties of Rieske Fe-S proteins". Structure 8, 1267-78.

(19)      Ellis, P. J., Conrads, T., Hille, R., and Kuhn, P. (2001) "Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 and 2.03 . Structure 9, 125-32.

(20)      Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000) "Structure at 2.3 A resolution of the cytochrome bc1 complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment". Structure 8, 669-84.

(21)      Kauppi, B., Lee, K., Carredano, E., Parales, R. E., Gibson, D. T., Eklund, H., and Ramaswamy, S. (1998) "Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase". Structure 6, 571-86.

(22)      Bonisch, H., Schmidt, C. L., Schafer, G., and Ladenstein, R. (2002) "The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 resolution". J. Mol. Biol. 319, 791-805.

(23)      Leggate, E. J., and Hirst, J. (2005) "Roles of the Disulfide Bond and Adjacent Residues in Determining the Reduction Potentials and Stabilities of Respiratory-Type Rieske Clusters". Biochemistry 44, 7048-7058.

(24)      Denke, E., Merbitz-Zahradnik, T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (1998) "Alteration of the midpoint potential and catalytic activity of the rieske iron-sulfur protein by changes of amino acids forming hydrogen bonds to the iron-sulfur cluster". J. Biol. Chem. 273, 9085-93.

(25)      Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B., and Link, T. A. (1998) "Mutational analysis of residues forming hydrogen bonds in the Rieske [2Fe-2S] cluster of the cytochrome bc1 complex in Paracoccus denitrificans". Eur. J. Biochem. 255, 100-6.