Induction of all constructs resulted in yellow-brown to brown pellets. The cleared lysate was also yellow-brown to brown. Over the period of an hour the color of the solution would fade when working in an aerobic environment. The induction levels varied greatly from prep to prep. The best results were attained from fresh transformations not more than two days old. Purification by Strep-Tactin resin did not work in a preparative manner. Miniscule quantities of protein adsorbed to the column even with incubation times greater than two hours.
Here we have begun an investigation of two class II hybrid cluster proteins and their associated reductases from S. oneidensis and E. coli. Our initial efforts have a lot of room for improvement. The loss of color during the purification process leads to the hypothesis that these enzymes are less oxygen tolerant than previously reported. The loss of color in iron-sulfur proteins often corresponds to cofactor degradation. The strep-tag does not appear to be very effective with this construct and another may prove to be more effective.
In this appendix to the larger dissertation we have described the initial attempts to study hybrid cluster proteins and reductases from E. coli and S. oneidensis. While our initial goal of characterizing these enzymes and investigating their electrochemical
properties has fallen short, we have effectively laid a foundation of future constructs. From here optimization of growth condition, anaerobic purification, and possibly using different expression vectors will lead to stable loaded protein to study. We have recently acquired the E. coli ∆iscR strain from Patrik Jones’ Lab, which has been shown to help optimize expression of H2ase proteins (213). This strain has a deletion of the repressor which controls the iron-sulfur cluster maturation operon. Lacking the repressor causes the machinery to continually be produce for cluster maturation.
References
1. Imlay, J. A. (2002) How Oxygen Damages Microbes: Oxygen Tolerance and Obligate Anaerobiosis, Advances in Microbial Physiology, Vol 46 46, 111-153. 2. Imlay, J. A., and Linn, S. (1988) DNA Damage and Oxygen Radical Toxicity,
Science 240, 1302-1309.
3. Imlay, J. A., Chin, S. M., and Linn, S. (1988) Toxic DNA Damage by Hydrogen- Peroxide through the Fenton Reaction Invivo and Invitro, Science 240, 640-642. 4. Mittler, R. (2002) Oxidative Stress, Antioxidants and Stress Tolerance, Trends in
Plant Science 7, 405-410.
5. Massey, V. (1994) Activation of Molecular-Oxygen by Flavins and Flavoproteins, Journal of Biological Chemistry 269, 22459-22462.
6. Ray, P. D., Huang, B. W., and Tsuji, Y. (2012) Reactive Oxygen Species (Ros) Homeostasis and Redox Regulation in Cellular Signaling, Cellular Signalling 24, 981-990.
7. Apel, K., and Hirt, H. (2004) Reactive Oxygen Species: Metabolism, Oxidative Stress, and Signal Transduction, Annual Review of Plant Biology 55, 373-399. 8. Jang, S. J., and Imlay, J. A. (2007) Micromolar Intracellular Hydrogen Peroxide
Disrupts Metabolism by Damaging Iron-Sulfur Enzymes, Journal of Biological Chemistry 282, 929-937.
9. Kaim, W., and Wolfgang, K. (1996) Bioinorganic Chemistry : Inorganic Elements in the Chemistry of Life : An Introduction and Guide, Wiley, New York. 10. DeRosa, M. C., and Crutchley, R. J. (2002) Photosensitized Singlet Oxygen and
Its Applications, Coordination Chemistry Reviews 233–234, 351-371.
11. Brillas, E., Sirés, I., and Oturan, M. A. (2009) Electro-Fenton Process and Related Electrochemical Technologies Based on Fenton’s Reaction Chemistry, Chemical Reviews 109, 6570-6631.
12. Fenton, H. J. H. (1894) Lxxiii.-Oxidation of Tartaric Acid in Presence of Iron, Journal of the Chemical Society, Transactions 65, 899-910.
13. Haber, F., and Weiss, J. (1934) The Catalytic Decomposition of Hydrogen Peroxide by Iron Salts, Proceedings of the Royal Society of London. Series A - Mathematical and Physical Sciences 147, 332-351.
14. Ma, Z., Jacobsen, F. E., and Giedroc, D. P. (2009) Coordination Chemistry of Bacterial Metal Transport and Sensing, Chemical Reviews 109, 4644-4681.
15. Stubbe, J., and van der Donk, W. A. (1998) Protein Radicals in Enzyme Catalysis, Chemical Reviews 98, 705-762.
16. Shaik, S., Cohen, S., Wang, Y., Chen, H., Kumar, D., and Thiel, W. (2010) P450 Enzymes: Their Structure, Reactivity, and Selectivity-Modeled by Qm/Mm Calculations, Chemical Reviews 110, 949-1017.
17. Stubbe, J., Nocera, D. G., Yee, C. S., and Chang, M. C. Y. (2003) Radical Initiation in the Class I Ribonucleotide Reductase: Long-Range Proton-Coupled Electron Transfer?, Chemical Reviews 103, 2167-2201.
18. Messner, K. R., and Imlay, J. A. (2002) Mechanism of Superoxide and Hydrogen Peroxide Formation by Fumarate Reductase, Succinate Dehydrogenase, and Aspartate Oxidase, Journal of Biological Chemistry 277, 42563-42571.
19. Messner, K. R., and Imlay, J. A. (2002) In Vitro Quantitation of Biological Superoxide and Hydrogen Peroxide Generation, Superoxide Dismutase 349, 354- 361.
20. Imlay, J. A. (2008) Cellular Defenses against Superoxide and Hydrogen Peroxide, Annual review of biochemistry 77, 755-776.
21. Fridovich, I. (1995) Superoxide Radical and Superoxide Dismutases, Annual review of biochemistry 64, 97-112.
22. Chelikani, P., Fita, I., and Loewen, P. C. (2004) Diversity of Structures and Properties among Catalases, Cellular and Molecular Life Sciences 61, 192-208. 23. Smulevich, G., Jakopitsch, C., Droghetti, E., and Obinger, C. (2006) Probing the
Structure and Bifunctionality of Catalase-Peroxidase (Katg), Journal of Inorganic Biochemistry 100, 568-585.
24. Atack, J. M., and Kelly, D. J. (2007) Structure, Mechanism and Physiological Roles of Bacterial Cytochrome C Peroxidases, Advances in Microbial Physiology, Vol 52 (Poole, R. K., Ed.), pp 73-106, Academic Press Ltd-Elsevier Science Ltd, London.
25. Gajhede, M., Schuller, D. J., Henriksen, A., Smith, A. T., and Poulos, T. L. (1997) Crystal Structure of Horseradish Peroxidase C at 2.15 a Resolution, Nature Structural Biology 4, 1032-1038.
26. Poulos, T. L., Freer, S. T., Alden, R. A., Edwards, S. L., Skogland, U., Takio, K., Eriksson, B., Xuong, N., Yonetani, T., and Kraut, J. (1980) The Crystal Structure of Cytochrome C Peroxidase, The Journal of biological chemistry 255, 575-580. 27. Welinder, K. G., Mauro, J. M., and Norskovlauritsen, L. (1992) Structure of Plant
and Fungal Peroxidases, Biochemical Society Transactions 20, 337-340.
28. Moss, T. H., Ehrenberg, A., and Bearden, A. J. (1969) Mossbauer Spectroscopic Evidence for the Electronic Configuration of Iron in Horseradish Peroxidase and Its Peroxide Derivatives, Biochemistry 8, 4159-4162.
29. Finzel, B. C., Poulos, T. L., and Kraut, J. (1984) Crystal Structure of Yeast Cytochrome C Peroxidase Refined at 1.7-a Resolution, The Journal of biological chemistry 259, 13027-13036.
30. Sivaraja, M., Goodin, D. B., Smith, M., and Hoffman, B. M. (1989) Identification by Endor of Trp191 as the Free-Radical Site in Cytochrome C Peroxidase Compound Es, Science 245, 738-740.
31. Meunier, B., de Visser, S. P., and Shaik, S. (2004) Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 Enzymes, Chemical Reviews 104, 3947-3980.
32. Ellfolk, N., and Soininen, R. (1970) Pseudomonas Cytochrome C Peroxidase. I. Purification Procedure, Acta Chem Scand 24, 2126-2136.
33. Ellfolk, N., and Soininen, R. (1971) Pseudomonas Cytochrome C Peroxidase. 3. The Size and Shape of the Enzyme Molecule, Acta Chem Scand 25, 1535-1540. 34. Ronnberg, M., and Ellfolk, N. (1978) Pseudomonas Cytochrome C Peroxidase.
Initial Delay of the Peroxidatic Reaction. Electron Transfer Properties, Biochimica et biophysica acta 504, 60-66.
35. Ronnberg, M., and Ellfolk, N. (1979) Heme-Linked Properties of Pseudomonas Cytochrome C Peroxidase. Evidence for Non-Equivalence of the Hemes, Biochimica et biophysica acta 581, 325-333.
36. Ronnberg, M., and Ellfolk, N. (1975) Pseudomonas Cytochrome C Peroxidase Xi. Kinetics of the Peroxidatic Oxidation of Pseudomonas Respiratory Chain
Components, Acta Chemica Scandinavica. Series B. Organic Chemistry and Biochemistry 29, 719-727.
37. Ronnberg, M., Araiso, T., Ellfolk, N., and Dunford, H. B. (1981) The Catalytic Mechanism of Pseudomonas Cytochrome C Peroxidase, Archives of Biochemistry and Biophysics 207, 197-204.
38. Ellfolk, N., Ronnberg, M., Aasa, R., Andreasson, L. E., and Vanngard, T. (1983) Properties and Function of the Two Hemes in Pseudomonas Cytochrome C Peroxidase, Biochimica et biophysica acta 743, 23-30.
39. Pettigrew, G. W. (1991) The Cytochrome C Peroxidase of Paracoccus Denitrificans, Biochimica et biophysica acta 1058, 25-27.
40. Ellfolk, N., Ronnberg, M., and Osterlund, K. (1991) Structural and Functional Features of Pseudomonas Cytochrome C Peroxidase, Biochimica et biophysica acta 1080, 68-77.
41. Arciero, D. M., and Hooper, A. B. (1994) A Di-Heme Cytochrome C Peroxidase from Nitrosomonas Europaea Catalytically Active in Both the Oxidized and Half- Reduced States, Journal of Biological Chemistry 269, 11878-11886.
42. Shimizu, H., Schuller, D. J., Lanzilotta, W. N., Sundaramoorthy, M., Arciero, D. M., Hooper, A. B., and Poulos, T. L. (2001) Crystal Structure of Nitrosomonas Europaea Cytochrome C Peroxidase and the Structural Basis for Ligand Switching in Bacterial Di-Heme Peroxidases†, Biochemistry 40, 13483-13490. 43. Zahn, J. A., Arciero, D. M., Hooper, A. B., Coats, J. R., and DiSpirito, A. A.
(1997) Cytochrome C Peroxidase from Methylococcus Capsulatus Bath, Archives of microbiology 168, 362-372.
44. Elliott, S. J., Bradley, A. L., Arciero, D. M., and Hooper, A. B. (2007) Protonation and Inhibition of Nitrosomonas Europaea Cytochrome C Peroxidase Observed with Protein Film Voltammetry, Journal of Inorganic Biochemistry 101, 173-179.
45. Bradley, A. L., Chobot, S. E., Arciero, D. M., Hooper, A. B., and Elliott, S. J. (2004) A Distinctive Electrocatalytic Response from the Cytochrome C Peroxidase of Nitrosomonas Europaea, Journal of Biological Chemistry 279, 13297-13300.
46. Bertini, I., Cavallaro, G., and Rosato, A. (2006) Cytochrome C: Occurrence and Functions, Chemical Reviews 106, 90-115.
47. Marcus, R. A., and Sutin, N. (1985) Electron Transfers in Chemistry and Biology, Biochimica Et Biophysica Acta 811, 265-322.
48. Paoli, M., Marles-Wright, J., and Smith, A. (2002) Structure-Function Relationships in Heme-Proteins, DNA and Cell Biology 21, 271-280.
49. Winkler, J. R. (2004) Cytochrome C Folding Dynamics, Current Opinion in Chemical Biology 8, 169-174.
50. Tezcan, F. A., Winkler, J. R., and Gray, H. B. (1998) Effects of Ligation and Folding on Reduction Potentials of Heme Proteins, Journal of the American Chemical Society 120, 13383-13388.
51. Bixler, J., Bakker, G., and McLendon, G. (1992) J. Am. Chem. Soc. 114, 6938. 52. Gunner, M. R., and Honig, B. (1991) Proceedings of the National Academy of
Sciences of the United States of America 88, 9151.
53. Moore, G. R., and Pettigrew, G. W. (1990) Cytochromes C : Evolutionary, Structural, and Physicochemical Aspects, Springer-Verlag, Berlin ; New York. 54. Mao, J. J., Hauser, K., and Gunner, M. R. (2003) How Cytochromes with
Different Folds Control Heme Redox Potentials, Biochemistry 42, 9829-9840. 55. Bowman, S. E. J., and Bren, K. L. (2010) Variation and Analysis of Second-
Sphere Interactions and Axial Histidinate Character in C-Type Cytochromes, Inorganic Chemistry 49, 7890-7897.
56. Michel, L. V., Ye, T., Bowman, S. E. J., Levin, B. D., Hahn, M. A., Russell, B. S., Elliott, S. J., and Bren, K. L. (2007) Heme Attachment Motif Mobility Tunes Cytochrome C Redox Potential, Biochemistry 46, 11753-11760.
57. Ye, T., Kaur, R., Senguen, F. T., Michel, L. V., Bren, K. L., and Elliott, S. J. (2008) Methionine Ligand Lability of Type I Cytochromes C: Detection of Ligand Loss Using Protein Film Voltammetry, Journal of the American Chemical Society 130, 6682-+.
58. Massari, A. M., Finkelstein, I. J., McClain, B. L., Goj, A., Wen, X., Bren, K. L., Loring, R. F., and Fayer, M. D. (2005) The Influence of Aqueous Versus Glassy Solvents on Protein Dynamics: Vibrational Echo Experiments and Molecular Dynamics Simulations, Journal of the American Chemical Society 127, 14279- 14289.
59. Wen, X., Patel, K. M., Russell, B. S., and Bren, K. L. (2007) Effects of Heme Pocket Structure and Mobility on Cytochrome C Stability, Biochemistry 46, 2537- 2544.
60. Scott, R. A., and Mauk, A. G. (1996) Cytochrome C : A Multidisciplinary Approach, University Science Books, Sausalito, Calif.
61. Travaglini-Allocatelli, C., Gianni, S., Dubey, V. K., Borgia, A., Di Matteo, A., Bonivento, D., Cutruzzola, F., Bren, K. L., and Brunori, M. (2005) An Obligatory Intermediate in the Folding Pathway of Cytochrome C(552) from Hydrogenobacter Thermophilus, Journal of Biological Chemistry 280, 25729- 25734.
62. Russell, B. S., and Bren, K. L. (2002) Denaturant Dependence of Equilibrium Unfolding Intermediates and Denatured State Structure of Horse Ferricytochrome C, Journal of Biological Inorganic Chemistry 7, 909-916.
63. Russell, B. S., Melenkivitz, R., and Bren, K. L. (2000) Nmr Investigation of Ferricytochrome C Unfolding: Detection of an Equilibrium Unfolding Intermediate and Residual Structure in the Denatured State, Proceedings of the National Academy of Sciences of the United States of America 97, 8312-8317. 64. Wen, X., and Bren, K. L. (2005) Heme Axial Methionine Fluxion in
Pseudomonas Aeruginosa Asn64gln Cytochrome C(551), Inorganic Chemistry 44, 8587-8593.
65. Wen, X., and Bren, K. L. (2005) Suppression of Axial Methionine Fluxion in Hydrogenobacter Thermophilus Gln64asn Cytochrome C(552), Biochemistry 44, 5225-5233.
66. Zhong, L. H., Wen, X., Rabinowitz, T. M., Russell, B. S., Karan, E. F., and Bren, K. L. (2004) Heme Axial Methionine Fluxionality in Hydrogenobacter Thermophilus Cytochrome C-552, Proceedings of the National Academy of Sciences of the United States of America 101, 8637-8642.
67. Paes de Sousa, P., Pauleta, S., Simões Gonçalves, M., Pettigrew, G., Moura, I., Correia dos Santos, M., and Moura, J. (2007) Mediated Catalysis of Paracoccus Pantotrophus Cytochrome C Peroxidase by P. Pantotrophus Pseudoazurin: Kinetics of Intermolecular Electron Transfer, Journal of Biological Inorganic Chemistry 12, 691-698.
68. Creutz, C., and Sutin, N. (1974) Mechanisms of Reactions of Cytochrome-C4 Kinetics of Ligand-Binding and Oxidation-Reduction Reactions of Cytochrome-C from Horse Heart and Candida-Krusei, Journal of Biological Chemistry 249, 6788-6795.
69. Schejter, A., and Aviram, I. (1969) Reaction of Cytochrome C with Imidazole, Biochemistry 8, 149-&.
70. Schejter, A., Plotkin, B., and Vig, I. (1991) The Reactivity of Cytochrome-C with Soft Ligands, Febs Letters 280, 199-201.
71. Schejter, A., Ryan, M. D., Blizzard, E. R., Zhang, C. Y., Margoliash, E., and Feinberg, B. A. (2006) The Redox Couple of the Cytochrome C Cyanide Complex: The Contribution of Heme Iron Ligation to the Structural Stability, Chemical Reactivity, and Physiological Behavior of Horse Cytochrome C., Protein Science 15, 234-241.
72. Viola, F., Aime, S., Coletta, M., Desideri, A., Fasano, M., Paoletti, S., Tarricone, C., and Ascenzi, P. (1996) Azide, Cyanide, Fluoride, Imidazole and Pyridine Binding to Ferric and Ferrous Native Horse Heart Cytochrome C and to Its Carboxymethylated Derivative: A Comparative Study, Journal of Inorganic Biochemistry 62, 213-222.
73. Assfalg, M., Bertini, I., Dolfi, A., Turano, P., Mauk, A. G., Rosell, F. I., and Gray, H. B. (2003) Structural Model for an Alkaline Form of Ferricytochrome C, Journal of the American Chemical Society 125, 2913-2922.
74. Banci, L., Bertini, I., Liu, G. H., Lu, J., Reddig, T., Tang, W. X., Wu, Y. B., Yao, Y., and Zhu, D. X. (2001) Effects of Extrinsic Imidazole Ligation on the Molecular and Electronic Structure of Cytochrome C, Journal of Biological Inorganic Chemistry 6, 628-637.
75. Dumortier, C., Meyer, T. E., and Cusanovich, M. A. (1999) Protein Dynamics: Imidazole Binding to Class I C-Type Cytochromes, Archives of Biochemistry and Biophysics 371, 142-148.
76. Spooner, P. J. R., and Watts, A. (1991) Reversible Unfolding of Cytochrome-C Upon Interaction with Cardiolipin Bilayers .1. Evidence from Deuterium Nmr Measurements, Biochemistry 30, 3871-3879.
77. Spooner, P. J. R., and Watts, A. (1991) Reversible Unfolding of Cytochrome-C Upon Interaction with Cardiolipin Bilayers .2. Evidence from P-31 Nmr Measurements, Biochemistry 30, 3880-3885.
78. Battistuzzi, G., Borsari, M., Cowan, J. A., Ranieri, A., and Sola, M. (2002) Control of Cytochrome C Redox Potential: Axial Ligation and Protein Environment Effects, Journal of the American Chemical Society 124, 5315-5324. 79. Battistuzzi, G., Borsari, M., De Rienzo, F., Di Rocco, G., Ranieri, A., and Sola,
M. (2007) Free Energy of Transition for the Individual Alkaline Conformers of Yeast Iso-1-Cytochrome C, Biochemistry 46, 1694-1702.
80. Barker, P. D., and Mauk, A. G. (1992) Ph-Linked Conformational Regulation of a Metalloprotein Oxidation-Reduction Equilibrium: Electrochemical Analysis of the Alkaline Form of Cytochrome C, Journal of the American Chemical Society 114, 3619-3624.
81. Dopner, S., Hildebrandt, P., Rosell, F. I., and Mauk, A. G. (1998) Alkaline Conformational Transitions of Ferricytochrome C Studied by Resonance Raman Spectroscopy, Journal of the American Chemical Society 120, 11246-11255. 82. Ferrer, J. C., Guillemette, J. G., Bogumil, R., Inglis, S. C., Smith, M., and Mauk,
A. G. (1993) Identification of Lys79 as an Iron Ligand in One Form of Alkaline Yeast Iso-1-Ferricytochrome-C, Journal of the American Chemical Society 115, 7507-7508.
83. Pollock, W. B. R., Rosell, F. I., Twitchett, M. B., Dumont, M. E., and Mauk, A. G. (1998) Bacterial Expression of a Mitochondrial Cytochrome C. Trimethylation of Lys72 in Yeast Iso-1-Cytochrome C and the Alkaline Conformational Transition, Biochemistry 37, 6124-6131.
84. Rosell, F. I., Ferrer, J. C., and Mauk, A. G. (1998) Proton-Linked Protein Conformational Switching: Definition of the Alkaline Conformational Transition of Yeast Iso-1-Ferricytochrome C, Journal of the American Chemical Society 120, 11234-11245.
85. Dumortier, C., Fitch, J., Meyer, T. E., and Cusanovich, M. A. (2002) Protein Dynamics: Imidazole and 2-Mercaptoethanol Binding to the Rhodobacter Capsulatus Cytochrome C(2) Mutant, Glycine 95 Proline, Archives of Biochemistry and Biophysics 405, 154-162.