The ability to clone, express, and manipulate the sequences of DNA molecules has provided a powerful new set of tools for studying the relationship between protein structure and function. One can now study the effects of specific amino acid replacements of function and more precisely determine the specific role of individual residues in an enzyme mechanisms, stability, and folding properties of proteins. The goal of this proposal is to apply these protein engineering techniques to the study of heme enzyme structure and function. Heme proteins have provided some valuable insights into the relationship between protein structure and function for the following reason. Most well studied heme proteins (globins, c- and b-type cytochromes, peroxidases, P450) all have exactly the same heme prosthetic group yet each exhibits very different and clearly defined functions. These difference are dictated by the interaction between protein and heme much of which has been deciphered through a variety of spectral probes and x-ray crystallography. Therefore, there are some specific questions that can be asked regarding the relationship between structure and function in heme proteins. The research in this proposal will focus on yeast cytochrome c peroxidase (CCP) where the reaction mechanism with peroxides, the interaction between CCP and cyt.c, and the stability of the two CCP structural domains will be studied using mutagenesis methods. CCP was chosen for the following reasons: 1) CCP has been cloned and expressed at high levels in E. coli; 2) the crystal structure is known; 3) CCP forms a tight, 1:1 electrostatic complex with cyt.c and has provided some of the most detailed structural information on electrostatic interprotein electron transfer complexes; and 4) the PI's laboratory has had extensive experience working with CCP and related enzymes.
| Poulos, Thomas L (2014) Heme enzyme structure and function. Chem Rev 114:3919-62 |
| Tripathi, Sarvind; O'Neill, Maura J; Wilks, Angela et al. (2013) Crystal structure of the Pseudomonas aeruginosa cytoplasmic heme binding protein, Apo-PhuS. J Inorg Biochem 128:131-6 |
| Martell, Jeffrey D; Deerinck, Thomas J; Sancak, Yasemin et al. (2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol 30:1143-8 |
| Jasion, Victoria S; Doukov, Tzanko; Pineda, Stephanie H et al. (2012) Crystal structure of the Leishmania major peroxidase-cytochrome c complex. Proc Natl Acad Sci U S A 109:18390-4 |
| Jensen, Lyndal M R; Meharenna, Yergalem T; Davidson, Victor L et al. (2012) Geometric and electronic structures of the His-Fe(IV)=O and His-Fe(IV)-Tyr hemes of MauG. J Biol Inorg Chem 17:1241-55 |
| Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan et al. (2012) Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA. J Biol Chem 287:21729-40 |
| Jasion, Victoria S; Poulos, Thomas L (2012) Leishmania major peroxidase is a cytochrome c peroxidase. Biochemistry 51:2453-60 |
| Jasion, Victoria S; Polanco, Julio A; Meharenna, Yergalem T et al. (2011) Crystal structure of Leishmania major peroxidase and characterization of the compound i tryptophan radical. J Biol Chem 286:24608-15 |
| Meharenna, Yergalem T; Doukov, Tzanko; Li, Huiying et al. (2010) Crystallographic and single-crystal spectral analysis of the peroxidase ferryl intermediate. Biochemistry 49:2984-6 |
| Sundaramoorthy, Munirathinam; Gold, Michael H; Poulos, Thomas L (2010) Ultrahigh (0.93A) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: implications for the catalytic mechanism. J Inorg Biochem 104:683-90 |
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