Electron transfer and catalysis in bacterial diheme cytochrome c peroxidases and peroxidase paralogues: developing a structure function relationship
Ellis, Katie E.
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The major source of energy within aerobic orgamsms results from the direct reduction of molecular oxygen. This four-electron four-proton coupled reaction produces toxic intermediates before producing the benign product, water. These intermediates, reactive oxygen species (ROS), are strong oxidants and with the ability to oxidize proteins and damage DNA. In gram-negative bacteria cytochrome c peroxidases are used as a regulatory mechanism in removing hydrogen peroxide, a ROS, from the organism. The diheme cytochrome c peroxidases found in bacteria catalyze the two-electron reduction of hydrogen peroxide to water with the aid of an endogenous electron donor. Two main classes of bacterial peroxidases exist based on their necessity for an electron transfer to confer activity to the enzyme. The canonical class of enzymes, like the enzyme from Pseudomonas aeruginosa, requires a prereduction step before activity is seen. The second class contains the more unique enzymes like those from Nitrosomonas europaea that are active in the as-isolated state. Here, investigations and classifications of the mechanistic chemistry of bacterial cytochrome c peroxidases from Shewanella oneidensis and Geobacter sulfurreducens and MacA a cytochrome c peroxidase paralog from Geobacter sulfurreducens are described. Properties of these enzymes are reported using various biophysical techniques. Specifically, the technique of protein film voltammetry is used to further elucidate the catalytic mechanism of each peroxidase, probing insights into rate-limiting electron transfer kinetics, protein:protein interactions, as well as conformational changes in protein structure that are involved in enzyme activation. An additional question of quaternary structure is pursued in Chapter 2 of this thesis. To date all studied bacterial diheme cytochrome c peroxidases are purified with a dimeric quaternary structure, as is the enzyme from S. oneidensis. We use biophysical techniques, including electrochemistry to investigate the necessity for this dimeric state by preparing two forms of a monomeric S. oneidensis peroxidase. We demonstrate that the monomeric enzymes have different kinetic characteristics than the native dimeric enzyme. The data support a model where the monomer can catalyze the reduction of peroxide, but with diminished efficiency associated with decreased stability of the activated forms of the bCcP enzyme.
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