This study uses the electrochemical tool of protein film voltammetry (PFV) to uniquely report upon the catalytic chemistry, redox properties, and activation and deactivation reactions of bacterial CCP enzymes. Bacterial organisms, like all organisms, destroy toxic hydrogen peroxide by the use of specific enzymes. In the case of bacteria, diheme peroxidases (CCPs) take electrons from cytochrome c and use them to reduce hydrogen peroxide to water. This reaction is crucial to survival for the microbes, as it defends the organism against oxidizing conditions, such as those engendered by a host's natural defense systems. In this proposal, we will study the mechanisms of electron transfer and peroxide reduction in CCPs from bacteria. Interestingly some CCPs become easily inactivated when they are fully oxidized;others do not have this trait. Bacterial CCPs seem to be homologous in sequence and structure, which makes the molecular cause for this difference amongst the peroxidases intriguing. The long-range goals of our study are to understand the molecular details that determine if a CCP reactivity, and how the redox state of a peroxidase relates to activation and inactivation. We hypothesize that there are a small number of determinants in the primary sequence of CCPs, indicating the requirements for activation. We will (1) measure the reduction potentials and electrochemical characteristics of the wild type Nitrosomonas europaea enzyme, which does not require redox-linked activation;(2) study the activation/deactivation reaction within the CCP from Paracoccus denitrificans, which is known to require activation;(3) generate an overexpression system of the Shewanella oneidensis enzyme, that will allow us to engage in site-directed mutagenesis studies;and (4) characterize a novel sub-class of triheme CCPs that have yet to be described in the literature. Biomedical impact: The proposed experiments will yield a detailed understanding of how Biology defends itself against reactive oxygen species such as hydrogen peroxide, by understanding the interplay between redox chemistry and enzyme mechanism. Further, our study of triheme CCPs will elucidate the CCP machinery which is unique to pathogens such as Salmonella enterica and Yersinia pestis, providing new insights into their biochemical pathways.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072663-04
Application #
7681465
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2006-09-01
Project End
2011-08-31
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
4
Fiscal Year
2009
Total Cost
$201,180
Indirect Cost
Name
Boston University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
049435266
City
Boston
State
MA
Country
United States
Zip Code
02215
Bak, Daniel W; Elliott, Sean J (2013) Conserved hydrogen bonding networks of MitoNEET tune Fe-S cluster binding and structural stability. Biochemistry 52:4687-96
Bewley, Kathryn D; Ellis, Katie E; Firer-Sherwood, Mackenzie A et al. (2013) Multi-heme proteins: nature's electronic multi-purpose tool. Biochim Biophys Acta 1827:938-48
Pulcu, Gokce Su; Frato, Katherine E; Gupta, Rupal et al. (2012) The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation. Biochemistry 51:974-85
Ellis, Katie E; Frato, Katherine E; Elliott, Sean J (2012) Impact of quaternary structure upon bacterial cytochrome c peroxidases: does homodimerization matter? Biochemistry 51:10008-16
Seidel, Julian; Hoffmann, Maren; Ellis, Katie E et al. (2012) MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens. Biochemistry 51:2747-56
Hamill, Michael J; Jost, Marco; Wong, Cintyu et al. (2012) Electrochemical characterization of Escherichia coli adaptive response protein AidB. Int J Mol Sci 13:16899-915
Judd, Evan T; Youngblut, Matthew; Pacheco, A Andrew et al. (2012) Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence of interactions across the dimeric interface. Biochemistry 51:10175-85
Levin, Benjamin D; Can, Mehmet; Bowman, Sarah E J et al. (2011) Methionine ligand lability in bacterial monoheme cytochromes c: an electrochemical study. J Phys Chem B 115:11718-26
Hamill, Michael J; Jost, Marco; Wong, Cintyu et al. (2011) Flavin-induced oligomerization in Escherichia coli adaptive response protein AidB. Biochemistry 50:10159-69
Ellis, Katie E; Seidel, Julian; Einsle, Oliver et al. (2011) Geobacter sulfurreducens cytochrome c peroxidases: electrochemical classification of catalytic mechanisms. Biochemistry 50:4513-20

Showing the most recent 10 out of 14 publications