The aim of the proposed work is to use a combination of structural, biochemical and genetic approaches to study structure/function relationships in flavoproteins. The work will focus on two structurally homologous FAD-containing enzymes, putidaredoxin reductase from Pseudomonas putida and apoptosis inducing factor from mice. Putidaredoxin reductase catalyzes electron transfer from NADH to an iron-sulfur protein, putidaredoxin, in cytochrome P450cam monooxygenase. The mechanism of complex formation and electron transfer between putidaredoxin reductase and putidaredoxin is not well understood. We have recently demonstrated that putidaredoxin reductase has redox active cysteines and can function as dithiol/disulfide oxidoreductase. Since there are no putative disulfide redox centers encoded by CysXXCys or CysXXXXCys motifs characteristic for traditional disulfide reductases, the mechanism of dithiol/disulfide oxidoreduction catalyzed by putidaredoxin reductase seems to be unique and needs to be elucidated. Apoptosis inducing factor is a phylogenetically old mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus where it causes chromatin condensation and DNA fragmentation. Neither physiological function nor mechanism of apoptosis induced by this protein is known. ? ? Our research will address the question of how the structures of putidaredoxin reductase and ? apoptosis inducing factor are related to their catalytic function. Crystal structures of putidaredoxin reductase and apoptosis inducing factor will be solved, compared, and related to the catalytic properties of the enzymes. From structural interpretations, these relationships will be further probed and modified by genetic engineering and the effect of specific structural changes on catalytic function of the proteins will be assessed. The study will explain the mechanisms of electron transfer and dithiol/disulfide oxidoreduction catalyzed by putidaredoxin reductase and will provide an insight into the mechanism and function of apoptosis inducing factor. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067637-03
Application #
6938566
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
2003-08-01
Project End
2008-07-31
Budget Start
2005-08-01
Budget End
2006-07-31
Support Year
3
Fiscal Year
2005
Total Cost
$228,500
Indirect Cost
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Rinaldi, Carlo; Grunseich, Christopher; Sevrioukova, Irina F et al. (2012) Cowchock syndrome is associated with a mutation in apoptosis-inducing factor. Am J Hum Genet 91:1095-102
Miseviciene, Lina; Anusevicius, Zilvinas; Sarlauskas, Jonas et al. (2011) Redox reactions of the FAD-containing apoptosis-inducing factor (AIF) with quinoidal xenobiotics: a mechanistic study. Arch Biochem Biophys 512:183-9
Sevrioukova, Irina F; Poulos, Thomas L (2011) Structural biology of redox partner interactions in P450cam monooxygenase: a fresh look at an old system. Arch Biochem Biophys 507:66-74
Churbanova, Inna Y; Poulos, Thomas L; Sevrioukova, Irina F (2010) Production and characterization of a functional putidaredoxin reductase-putidaredoxin covalent complex. Biochemistry 49:58-67
Sevrioukova, Irina F; Poulos, Thomas L (2010) Arginines 65 and 310 in putidaredoxin reductase are critical for interaction with putidaredoxin. Biochemistry 49:5160-6
Sevrioukova, Irina F; Poulos, Thomas L; Churbanova, Inna Y (2010) Crystal structure of the putidaredoxin reductase x putidaredoxin electron transfer complex. J Biol Chem 285:13616-20
Sevrioukova, Irina F (2009) Redox-linked conformational dynamics in apoptosis-inducing factor. J Mol Biol 390:924-38
Churbanova, Inna Y; Sevrioukova, Irina F (2008) Redox-dependent changes in molecular properties of mitochondrial apoptosis-inducing factor. J Biol Chem 283:5622-31
Kuznetsov, Vadim Yu; Poulos, Thomas L; Sevrioukova, Irina F (2006) Putidaredoxin-to-cytochrome P450cam electron transfer: differences between the two reductive steps required for catalysis. Biochemistry 45:11934-44
Kuznetsov, Vadim Yu; Blair, Emek; Farmer, Patrick J et al. (2005) The putidaredoxin reductase-putidaredoxin electron transfer complex: theoretical and experimental studies. J Biol Chem 280:16135-42