IDENTIFYING REDOX PARTNERS AND A ROLE IN OXIDATIVE STRESS FOR IRON-SULFUR FLAVOPROTEIN (ISF) FROM METHANOSARCINA THERMOPHILA

Open Access
- Author:
- Cruz, Francisco
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 16, 2005
- Committee Members:
- James Gregory Ferry, Committee Chair/Co-Chair
J Martin Bollinger Jr, Committee Member
Jean Elnora Brenchley, Committee Member
Squire J Booker, Committee Member
Christopher Falzone, Committee Member - Keywords:
- Iron-sulfur flavoprotein
oxidative stress
methanosarcina thermophila - Abstract:
- Abstract Iron-sulfur flavoprotein from Methanosarcina thermophila is the prototype for a widely-distributed family of proteins existing amongst distantly-related, strict anaerobes. This family traverses all three domains of life and members containing an Isf homolog usually contain multiple copies. Isf homologs contain one FMN cofactor and one 4Fe-4S cluster per monomer. Also, the 4Fe-4S cluster is ligated by a unusually compact cysteine motif. Previous works have suggested that Isf participates as an electron mediator in the fermentation of acetate to methane. Indeed, Isf is capable of reducing membrane fractions isolated from acetate-grown M. thermophila. The absolute function of Isf, however, remains enigmatic. A recent crystal structure of Isf reveals the protein to exist as a homodimeric tetramer. Isf also bears strong structural homology with the flavodoxin domain of Rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas. ROO is an oxidative-stress protein which detoxifies O2 to water. In addition to this observation, isf homologs were found in operons containing annotated oxidative stress genes. Since the function and electron acceptor(s) for Isf are unknown, various electron acceptors were tested including: O2, H2O2, O2-, cytochrome c, benzoquinone, ferricyanide, and dihydroquinone. In these studies, the ability of Isf to reduce O2, H2O2, and O2- to water was revealed. In addition, the oxidative half-reaction kinetic parameters of Isf with these compounds was determined. During these studies, new redox properties are revealed suggesting the 4Fe-4S cluster is the site of Isf reduction and the FMN is the site of Isf oxidation. The kinetic parameters obtained by the reduction of Cytochrome c and O2 were catalytically efficient enough to merit physiological consideration. A role for Isf in oxidative stress is considered as well as a role in electron-transfer mediation.