Biochemical, spectroscopic, and thermodynamic properties of flavodiiron proteins.
The flavodiiron proteins (FDPs), present in Archaea, Bacteria, and some protozoan pathogens (mostly anaerobes or microaerophiles), have been proposed to afford protection to microbes against nitric oxide and/or oxygen (toxic for anaerobes). The structural prototype of this protein family is a homodimer assembled in a "head-to-tail" configuration, with each monomer being composed of two domains: an N-terminal metallo-beta-lactamase module harboring a nonheme diiron center (active site of NO/O(2) reduction) and a C-terminal flavodoxin module, where a flavin mononucleotide moiety is embedded. Several FDPs bear C-terminal extra domains, which influence the composition of the respective electron transfer chains that couple NAD(P)H oxidation to NO/O(2) reduction. Herein are described methodologies employed to successfully produce, isolate, and characterize fully operative recombinant flavodiiron proteins. Spectroscopic techniques, namely absorption (visible and near-ultraviolet) and electron paramagnetic resonance spectroscopies, allowed redox-sensitive spectral fingerprints to be obtained, used further in the functional characterization of isolated flavodiiron proteins. Altogether, these studies on pure proteins contribute to understanding the molecular determinants that govern the in vivo function of the FDPs.
Vicente
João B
JB
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Justino
Marta C
MC
Gonçalves
Vera L
VL
Saraiva
Lígia M
LM
Teixeira
Miguel
M
D003001:Cloning, Molecular; D044927:Electron-Transferring Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D008954:Models, Biological; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D013057:Spectrum Analysis; D013816:Thermodynamics
D044927:Electron-Transferring Flavoproteins; D011994:Recombinant Proteins; D007501:Iron