title: Rubredoxin as a paramagnetic relaxation-inducing probe.
abstract: The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c(3) was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2(1) and M18(1). The rubredoxin binding surface in the complex with cytochrome c(3) was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c(3) are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in (1)HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes.
authors: REQUIMTE/CQFB, Departamento de Química, Universidade Nova de Lisboa, Caparica, Portugal.
authors: Rui M
authors: RM
authors: Almeida
authors: Sofia R
authors: SR
authors: Pauleta
authors: Isabel
authors: I
authors: Moura
authors: José J G
authors: JJ
authors: Moura
mesh_terms: D000465:Algorithms; D001665:Binding Sites; D003198:Computer Simulation; D003574:Cytochrome c Group / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003580:Cytochromes / Q000378:metabolism; D045286:Desulfovibrio gigas / Q000201:enzymology; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry; D007553:Isotope Labeling; D016018:Least-Squares Analysis; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D009587:Nitrogen Isotopes / Q000378:metabolism; D019159:Nonheme Iron Proteins / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012416:Rubredoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017186:Titrimetry; D015032:Zinc / Q000378:metabolism
chemical_list: D003574:Cytochrome c Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D009587:Nitrogen Isotopes; D019159:Nonheme Iron Proteins; D011994:Recombinant Proteins; D012416:Rubredoxins; C017165:cytochrome c(3); D015032:Zinc