31P nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase.
SiR-FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has been analysed by 31P-NMR spectroscopy. This protein was reported previously as a reliable simplified model for native SiR-FP [Zeghouf, M., Fontecave, M., Macherel, D., & Covès, J. (1998) Biochemistry 37, 6117-6123]. SiR-FP60 was examined in its native form, as a complex with NADP+ and after monoelectronic reduction either with NADPH or dithionite. In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe. The paramagnetic effect of added manganese was also studied. In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase. Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR-FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface. This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle.
Evrard
A
A
Laboratoire de Résonance Magnétique en Biologie Métabolique, CEA-Grenoble and Université Joseph Fourier, Grenoble, France.
Zeghouf
M
M
Fontecave
M
M
Roby
C
C
Covès
J
J
D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004227:Dithionite / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000737:chemistry; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry*; D050864:Sulfite Reductase (NADPH)
D005420:Flavoproteins; D009243:NAD; D004227:Dithionite; C043609:flavin mononucleotide semiquinone; D008345:Manganese; D009249:NADP; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D050862:Oxidoreductases Acting on Sulfur Group Donors; D050864:Sulfite Reductase (NADPH); C494314:sulfite reductase (NADPH), E coli