Sequential resonance assignments of oxidized high-potential iron-sulfur protein from Chromatium vinosum.
2D NMR spectra of the high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum have been used to obtain partial resonance assignments for the oxidized paramagnetic redox state of the protein. Sequence-specific assignments were made using NOESY and COSY spectra in H2O and D2O of the following backbone segments: Asn-5-Arg-33, Glu-39-Asp-45, Gly-55-Cys-63, Gly-68-Ala-78, and Leu-82-Gly-85. NOESY spectra with a spectral width wide enough to include the hyperfine-shifted resonances revealed numerous NOE contacts between these signals and those in the main envelope of the proton spectrum. With the aid of the X-ray crystal structure [Carter, C.W., Kraut, J., Freer, S. T., Xuong, N. H., Alden, R. A., & Bartsch, R. G. (1974) J. Biol. Chem. 249, 4212], these NOEs permitted seven of the nine hyperfine-shifted signals to be assigned to three of the cysteine residues liganded to the metal cluster (Cys-43, Cys-46, and Cys-77). The other two hyperfine-shifted signals produced no detectable NOEs to other resonances in the spectrum and were tentatively assigned to the remaining cysteinyl ligand (Cys-63). These assignments, in conjunction with recent theoretical models of the electronic structure of the Fe4S4 cluster [Noodleman, L. (1988) Inorg. Chem. 27, 3677; Bertini, I., Briganti, F., Luchinat, C., Scozzafava, A., & Sola, M. (1991) J. Am. Chem. Soc. 113, 1237], indicate that the iron atoms coordinated to Cys-63 and Cys-77 are those of the mixed-valence Fe(3+)-Fe2+ pair whereas Cys-43 and Cys-46 are ligands to the Fe(3+)-Fe3+ metal pair.
Nettesheim
D G
DG
Department of Chemistry, University of Wisconsin-Milwaukee 53201.
Harder
S R
SR
Feinberg
B A
BA
Otvos
J D
JD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D002844:Chromatium / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D010455:Peptides; D014443:Tyrosine; D014364:Tryptophan; D003545:Cysteine