Sequence-specific assignments of the 1H nuclear magnetic resonance spectra of reduced high-potential ferredoxin (HiPIP) from Chromatium vinosum.
The 1H resonances of the high-potential [4Fe-4S]2+ ferredoxin from Chromatium vinosum have been assigned through conventional sequential methodology applied to 2D NMR spectra. Almost 80% of the residues were identified using standard 2D COSY, HOHAHA, and NOESY pulse sequences. These residues correspond to four segments of the primary structure that do not interact strongly with the iron-sulfur cluster. A minor correction to the amino acid sequence is strongly suggested by these NMR data. Additional protons more sensitive to the proximity of the cluster were assigned by a combination of NOESY experiments with fast repetition rates and short mixing times and of HOHAHA spectra recorded with reduced spin-lock duration aimed at compensating for the short relaxation rates. Hence, the contributions of 79 residues out of 85 were identified in NMR spectra, among which the assignments of 64 residues were completed. Even the fastest relaxing protons, like those of the cysteine ligands, could be correlated, partly because the strong hyperfine shifts isolate them from the crowded diamagnetic region. However, other protons, in particular those involved in NH-S hydrogen bonds with the iron-sulfur cluster, were more difficult to identify, most probably because their relatively broad signals overlapped with those of protons not or less perturbed by the active site. The availability of the major part of the 1H NMR assignments has enabled the detection and identification of many interresidue NOESY cross peaks. These data are in full agreement with the elements of secondary structure previously revealed by X-ray crystallographic analysis of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Gaillard
J
J
DRFMC-SESAM-SCPM, Grenoble, France.
Albrand
J P
JP
Moulis
J M
JM
Wemmer
D E
DE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D002844:Chromatium / Q000737:chemistry*; D005288:Ferredoxins / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins
D001426:Bacterial Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; C007943:high potential iron-sulfur protein