Isolation and sequences of the cysteinyl tryptic peptides from the MoFe-protein of Azotobacter vinelandii nitrogenase.
The cysteinyl residues in the alpha and beta subunits of the MoFe-protein from Azotobacter vinelandii nitrogenase were radiolabeled by carboxymethylation with iodo[14C]acetic acid. The tryptic peptides from the isolated subunits were separated by ion-exchange chromatography on DEAE-Sephadex and SP-Sephadex. The radiolabeled (cysteinyl) peptides were sequenced by Edman degradation. The isolation procedure and sequences of the peptides provide a method for the identification of the potential cysteinyl ligands for the protein Fe:S and Mo:Fe:S centers. Although the cysteinyl peptides account for approximately 20% of the total sequence, little sequence homology was observed between the peptides from the two subunits. In contrast to the ferredoxins, the cysteinyl residues are not grouped within the protein. Only one peptide from the beta subunit has sequence homology with other Fe:S proteins. This peptide has 15 of 22 residues identical or conservative with residues 11 to 32 of the bacterial ferredoxins. The conservation of this region suggests that it may contain an important secondary structure unique to 4Fe:4S proteins, namely, the sequence (formula, see text) where (formula, see text) represents a variable residue. One peptide from the alpha subunit has a sequence identical with the conserved residues 7 to 11 of the bacterial ferredoxins. This alpha-peptide has the correct sequence, -Glu-Pro-Val-Ser-Cys-Val-Ser-Asp-Ser-, for the glutamyl, cysteinyl, and aspartyl residues to ligand a single 4Fe:4S cluster.
D000595:Amino Acid Sequence; D001395:Azotobacter / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D003545:Cysteine; D005288:Ferredoxins / Q000302:isolation & purification*; D007461:Iodoacetates; D008983:Molybdoferredoxin / Q000302:isolation & purification*; D009591:Nitrogenase / Q000032:analysis*; D010446:Peptide Fragments / Q000302:isolation & purification*; D014357:Trypsin
D005288:Ferredoxins; D007461:Iodoacetates; D008983:Molybdoferredoxin; D010446:Peptide Fragments; D009591:Nitrogenase; D014357:Trypsin; D003545:Cysteine