Derivatives of Clostridium acidi-urici ferredoxin containing altered amino acid sequences. Semisynthetic synthesis, biological activity, and stability.

The semisynthetic syntheses and some properties of derivatives of Clostridium acidi-urici ferredoxin that contain amino acid deletions or replacements in the peptide chain are described. All 16 stable derivatives prepared, with the exception of Trp2]ferredoxin, were fully active as electron carriers in the two enzymatic assay systems tested: the phosphoroclastic system and the ferrodoxin-dependent reduction of cytochrome c. E1Trp1]Ferredoxin had 70% of the activity of native ferredoxin in both assay systems. The stability in aerobic solution of Ala1]ferredoxin, which had had its natural alanyl NH2-terminal residue removed and then replaced chemically, is the same as that of the native ferrodoxin (half-life of approximately 54 days). The relative stabilities of derivatives with a replacement or deletion of the NH2-terminal residue are as follows: Ala1]- greater than or equal to Phe1]-, Lys1]-, Pro1]-, Leu1]- greater than Met1]- greater than Gly1]- greater than Glu1]- greater than des-Ala1-ferrodoxin. The data indicate that a large bulky residue, but not a negatively charged residue, is tolerated in position 1 of the peptide chain and the greatly decreased stability (half-life = 1 day) of des-Ala1-ferredoxin confirms the importance of the NH2-terminal residue for the stability of the protein. The relative stabilities of derivatives containing Ala1, but including a replacement for the normal Tyr2, are as follows: Native greater than Trp2]- greater than or equal to Phe2]- greater than His2]- greater than Leu2]- greater than Pro2]ferredoxin. Gly2]- and des-Ala1-Tyr2-apoferredoxin did not form stable derivatives upon reconstitution with iron and sulfide, nor did 3-NO2-Tyr2, 30]- and Leu2,3-NO2-Tyr30]apoferredoxins. Other relatively stable and fully active derivatives prepared included: 3-NH2-Tyr30]-, 3-F-Phe2]-, and 2-F-Phe2]ferredoxin. The behavior of these various derivatives demonstrates the importance of the peptide chain for the stability of C. acidi-urici ferredoxin and shows that the activity of ferredoxin can be altered by a single amino acid substitution in the peptide chain.