Apparent oxidation-reduction potential of Clostridium acidi-urici ferredoxin. Effect of pH, ionic strength, and amino acid replacements.

The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase. The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V. In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase. A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes. This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate. Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts. This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables. The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V. Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C. acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined. Under similar conditions, the Emp of most of the 13 derivatives examined, including those of [Leu2]- and[3-NH2-Tyr30]ferredoxin, is approximately the same as that of native ferredoxin. However, the Emp of [His2]ferredoxin is approximately 15 mv more positive, whereas that of [Trp2]ferredoxin is 22 mv more negative than that of native C. acidi-urici ferredoxin. Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives. It is suggested that the changes observed in the Emp of C. acidi-urici ferredoxin are caused by protein conformational changes.

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.

Synthesis and properties of Clostridium acidi-urici (Leu2)-ferredoxin: a function of the peptide chain and evidence against the direct role of the aromatic residues in electron transfer.

Tyrosyl or other aromatic residues generally occur in two conserved positions in the peptide chain of clostridial-type ferredoxins and have been implicated in the electron transfer function of these iron-sulfur proteins. We have prepared and determined some of the properties of a derivative of Clostridium acidi-urici ferredoxin, [Leu(2)]-ferredoxin, in which a leucyl residue has been substituted for the tyrosyl residue in position 2 from the amino terminus. [Leu(2)]-ferredoxin is fully active as an electron carrier in two biological assays, the phosphoroclastic enzyme system and the ferredoxin-dependent reduction of cytochrome c in the presence of ferredoxin-TPN reductase and TPNH. Quantitative electron paramagnetic resonance experiments indicate that [Leu(2)]-ferredoxin accepts nearly two electrons upon enzymatic reduction by pyruvate-ferredoxin oxidoreductase and an excess of pyruvate. If electron transfer to an iron-sulfur cluster is the rate-limiting step in the assays used, and if the rate of electron transfer through Tyr(30) is not much faster than through Tyr(2), these results indicate that the primary pathway of electron transfer in clostridial-type ferredoxins is not via Tyr or other aromatic amino-acid residues. The syntheses of other ferredoxin derivatives with amino-acid substitutions or deletions in positions 1 and 2 indicate that a large bulky residue, but not necessarily an aromatic residue, is needed in position 2 for the stability of this ferredoxin. The residue in position 2, therefore, appears to act as a hydrophobic shield for an iron-sulfur cluster.