Enhancing Effect of Nucleic Acids and Their Derivatives in the Reduction of Cytochrome c by Ferrous Ions

Abstract Additional properties of the Ferrobacillus ferrooxidans heat-stable iron-cytochrome c "reductase," in view of the doubts of its protein or enzymatic nature, were studied and compared with those of various nucleic acids and their derivatives in enhancing the reduction of cytochrome c by ferrous ions with the following results. Calf thymus deoxyribonucleic acid, salmon sperm DNA, yeast RNA, and the derivatives, RNA core and apurinic acid, enhance the reduction of cytochrome c by ferrous ions at physiological pH values (pH 5.7 to 6.0). The action of these compounds is inhibited by atebrin, reduced glutathione, p-hydroxymercuribenzoate, and cysteine, and also by several metal ions including Fe+++, Co++, Ni++, Cu++, and Zn++. The products of the reaction, namely reduced cytochrome c and ferric ions, inhibited 60 and 100% at concentrations of 8 x 10-5 m and 10-3 m, respectively. All of the above nucleic acids and derivatives showed substrate saturation curves for both reactants with Km values ranging from 10-4 m to 7.5 x 10-4 m for FeSO4·7H2O and from 1.8 x 10-5 m to 8 x 10-5 m for oxidized cytochrome c. In all of the above respects their properties resembled closely those displayed by the heat-stable iron-cytochrome c "reductase" from F. ferrooxidans. The prolonged action of DNase on calf thymus DNA and on the heat-stable iron-cytochrome c "reductase" preparation from F. ferrooxidans rendered iron-cytochrome c reducing activity dialyzable but did not destroy it. RNAase had no effect on the F. ferrooxidans preparation but rendered the iron-cytochrome c reductase activity of yeast RNA dialyzable. This evidence indicates that DNA is associated with the heat-stable preparation from F. ferrooxidans. The action of DNAase on serum bovine albumin produced no dialyzable protein, suggesting that no protease is present in the DNAase that could account for its effect. The proteolytic enzyme Pronase partially inhibited the enhancement of cytochrome c reduction by iron with calf thymus DNA and the heat-stable preparation from F. ferrooxidans, an inhibition that could be reversed by heating in a boiling water bath for 5 min followed by rapid cooling. Bovine serum albumin also produced the same heat-reversible inhibition as Pronase suggesting that the inhibitory effect of Pronase in these instances was probably due to its physical binding with the nucleic acid, thus preventing the nucleic acid enhancement of cytochrome c reduction. Treatment by phenol subsequent to Pronase action removed up to 85% of the protein associated with DNA but did not affect the iron-cytochrome c reduction enhancing activity of the nucleic acid. Sonic disintegration of a calf thymus DNA solution for increasing periods of time resulted in a large initial decrease in viscosity followed by a gradual decrease in this property. This pattern of change was closely reflected in the ironcytochrome c reduction enhancing properties of the nucleic acid. Flavin mononucleotide, triphosphopyridine nucleotide, and phosphate ions stimulated the action of nucleic acids and their derivatives as well as the heat-stable preparation from F. ferrooxidans in their capacity to enhance the reduction of cytochrome c by ferrous ions. The inhibition by atebrin could be definitely prevented or reversed by FMN and FAD. The stimulatory effects of FMN, TPN, and phosphate ions were not additive suggesting that they might all react at the same site. In view of the striking similarities of nucleic acids (and their derivatives) and the heat-stable iron-cytochrome c "reductase" (which apparently contains DNA) from F. ferrooxidans in enhancing the reduction of cytochrome c by ferrous ions, it is tentatively suggested that the activity of the latter is due to its DNA content. Whether this is of physiological significance is not certain at present.