Indirect determination of the rate of iron uptake into the apoprotein of the ribonucleotide reductase of E. coli

The second-order rate constant k(apo) for uptake of FeII by the apoprotein of Ribonucleotide Reductase R2 has been measured by letting that reaction compete with the uptake of FeII by ferrozine (rate constant k(Fz)). The rate of the FeII/ferrozine reaction was studied at high ferrozine concentrations, and an effective first-order rate constant k(Fz) for the disappearance of FeII determined in the presence of bovine serum albumin as a viscogen. Solutions of apoprotein and ferrozine in various ratios were mixed with FeII solutions in a stopped-flow apparatus, and the growth of the 562 nm FeII(ferrozine)3 absorbance monitored. Attempts to fit the data to a variety of kinetic schemes imply that uptake of the second FeII by apo is slower than uptake of the first, suggesting that the rate-determining step in the activation of R2 is a conformational change after the uptake of the first iron. The resulting value of k(apo) is 1.8(1) x 10(6) M(-1) x s(-1).