Figure 1.

Effect of increasing concentrations of IscX on the enzymatic kinetics of Fe-S cluster formation on IscU. (A) Kinetics of cluster formation at increasing concentrations of IscX as measured by absorbance at room temperature. (B) Plot of the initial rates at increasing concentrations of IscX. Negative values are due to imperfect subtraction from the baseline and should be considered as zero (full inhibition). Only the initial part of the kinetics for each IscX concentration point was considered. The slope of these linear regions (for each IscX concentration) was used as the initial velocity. A molar extinction coefficient of 10.5 mM⁻¹ cm⁻¹ was assumed to convert the absorbance in concentration of Fe-S cluster produced. (C) Kinetics followed by CD. The assays were carried out using 1 μM IscS, 50 μM IscU, 250 μM Cys, 2 mM DTT, and 25 μM Fe²⁺ and increasing concentrations of IscX as indicated. For comparison, the experiment was also repeated in the presence of 5 μM CyaY. The error bars are not shown for sake of clarity but were typically within 5% or less.