Time-dependent [2Fe-2S] cluster transfer from holo SyNfu to apo human FDX1 (A) and apo human FDX2 (C) recorded by CD in 50 mM HEPES, 100 mM NaCl, pH 7.5 at a 1:1 concentration ratio. Spectra were recorded every 2 min after the addition of holo SyNfu and converted to percent cluster transfer, (B) or (D), to calculate the second-order rate constant based on the concentration of the [2Fe-2S] cluster. The rate constants for cluster transfer to apo FDX1 and FDX2 were calculated to be 2015 M−1min−1 and 5642 M−1min−1, respectively. For both (A) and (C), the starting time (tinitial) is denoted by the (black) trace, and the tfinal is denoted by the (purple) trace.