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. 2018 Feb 7;293(12):4422–4433. doi: 10.1074/jbc.RA117.001592

Figure 7.

Figure 7.

Time course of cluster transfer from potential plastidial [Fe2S2]2+ cluster donor proteins to apo AtDHAD monitored by UV-visible CD spectroscopy. All Δϵ values are based on the concentrations of [Fe2S2]2+ clusters on the donor proteins in the reaction mixture, i.e. buffer A in the absence of 1 mm DTT unless otherwise as indicated. Arrows indicated the direction of change in CD intensity with time at selected wavelengths. The thick red lines and black lines in A, B, and D–F indicate the CD spectra of DHAD replete with [Fe2S2]2+ clusters and the CD spectra of the [Fe2S2]2+ cluster donors in the reaction mixture, prior to initiating the reaction by the addition of DHAD, respectively. A, CD spectra of a reaction mixture containing AtNFU2 (42 μm in [Fe2S2]2+ clusters) and apo AtDHAD (126 μm) at 4, 8, 13, 19, 24, 36, 55, and 100 min (thin gray lines). B, CD spectra of a reaction mixture containing AtNFU2 (42 μm in [Fe2S2]2+ clusters), apo AtDHAD (42 μm), and 1 mm DTT at 4, 13, 21, 31, 41, 51, 61, 91, and 120 min (thin gray lines). C, kinetic simulations of cluster transfer from [Fe2S2]-NFU2 to apo DHAD in the absence (black line) and presence (red line) of 1 mm DTT, based on second-order kinetics and the initial concentrations of [Fe2S2] clusters on NFU2 and of apo DHAD. Percent cluster transfer in the absence of DTT was assessed at 466 nm (black circles) and simulated with a second-order rate constant of 1000 m−1 min−1. Percent cluster transfer in the presence of DTT was assessed at 620 nm (red circles) and simulated with a second-order rate constant of 1050 m−1 min−1. D, CD spectra of a reaction mixture containing AtGRXS14 (45 μm in [Fe2S2]2+ clusters) and apo AtDHAD (45 μm in monomer) at 4 and 50 min (thin gray lines). E, CD spectra of a reaction mixture containing AtGRXS16 (45 μm in [Fe2S2]2+ clusters) and apo AtDHAD (45 μm in monomer) at 4 and 50 min (thin gray lines). F, CD spectra of a reaction mixture containing AtSUFA1 (42 μm in [Fe2S2]2+ clusters) and apo AtDHAD (42 μm in monomer) at 4 and 50 min (thin gray lines).