Fig. 4. H-Cluster assembly monitored by FTIR and X-band EPR spectroscopy. (A) FTIR spectrum recorded on 2.5 mM [2Fe]adt-CaHydF (reproduced from Fig. 2, panel B). (B) FTIR spectrum recorded on a mixture of apo-CrHydA1 (1.5 mM) and [2Fe]adt-CaHydF (300 μM) after 60 min incubation in 25 mM Tris-HCl pH 7.0, 50 mM KCl. Color coding: Hox, red; Hox-CO, purple. (C) X-band EPR spectra. Black line: apo-CrHydA1 (200 μM) incubated with [2Fe]adt-CaHydF (30 μM) for 60 min; Red line: [2Fe]adt-CaHydF (30 μM); Purple line: apo-CrHydA1 (200 μM). (Inset): Black line: apo-CrHydA1 (200 μM) treated with [2Fe]adt-CaHydF (30 μM); Green line: a linear combination of [4Fe4S]+-CaHydF (16 μM) and apo-CrHydA1 (200 μM). All EPR samples were prepared in 100 mM Tris-HCl pH 7.0 300 mM KCl, and recorded at a microwave power of 1 mW, at 10 K, microwave frequency: 9.28 GHz.
