Extended Data Fig. 7: EPR spectra of FeSII and the FeSII-nitrogenase mixtures.
From: Structural basis for the conformational protection of nitrogenase from O2
a, X-band EPR spectrum of chemically reduced FeSII collected at 40 K, 1 mW (black, top) and its simulated spectrum (blue, bottom) showing the S = 1/2 EPR signal of the [2Fe:2S]+-cluster with g = 2.04, 1.95, 1.89, which match those previously reported by Moshiri et al. (ref. 41). b, Overlaid X-band EPR spectra of chemically reduced FeSII (black trace) and the mixture of reduced MoFeP, reduced FeP, and oxidized FeSII (red trace) collected at 15 K, 63 µW. The difference spectrum (gray trace) demonstrates that both spectra are identical. Therefore, the EPR spectrum of the protein mixture does not have any contribution from reduced FeP. c, X-band EPR spectra (15 K) of a mixture of reduced FeP and oxidized FeSII in different incubation conditions: 15-s incubation prior to 90-s O2 exposure (top), 15-s incubation prior to 90-s O2 exposure in the presence of reduced MoFeP (middle), and 5-min incubation prior to 90-s O2 exposure (bottom). The spectra show that (1) electron transfer (ET) from FeP to FeSII can occur in the absence of MoFeP, and (2) MoFeP enhances ET between FeP and FeSII, allowing it to happen with minimal incubation time, in agreement with the formation of a ternary complex. The S = 3/2 signal in the bottom spectrum is assigned to “junk” Fe(III) which likely originates from O2 exposure of the oxidized FeP. d, The low-field region of EPR spectra (15 K) of a sample containing reduced MoFeP and oxidized FeSII (top), a separate sample containing reduced MoFeP, reduced FeP, and oxidized FeSII (middle), and the difference spectrum (grey, bottom) showing the presence of “junk” Fe(III) that originates from the O2 damage to MoFeP clusters when all three protein components are not present (i.e., the ternary complex cannot be formed).
