Fig. 5. g-2 region X-band EPR spectra of V-nitrogenase proteins. (A) 12 K spectra of resting states (black trace for VFe^Str and magenta trace for Fe protein) and Fe protein-VFe^Str freeze-trapped during turnover under Ar (red trace) and N₂ (blue trace) (B) 5.5 K (green trace) spectra of Ar-turnover Fe protein–VFe^Str along with simulation of turnover intermediate (cyan trace) with ⁵¹V hyperfine coupling, a_iso = 110 MHz, g = [2.18, 2.12, 2.09], and isotropic EPR linewidth of 75 MHz. Samples: all samples contain 5 μM of VFe^Str protein except the Fe protein resting state sample, and 40 μM of Fe protein except the VFe^Str resting state sample. All samples made in buffer with 200 mM MOPS at pH 7.3 and an ATP-regeneration system (20 mM ATP, 20 mM MgCl₂, 1 mg mL⁻¹ BSA, and 0.4 mg mL⁻¹ creatine phosphokinase) with a final dithionite concentration at ∼20 mM. EPR conditions: as in Fig. 3 except each spectrum is the sum of 10 scans.