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. Author manuscript; available in PMC: 2022 Nov 16.
Published in final edited form as: Nat Catal. 2022 May 16;5(5):443–454. doi: 10.1038/s41929-022-00782-7

Fig. 2. EPR and GC-MS analyses of various Av1 protein species.

Fig. 2.

Shown are the perpendicular (a-e) and parallel (f-j) mode EPR spectra of the resting-state Av1 (a, f), the N2-bound Av1* (b, g), the dithionite-reactivated Av1*(TOD) (c, h), the Eu(II)-EGTA/sulphite-reactivated Av1*(TOS) (d, i), and the Eu(II)-EGTA/selenite-reactivated Av1*(TOSe) (e, j). Reactivation of Av1* with dithionite, Eu(II)-EGTA/sulphite, or Eu(II)-EGTA/selenite was achieved under turnover conditions in the presence of Av2 and ATP. The perpendicular-mode spectra (a-e) were recorded at 10K (black), 15K (red), 20K (green) and 30K (blue) using a microwave power of 20 mW. Previously unobserved features appeared in the S = 1/2 region of the spectrum of Av1* at g = 2.22 (maximized at 10K) and g = 2.01 and 1.88 (maximized at 30K), respectively (b, inset, 4x enlarged). The relative intensities of the M-cluster-specific S = 3/2 signals are indicated, with the signal intensity of Av1 set at 100%. The parallel-mode spectra (f-j) were recorded at 10K using a microwave power of 50 mW. The relative intensities of the POX-specific signal at g = 11.8 are indicated, with the signal intensity of Av1* set at 100%. (k-o) GC-MS analysis of 15N2 released upon acid quenching of Av1 (k), Av1* (l), Av1*(TOD) (m), Av1*(TOS) (n), and Av1*(TOSe) (o). 15N2 was traced at m/z = 30.