Fig. 3: Characterization of ScNifH^Ht.

a UV–vis absorption spectra of as-isolated and air-exposed ScNifH^Ht. b Processing site (black arrow) of COX4 mitochondria targeting signal (blue) as determined by N-terminal sequencing of ScNifH^Ht. The conversion of Q to E (marked by *) could be due to deamination processes. c EPR signal of as-isolated ScNifH^Ht (190 μM) compared to NifH^Av (71.2 μM). Experimental data (black lines) and simulations (blue lines) of each protein are shown together with g values. d Schematic representation of NifH-dependent activities tested in e–h. Figure adapted from Burén et al.⁹. Copyright 2020 ACS under CC BY 4.0 http://creativecommons.org/licenses/by/4.0. e In vitro P-cluster maturation of apo-NifDK^Av present in CFE of A. vinelandii DJ77 (ΔnifH) after addition of ScNifH^Ht or NifH^Av as indicated (±). Tetrathiomolybdate was added (following step I + II) to inhibit further apo-NifDK^Av activation during the ARA (step III). Data represent mean values (n = 2 technical replicates). f NifB-co and NifB-dependent in vitro FeMo-co synthesis using ScNifH^Ht or NifH^Av. Tetrathiomolybdate was added (following step II) to inhibit further apo-NifDK^Av activation during the ARA (step III). A 20:1 molar ratio of ScNifH^Ht to NifDK^Av was used in the ARA (step III). Data represent mean values (n = 2 technical replicates (NifB-co + NifH^Av), n = 3 technical replicates (NifB-co + ScNifH^Ht), n = 4 technical replicates (NifB^Av + NifH^Av), n = 5 technical replicates (ScNifB^Mt + ScNifH^Ht), n = 3 technical replicates (ScNifB^Mt), n = 4 technical replicates (ScNifH^Ht)). Blue and red dots correspond to independent experiments. g Titration of NifDK^Av activity with ScNifH^Ht. Positive control reactions performed with NifH^Av and NifDK^Av at 40:1 molar ratio gave 1692 ± 4 units (nmol ethylene formed per min and mg of NifDK^Av). Reactions lacking NifH (negative control) gave 2.5 ± 0.8 units. Data represent mean values (n = 2 technical replicates). h ARA (red dots, left y-axis) and N₂-reduction assay (blue squares, right y-axis) using ScNifH^Ht and NifDK^Av (step III). NifH^Av was used as control. Data represent mean values (n = 4 technical replicates).