Protein methods, antibodies, UV–vis absorption spectrum, and electron paramagnetic resonance

Protein concentrations were measured using the BCA protein assay (PIERCE) in combination with iodoacetamide to eliminate the interfering effect of DTH⁴⁵. Colorimetric Fe determination was performed as reported⁴⁶, and the N-terminal amino acid sequences were determined by Edman degradation (Proteome Factory AG).

Antibodies used in this study and their dilutions for immunoblotting were as follows: polyclonal antibodies detecting NifU^Av (used at 1:2,000 in 5% BSA), NifS^Av (used at 1:1,000 in 5% BSA), NifH^Av (used at 1:5,000 in 5% BSA), NifM^Av (used at 1:2,000 in 5% BSA) were raised against purified preparations of the corresponding A. vinelandii proteins (generated in house). Strep-tag II (“Strep-MAB”, 2-1507-001, IBA Lifesciences, 1:2,000 in 5% BSA), Strep-Tactin conjutaged to HRP (“Strep-HRP”, 2-1502-001, IBA Lifesciences, 1:50,000 in TBS- T), GFP (sc-9996, Santa Cruz Biotechnology, 1:2,000 in 5% BSA), HSP60 (LK-2, ab59458, Abcam, 1:1,000 in 5% BSA), and Tubulin (3H3087, sc-69971, Santa Cruz Biotechnology, 1:500 in 5% BSA) specific antibodies are commercially available.

The UV–vis absorption spectra were recorded after removal of the DTH from the protein samples using PD-10 desalting columns (GE Healthcare) equilibrated with the corresponding protein buffer wihtout DTH. DTH-free protein samples were then diluted in the same buffers and transferred to a Q6 spectroscopy cuvettes with sealing closures. Absorption (280 nm to 800 nm) was recorded using a UV-2600 spectrophotometer (Shimadzu).

EPR measurements were performed in a Bruker E500 spectrometer equipped with a resonator operating in the TE₁₀₂ mode at 9.47 GHz. Temperature was set and stabilized to 10 K by an Oxford temperature controller regulating a gas-flow cryostat refrigerated with helium. For measurements, a microwave power of 2.5 mW and a magnetic field modulation amplitude of 1 mT was used. Experimental conditions were carefully monitored to avoid over-modulation or saturation effects. Simulations of the EPR spectra were performed using the Matlab toolbox Easyspin⁴⁷.