H2500

XAS spectra were recorded at the SUT-NANOTEC-SLRI XAS beamline (BL5.2) of the SLRI, Thailand. The synchrotron light had an electron energy of 1.2 GeV with a beam current of 80–150 mA. The X-ray light was produced from a bending magnet to obtain the energy range of 1,240–12,100 eV and a photon flux ranging from 10⁸ to 10¹⁰ photons/s/100 mA. Energy calibration was performed using a Fe foil in transmission mode. To scan the Fe K-edge XANES spectra, a Ge (220) double crystal monochromator was used to scan from −20 eV to 80 eV of E₀ at 7,112 eV, with a photon energy step of 0.2 eV. For EXAFS measurement, the photon energy scan was set up in a range of −150, –20, 30, and 13k of E₀ at 7,112 eV with a photon energy step of 5, 0.3, 0.05k eV, and a time step of 1, 3, and 5 s. The samples were measured up to five scans in fluorescent mode using a 4-element Si-drift detector. The IFEFFIT program, which incorporates Athena and Artemis (48 (link)) was used to preprocess and normalize the XANES data. Hemoglobin from bovine blood [lyophilized powder (H2500; SIGMA-ALDRICH)] was used as the Fe-porphyrin ring reference material. Subsequently, the FT-EXAFS was fitted with the structure of the Fe-porphyrin ring of the PubChem CID 139127581 with modified N6 to O atom and Fe–Mo cofactor (COD ID 4113594) of the nitrogenase enzyme.

Reductive methylation of bovine hemoglobin (Sigma-Aldrich Cat#H2500) was carried out in 1 ml reaction volumes containing 1 mg protein, 10 nmoles ³H formaldehyde (Isobio (Fleurus) 10 Ci/mmole Cat#ART-0311-1), 20 mM NaCNBH₃ (Thermo Fisher Scientific Cat#44892) and 100 mM Hepes, pH 7.5, according to the previously described protocol by Jentoft et al.^{81 (link)}. The mixture was incubated at room temperature for 24 h. The tritium-incorporation yield measured after 10% TCA precipitation varied between 7–20%. One-half of the sample was then removed and incubated with 50 mM H₂O₂ for 24 h, which caused discoloration of the light brown hemoglobin solution. The labeled hemoglobin treated or not with hydrogen peroxide was then dialyzed 3 times against 20 mM Tris–HCl, pH 7.5.
Samples (1.5 μg) of native or oxidized hemoglobin were separated in a denaturing 4–12% polyacrylamide gel and stained with the SilverQuest Silver Staining kit (Thermo Fisher Scientific Cat#LC6070). For each of the 2 conditions, a band corresponding to the 15 kDa hemoglobin of the two monomers was clearly visible and the intensities of the bands were similar.