¹H-NMR spectra of nonreactive substrate or amino acid substituents and interactions with paramagnetic Fe²⁺.

Fe²⁺ broadens the proton signal peaks of closely proximal molecules in solution (in this case solvent is 50 mM phosphate pH 5 in D₂O). (A) Chemical structures of glucose 6-phosphate (G6P) and methionine, with positions of resonant protons labeled. (B) Representative ¹H-NMR spectra of solutions containing 20 mM G6P + 20 mM cysteine or 20 mM 6PG + 20 mM methionine were compared with those of a solution with the key components of the reaction: 20 mM 6PG + 20 mM cysteine. Addition of 1 mM FeCl₂ (spectra in red) just slightly affected the individual species when analyzed separately, but extensively distorted the peaks of 6PG and cysteine when these were combined in the same solution, in contrast to the other 2 cases with either G6P or methionine where peak definition remained almost unaltered. This suggests a fairly specific cysteine–6PG interaction that makes them more likely to Fe²⁺ binding. Peak numbers correspond with proton labels in (A). In all cases, representative examples are shown from at least N = 3 independent experiments (for visual comparison, spectra are shown normalized to the maximum peak intensity in each case) (see S1 Data). 6PG, 6-phosphogluconate.

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