Adenosyl coenzyme and pH dependence of the [4Fe–4S]2+/1+ transition in lysine 2,3-aminomutase

5′-[N-[(3S)-3-Amino-carboxypropyl]-N-methylamino]-5′-deoxyadenosine (azaSAM), an analog of S-adenosyl-l-methionine (SAM), was used to study the cofactor-dependent reduction of the [4Fe–4S]2+ center in lysine 2,3-aminomutase to the +1 oxidation state. azaSAM has a tertiary nitrogen in place of the sulfonium center of SAM. The analog binds to lysine 2,3-aminomutase with Kds of 1.4±0.3 μM at pH 8.0 and 2.2±0.6 μM at pH 6.5. Reduction of the [4Fe–4S]2+ center in the presence of this analog gives a 10K [4Fe–4S]1+ electron paramagnetic resonance (EPR) signal similar to that seen with SAM or S-adenosyl-l-homocysteine (SAH). The pH dependence of cofactor-induced reduction was examined to determine whether ionization of the tertiary nitrogen (pKa=7.08) might affect reduction of the [4Fe–4S]2+ center. The results show similar behavior in azaSAM and SAH, demonstrating that ionization of the aza group in azaSAM does not account for pH dependence in cofactor-dependent reduction of the [4Fe–4S]2+ center. The signal shape of the low-temperature EPR signal for the [4Fe–4S]1+ center in the SAM-induced reduction displayed a pH dependence that was not observed in the azaSAM- or SAH-induced spectra. Unique features of the signal are at a maximum at the pH activity optimum of pH 8 and are diminished as the pH is lowered or raised. These features are also absent in the spectra at all pHs examined when reduction is induced by azaSAM or SAH.