Pyridoxal 5'-phosphate (PLP), in the active site of ornithine 4,5-aminomutase (OAM), forms a Schiff base with N(δ) of the d-ornithine side chain and facilitates interconversion of the amino acid to (2R, 4S) 2,4-diaminopentanoic acid via a radical-based mechanism. The crystal structure of OAM reveals that His225 is within hydrogen bond distance to the PLP phenolic oxygen, and may influence the pK(a) of the Schiff base during radical rearrangement. To evaluate the role of His225 in radical stabilization and catalysis, the residue was substituted with a glutamine and alanine. The H225Q and H225A variants have a 3- and 10-fold reduction in catalytic turnover, respectively, and a decrease in catalytic efficiency (7-fold for both mutants). Diminished catalytic performance is not linked to an increase in radical-based side reactions leading to enzyme inactivation. pH-dependence studies show that k(cat) increases with the ionization of a functional group, but it is not attributed to His225. Binding of 2,4-diaminobutyric acid to native OAM leads to formation of an overstabilized 2,4-diaminobutyryl-PLP derived radical. In the H225A and the H225Q mutants, the radical forms and then decays, as evidenced by accumulation of cob(III)alamin. From these data, we propose that His225 enhances radical stability by acting as a hydrogen bond acceptor to the phenolic oxygen, which favors the deprotonated state of the imino nitrogen and leads to greater resonance stabilization of the 2,4-diaminobutyryl-PLP radical intermediate. The potential role of His225 in lowering the activation energy barrier to mediate PLP-dependent radical rearrangement is discussed.