The Role of the Unusual 2-Tyr-1-carboxylate Nonheme Iron Motif in the Mechanism of <i>N</i>,<i>N</i>-Dimethylformamidase

N,N-Dimethylformamidase (DMFase) is a nonheme iron enzyme that catalyzes the hydrolysis of N,N-dimethylformamide (DMF) using a noncanonical Fe(III)-2Tyr-1Glu coordination motif. The precise role that this nonconventional active site plays in catalysis remains poorly understood. We performed an extensive computational investigation of DMFase catalysis, combining reaction pathway analysis with quantum mechanical cluster models, charge shift analysis, and energy decomposition analysis to identify the mechanistic role of the coordinating tyrosines/glutamate and second coordination sphere residues. We compared two mechanisms initiated by the key second coordination sphere residues Glu657 and His519. While both mechanisms generate a ferric hydroxide intermediate, the Glu657-initiated mechanism exhibits more favorable barriers and thermodynamics. These calculations reveal distinct catalytic roles for the second-sphere residues: Glu657 facilitates direct proton transfers, His519 and Asn547 stabilize the rate-determining transition state, and Lys567 stabilizes the anionic tyrosinate state of Tyr440. Mechanistic comparisons to canonical Fe(II)/Fe(III)-2His-1Glu variants reveal that coordination of Fe by tyrosine residues lowers the barrier for deprotonation of a water ligand and subsequent nucleophilic attack on DMF. Attempts to tune the active site through fluorination of coordinating tyrosinate residues yield minimal additional benefits, indicating that the native motif has finely tuned electronic characteristics. These results demonstrate how the 2Tyr-1Glu motif and its second coordination sphere context enable hydrolytic reactivity in DMFase and suggest Glu657 and Lys567 as targets of future mutagenesis to validate their mechanistic roles.