Adduct Formation between the Cupric Site of Phenylalanine Hydroxylase from Chromobacterium violaceum and 6,7-Dimethyltetrahydropterin

The interaction of pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum with the cofactor analogue 5-deaza-6-methyltetrahydropterin and the cofactor 6,7-dimethyltetrahydropterin (DMPH₄) has been investigated by multifrequency electron spin resonance (ESR) spectroscopy. 5-Deaza-6-methyltetrahydropterin, which lacks the N-5 nitrogen present in the pyrazine ring of DMPH₄, binds tightly to the cupric form of the enzyme; however, no changes are observed in the ESR parameters of the copper center. In contrast, the binding of DMPH₄ (or 6-methyltetrahydropterin) shifts the ESR parameters and Ay) associated with the cupric enzyme. In addition, superhyperfine transitions were resolved and assigned to hyperfine splitting from nitrogen ligands. ESR spectra of the enzyme recorded in the presence of [5-¹⁴N]DMPH₄ or [5-¹⁵N]DMPH₄ were computer simulated and found to be consistent with pterin serving as a direct donor ligand to the copper center through the N-5 position.