Reduction and oxidation of the active site iron in tyrosine hydroxylase: Kinetics and specificity

Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 ± 0.1 mM^-1 s^-1. 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 ± 0.1 mM ^-1 s^-1 and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated K_m for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH₄ does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM^-1 s^-1. S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.