Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evidence that the feedback inhibitors adrenaline and noradrenaline are coordinated to iron

Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a non-heme iron, tetrahydrohydropterin-dependent enzyme which catalyzes the rate-limiting step in the biosynthesis of catecholamines. The highly purified bovine adrenal enzyme contains an unusual blue-green chromophore with λ(max) at around 700 nm (ε = 1.3 (mM subunit enzyme)^-1cm^-1). On excitation at 605.2 nm, resonance-enhanced Raman vibrations are observed at 454, 494, 527, 604, 635, 835, 1130, 1271, 1320, 1426, and 1476 cm^-1. The excitation profiles of the modes of 1276 and 1476 cm^-1 (from 488 to 620 nm) follow the contour of the 700 nm absorption band. The vibrations observed strongly indicate the presence of a bidentate catecholamine-Fe(III) complex in the enzyme as isolated which gives rise to the characteristic charge-transfer transitions. This is further supported by the release of 0.11 ± 0.04 mol of noradrenaline and 0.25 ± 0.06 mol of adrenaline per mol of enzyme subunit on denaturation of the enzyme. The energies of the catecholate to Fe(III) charge-transfer transitions indicate a mixture of histidines and carboxylate(s) coordinated to the iron center in tyrosine hydroxylase. At neutral pH, the enzymatic activity was inhibited more than 50% by 10 μM dopamine, noradrenaline, and adrenaline. The high affinity of the catecholamines to the nonphosphorylated form of tyrosine hydroxylase may have significance in vivo since catecholamines are potent feedback inhibitors of the enzyme.