Spectral Studies of Bovine Dopamine β-Hydroxylase

Abstract

Bovine dopamine beta-hydroxylase was examined spectroscopically for the presence of covalently bound pyrroloquinoline quinone (PQQ). Pure dopamine beta-hydroxylase had a featureless UV-visible spectrum above 300 nm. An equimolar solution of dopamine beta-hydroxylase and exogenously added PQQ (1 PQQ/active site) had a strong absorption maximum at 333 nm. Dialysis removed the added PQQ, indicating that dopamine beta-hydroxylase does not bind PQQ irreversibly. Reaction of dopamine beta-hydroxylase with 6 mM phenylhydrazine in the presence of 15 mM ascorbate caused 96% inactivation within 20 min and did not produce any spectrally detectable amounts of the phenylhydrazone adduct of PQQ, as reported by van der Meer et al. (van der Meer, R.A., Jongejan, J.A., and Duine, J.A. (1988) FEBS Lett. 231, 303-307). The peptide profile of phenylhydrazine inactivated dopamine beta-hydroxylase was monitored at 316 nm and did not reveal any peptides that might contain a PQQ-phenylhydrazone adduct. Thus, the absence of any spectrally detectable PQQ-phenylhydrazone adducts under these conditions demonstrates that the mechanism of phenylhydrazine inactivation does not involve covalent modification of PQQ at the active site of dopamine beta-hydroxylase and provides strong evidence that the native enzyme does not contain PQQ.