br Abbreviations br Acknowledgements br Introduction Dopamin

Abbreviations
Acknowledgements
Introduction Dopamine β-hydroxylase (DbH) is the enzyme catalysing the conversion of dopamine to norepinephrine, two important neurotransmitters involved in the central nervous system [1]. This enzyme has been isolated from bovine chromaffin granules as a tetramer of 290 kDa [2], and exists as both membrane-associated and soluble forms. It is a copper protein [2] that contains two Type II copper atoms per active site, which both are Cu(II) in the resting state. No crystallographic data are available to date concerning DbH. However some indications about its structure have been given by EXAFS measurements [3], and by the examination of the recent publication of peptidyl glycine α-amidating monooxygenase hydroxylating domain (PHM) structure [4]. PHM, which was shown to possess mechanistic similarities with DbH [5], [6] and a sequence resembling that of DbH [7], contains two copper atoms 11 Å distant from each other, separated by a solvent pocket. In DbH, one copper (CuA) is bound to three histidine and a water molecule, while the other (CuB) is bound to two histidine residues and one additional methionine residue in the reduced form [3]. No magnetic coupling exists between the two copper ions, suggesting a distance more than 6 Å [8]. This would exclude the possibility of a transient Cu2-bis(μ-oxo) active species, which is observed in various model compounds [9]. During catalysis, DbH copper atoms RN486 mg between Cu(II) and Cu(I), as shown by EPR measurements [10]. A commonly used reducing cofactor is ascorbate, also present in chromaffin granules, and suggested to be a cofactor of DbH in vivo. Oxygen is the second cofactor for DbH, which accepts dopamine, tyramine or 2-phenyl-ethylamine as substrates. As early as 1961, Levin and Kaufman showed that incubation of the enzyme with either reducer ascorbate in the presence of air or oxidant H2O2 was responsible for a loss of catalytic activity [11]. Kinetic measurements on the influence of inactivating species concentration onto activity have later been realised by Skotland and Ljones [12]. Up to now, no mechanistic study was able to provide an explanation for such losses of activity. Reduction of the two copper atoms results in the activation of dioxygen by DbH and the generation of a peroxo-copper intermediate at CuB, which undergoes a cleavage of the oxygen–oxygen bond [13]. As a next step, a hydrogen atom is abstracted from the substrate, before incorporation of the oxygen atom from the oxo-copper radical. The species responsible for this abstraction is not identified to date. Moreover it is not clear yet whether catalysis requires two copper atoms [14] or only one [15]. A tyrosinyl radical was proposed by Klinman and co-workers to form transiently during catalysis [13] (Scheme 1A). Therefore we decided to investigate the degradation of a tyrosine residue following inactivation by ascorbate/oxygen or H2O2. In this article, we characterise the inactivated form of the enzyme produced by reaction with H2O2 or ascorbate/oxygen. We show that inactivation of DbH by hydrogen peroxide is partially prevented by substrate tyramine. Using visible spectroscopy and the quinone staining method by Paz et al. [16], we prove that inactivated DbH contains a protein-bound quinone derivative.