Mechanism of p-hydroxyphenylacetate-3-hydroxylase. A two-protein enzyme.
p-Hydroxyphenylacetate-3-hydroxylase purified from Pseudomonas putida is a two-protein enzyme requiring a flavoprotein and a coupling protein for productive hydroxylation (Arunachalam, U., Massey, V., and Vaidyanathan, C. S. (1992) J. Biol. Chem. 267, 25848-25855). This paper presents information on the mechanism of the enzyme from absorbance and fluorescence stopped-flow studies. The reduction of the substrate-free flavoprotein by NADH was slow and was not altered by the presence of the coupling protein. In contrast, the coupling protein has a dramatic effect in the oxidative half-reaction. The flavoprotein when present alone, both in the absence and presence of the aromatic substrate, reacts in a second-order fashion with oxygen to form oxidized flavoprotein, with no indication of flavin-oxygen intermediates. However, an intermediate identified as the C4a-flavin hydroperoxide is stabilized when the flavoprotein-coupling protein complex reacts with oxygen in the absence of the aromatic substrate, p-hydroxyphenylacetate, and at least three flavin-oxygen intermediates, attributed to the anionic (I) and protonated (I*) forms of the flavin hydroperoxide and the hydroxyflavin (III), are formed in the oxidative half-reaction in the presence of the aromatic substrate. A reaction mechanism for the two-protein complex is proposed in which the aromatic substrate has little effect on the rate of reduction of the enzyme flavin but has strict control in the oxidative half-reaction. In this phase the flavin hydroperoxide is remarkably stable in the absence of the substrate but disappears rapidly upon encountering the aromatic substrate.
Arunachalam
U
U
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Massey
V
V
Miller
S M
SM
D001386:Azides / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010648:Phenylacetates / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology; D013050:Spectrometry, Fluorescence
D001386:Azides; D005420:Flavoproteins; D010648:Phenylacetates; C008070:4-hydroxyphenylacetic acid; D006899:Mixed Function Oxygenases; C026252:4-hydroxyphenylacetate 3-monooxygenase; D010100:Oxygen