Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-depe ndent monooxygenase that attacks a broad spectrum of phenolic compounds. Ho wever, the function of each component in catalysis is unclear. The large co mponent (HpaB) was demonstrated here to be a reduced FAD (FADH(2))-utilizin g monooxygenase, When an E. coil flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH(2) in vitro, HpaB was able to use FADH(2) and O-2 for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH(2) for 4-hydroxyphenylacetate oxidation, furt her demonstrating that HpaB is an FADH(2)-utilizing monooxygenase, FADH(2) generated by Fre was rapidly oxidized by O-2 to form H2O2 in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphen ylacetate, HpaB bound FADH(2) and transitorily protected it from rapid auto xidation by O-2. When 4-hydroxyphenylacetate was also present, HpaB effecti vely competed with O-2 for FADH(2) utilization, leading to 4-hydroxyphenyla cetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH(2) produced by Fre was mainly used by HpaB for the oxidation of 4-hydr oxyphenylacetate. At low HpaB concentrations, most FADH(2) was autoxidized by O-2, causing uncoupling. However, the coupling of the two enzymes' activ ities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in b oth bacteria and archaea. This is the first report of an FADH(2)-utilizing monooxygenase that uses FADH(2) as a substrate rather than as a cofactor.