Kinetics of a Two-Component p-Hydroxyphenylacetate Hydroxylase Explain How Reduced Flavin Is Transferred from the Reductase to the Oxygenase

Abstract

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). HPAH is composed of two proteins: a flavin mononucleotide (FMN) reductase (C₁) and an oxygenase (C₂). C₁ catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH^-) for use by C₂ in the hydroxylation reaction. C₁ is unique among the flavin reductases in that the substrate HPA stimulates the rates of both the reduction of FMN and release of FMNH^- from the enzyme. This study quantitatively shows the kinetics of how the C₁-bound FMN can be reduced and released to be used efficiently as the substrate for the C₂ reaction; additional FMN is not necessary. Reactions in which O₂ is rapidly mixed with solutions containing C₁−FMNH^- and C₂ are very similar to those in which solutions containing O₂ are mixed with one containing the C₂−FMNH^- complex. This suggests that in a mixture of the two proteins FMNH^- binds more tightly to C₂ and has already been completely transferred to C₂ before it reacts with oxygen. Rate constants for the transfer of FMNH^- from C₁ to C₂ were found to be 0.35 and ≥74 s^-1 in the absence and presence of HPA, respectively. The reduction of cytochrome c by FMNH^- was also used to measure the dissociation rate of FMNH^- from C₁. In the absence of HPA, FMNH^- dissociates from C₁ at 0.35 s^-1, while with HPA present it dissociates at 80 s^-1; these are the same rates as those for the transfer from C₁ to C₂. Therefore, the dissociation of FMNH^- from C₁ is rate-limiting in the intermolecular transfer of FMNH^- from C₁ to C₂, and this process is regulated by the presence of HPA. This regulation avoids the production of H₂O₂ in the absence of HPA. Our findings indicate that no protein−protein interactions between C₁ and C₂ are necessary for efficient transfer of FMNH^- between the proteins; transfer can occur by a rapid-diffusion process, with the rate-limiting step being the release of FMNH^- from C₁.