Kinetics of a two-component p-hydroxyphenylacetate hydroxylase explain how reduced flavin is transferred from the reductase to the oxygenase

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 (C-1) and an oxygenase (C-2). C-1 catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH-) for use by C-2 in the hydroxylation reaction. C-1 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 C-1-bound FMN can be reduced and released to be used efficiently as the substrate for the C-2 reaction; additional FMN is not necessary. Reactions in which O-2 is rapidly mixed with solutions containing C-1-FMNH- and C-2 are very similar to those in which solutions containing O-2 are mixed with one containing the C-2-FMNH- complex. This suggests that in a mixture of the two proteins FMNH- binds more tightly to C-2 and has already been completely transferred to C-2 before it reacts with oxygen. Rate constants for the transfer of FMNH- from C-1 to C-2 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 C-1. In the absence of HPA, FMNH- dissociates from C-1 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 C-1 to C-2. Therefore, the dissociation of FMNH- from C-1 is rate-limiting in the intermolecular transfer of FMNH- from C-1 to C-2, 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 C-1 and C-2 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 C-1.