The reductase of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii requires p-hydroxyphenylacetate for effective catalysis

p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) from Acinetobacter baumannii catalyzes hydroxylation of HPA to form 3,4-dihydroxyphenylacetate. It is a two-protein system consisting of a smaller reductase component (C-1) and a larger oxygenase component (C-2). C-1 is a flavoprotein containing FMN, and its function is to provide reduced flavin for C-2 to hydroxylate HPA. We have shown here that HPA plays important roles in the reaction of C-1. The apoenzyme of C-1 binds to oxidized FMN tightly with a K-d of 0.006 mu M at 4 degrees C, but with a K-d of 0.038 mu M in the presence of HPA. Reduction of C-1 by NADH occurs in two phases with rate constants of 11.6 and 3.1 s(-1) and K-d values for NADH binding of 2.1 and 1.5 mM, respectively. This result indicates that C-1 exists as a mixture of isoforms. However, in the presence of HPA, the reduction of C, by NADH occurred in a single phase at 300 s-1 with a Kd of 25 mu M for NADH binding at 4 degrees C. Formation of the C-1-HPA complex prior to binding of NADH was required for this stimulation. The redox potentials indicate that the rate enhancement is not due to thermodynamics (E degrees(m) of the C-1-HPA complex is -245 mV compared to an E degrees(m) of C-1 of -236 mV). When the C-1-HPA complex was reduced by 4(S)-NADH, the reduction rate was changed from 300 to 30 s(-1), giving a primary isotope effect of 10 and indicating that C-1 is specifically reduced by the pro-(S)-hydride. In the reaction of reduced C-1 with oxygen, the reoxidation reaction is also biphasic, consistent with reduced C-1 being a mixture of fast and slow reacting species. Rate constants for both phases were the same in the absence and presence of HPA, but in the presence of HPA, the equilibrium shifted toward the faster reacting species.