The Reductase of 'p'-Hydroxyphenylacetate 3-Hydroxylase from 'Acinetobacter baumannii' Requires 'p'-Hydroxyphenylacetate for Effective Catalysis

Abstract

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 (C₁) and a larger oxygenase component (C₂). C₁ is a flavoprotein containing FMN, and its function is to provide reduced flavin for C₂ to hydroxylate HPA. We have shown here that HPA plays important roles in the reaction of C₁. The apoenzyme of C₁ binds to oxidized FMN tightly with a Kd of 0.006 μM at 4 °C, but with a Kd of 0.038 μM in the presence of HPA. Reduction of C₁ by NADH occurs in two phases with rate constants of 11.6 and 3.1 s⁻¹ and Kd values for NADH binding of 2.1 and 1.5 mM, respectively. This result indicates that C₁ exists as a mixture of isoforms. However, in the presence of HPA, the reduction of C₁ by NADH occurred in a single phase at 300 s⁻¹ with a Kd of 25 μM for NADH binding at 4 °C. Formation of the C₁-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°m of the C₁-HPA complex is -245 mV compared to an E°m of C₁ of -236 mV). When the C₁-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 C₁ is specifically reduced by the pro-(S)-hydride. In the reaction of reduced C₁ with oxygen, the reoxidation reaction is also biphasic, consistent with reduced C₁ 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.