Protein dynamics control proton transfers to the substrate on the His72Asnmutant of p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase (PHBH) hydroxylates activated benzoates using NADPH as a reductant and O-2 as an oxygenating substrate. Because the flav in, when reduced, will quickly react with oxygen in either the presence or absence of a phenolic substrate, it is important to regulate flavin reducti on to prevent the uncontrolled reaction of NADPH and oxygen to form H2O2 Re duction is controlled by the protonation state of the aromatic substrate p- hydroxybenzoate (pOHB), which when ionized to the phenolate facilitates the movement of flavin between two conformations, termed "in" and "out". When the hydrogen bond network that provides communication between the substrate and solvent is disrupted by changing its terminal residue, His72, to Asn, protons from solution no longer equilibrate rapidly with pOHB bound to the active site [Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Ma ssey, V. (1999) Biochemistry 38, 1153-1158]. Thus, one population of the Hi s72Asn enzyme reduces rapidly and has the phenolate form of pOHB bound at t he active site and the flavin in the out conformation. The remaining popula tion of the His72Asn enzyme reduces slowly and has the phenolic form of pOH B bound and the flavin in the in conformation. We have investigated the mec hanisms of proton transfer between solvent and pOHB bound to the His72Asn f orm of the enzyme by double-mixing and single-mixing stopped-flow experimen ts. We find that, depending on the initial ionization state of bound pOHB a nd the new pH of the solution, the ionization/protonation of pOHB proceeds through the direct reaction of hydronium or hydroxide with the enzyme-ligan d complex and leads to the conversion of one flavin conformation to the oth er. Our kinetic data indicate that the enzyme with the flavin in the in con formation reacts in two steps. Inspection of crystal structures suggests th at the hydroxide ion would react at the re-face of the flavin, and its reac tion with pOHB is limited by the movement of Pro293, a conserved residue in similar flavoprotein hydroxylases. We hypothesize that this type of breath ing mode by the protein may have been used to compensate for the lack of an efficient proton-transfer network in ancestral hydroxylases, permitting us eful catalysis prior to the emergence of specialized proton-transfer mechan isms.