| The degradation of phenols is important for the global carbon cycle and the detoxification of waste created by human activities. Phenol hydroxylase (PHHY) is an FAD-dependent monooxygenase that hydroxylates phenols at the ortho-position with the consumption of NADPH and O 2. The product, catechol, is metabolized further and eventually enters the citric acid cycle. The crystal structure of PHHY reveals two conformations of bound FAD. In one, the flavin is exposed to NADPH for reduction, while in the second, the flavin is buried in the protein for the hydroxylation of phenols. Pro364 and Tyr289 have been highlighted as critical to enzyme function on the basis of quantum and molecular mechanics calculations. This thesis describes detailed studies on specific mutant forms of PHHY (particularly Pro364Ser and Tyr289Phe) designed to test these proposals. It was shown that the mutation of Pro364 to Ser reduces the efficiency of phenol hydroxylation by 87%, probably due to the loss of stability of a transient intermediate involved in hydroxylation. In contrast, the mutation has little influence on the reaction of oxygen with resorcinol as substrate. Pro364 might not be directly involved in the reaction of resorcinol, because it is probably hydroxylated at a carbon atom distant from the residue. Mutation of Tyr289 to Phe results in a dramatic decrease in the rate of reduction of the enzyme by NADPH. It is concluded that the mutation disrupts a hydrogen bond between Tyr289 and the "exposed" FAD, and therefore destabilizes the flavin conformation favorable for reduction. However, the hydroxylation of phenols is hardly affected by the mutation, despite theoretical predictions. The enzyme-bound flavin conformations were also investigated by the photoreaction of 6-azido-FAD PHHY. Irradiation of the azido group leads to a reactive nitrene, which can insert into nearby covalent bonds and, thereby serve as a probe of the flavin environment. For substrate-free PHHY, 67% of the total flavin is cross-linked to the protein in the photoreaction, while the covalent linkage is only 15% in the presence of phenols. The binding of phenols must favor the "FAD exposed" conformation where the photoreactive flavin is not in the vicinity of any protein residues and therefore reacts with solvent. This result is consistent with the fact that PHHY is only reduced efficiently with its phenolic substrate bound, when the "exposed FAD" is the dominant conformation. |
