Mechanistic studies of p-hydroxybenzoate hydroxylase reconstituted with 2-Thio-FAD.
2-Thio-FAD (oxygen substituent at position 2 is replaced by sulfur) was used to reconstitute the apoenzyme of p-hydroxybenzoate hydroxylase. The 2-thio-FAD enzyme differs from native enzyme in several respects. While the native enzyme catalyzes the fully coupled hydroxylation of p-hydroxybenzoate, the 2-thio-FAD enzyme shows no hydroxylation of this substrate, instead reducing molecular oxygen to hydrogen peroxide. The rate of reduction of 2-thio-FAD p-hydroxybenzoate hydroxylase by NADPH in the presence of substrate was 7-fold faster than with the native enzyme. However, the oxygen reactivity of the reduced 2-thio-FAD enzyme was less than 1% that of native enzyme. This slow oxygen reaction results in the very high KmO2 observed in steady state kinetic studies of the modified enzyme. Stopped flow studies of the oxygen reaction of the reduced 2-thio-FAD enzyme in the presence of substrate confirmed the formation of a transient intermediate. The spectrum of this intermediate is very similar to those of the flavin-C(4a) adducts obtained with 2-thio-FMN lactate oxidase. This evidence suggests that reduced 2-thio-FAD p-hydroxybenzoate hydroxylase forms a flavin-C(4a)-hydroperoxide on reaction with oxygen in a reaction analogous to that with native enzyme, but that the resulting peroxyflavin is incompetent as an oxygenating species, breaking down instead to oxidized 2-thio-FAD enzyme and hydrogen peroxide.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013997:Time Factors
D005182:Flavin-Adenine Dinucleotide; C037737:2-thio-FAD; D009249:NADP; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; D010100:Oxygen