On the reaction mechanism of phenol hydroxylase. New information obtained by correlation of fluorescence and absorbance stopped flow studies.

Catalysis by calcium ion of the reoxidation of reduced PQQ by molecular oxygen

“…Since the direct reaction with O 2 to give a covalent bond is spin forbidden, it has been proposed that the reaction of 1,5-dihydroflavin with O 2 occurs via an electron-transfer mechanism, resulting in a radical-ion pair, which collapses to form the covalent flavin-4a-hydroperoxide after spin conversion. 7,8 In this context, we have recently demonstrated that Mg 2+ catalyses the electron transfer step from 1,5-dihydroflavin anion to O 2 via the complex formation of O 2 • 2 and Mg 2+ to accelerate the overall two-electron oxidation of 1,5-dihydroflavin anion. 9 Thus, Ca 2+ may also catalyse the electron transfer step from H 2 L 1 to O 2 .…”

Unique Biochemical and Sequence Features Enable BluB To Destroy Flavin and Distinguish BluB from the Flavin Monooxygenase Superfamily

“…A second-order rate constant was estimated from the linear portion of the curve to be 2.2 × 10 6 M −1 s −1 (at 25 °C), a value comparable to rate constants for C4a-OO(H) formation in other two-component flavin monooxygenases such as p-hydroxyphenylacetate 3hydroxylase (1.1 × 10 6 M −1 s −1 at 4 °C), tryptophan halogenase (6.3 × 10 5 M −1 s −1 at 25 °C), and phenol hydroxylase (1.1 × 10 5 M −1 s −1 at approximately 4 °C). 28,39,40 This suggests that the BluB-catalyzed flavin destructase reaction begins with canonical flavin−oxygen chemistry as in flavindependent hydroxylases, and the flavin destructase activity of BluB diverges after this step. The hyperbolic nature of the rate of C4a-OO(H) formation with an increasing O 2 concentration (Figure 2D, inset) is likely not due to a technical limitation of the instrument because a rate of >700 s −1 with the D32N mutant was observed under the same experimental conditions (Figure S2).…”

Flavoenzyme-Catalyzed Oxygenations and Oxidations of Phenolic Compounds

“…In the hydroxylation step, the product 3,4dihydroxybenzoate is formed together with the flavin C(4a)-hydroxide (intermediate III). The aromatic product initially is formed in its keto isomeric form (intermediate II) [46,47] which isomerizes to give the energetically favored dihydroxy isomer. Evidence for the initial formation of the quinoid form of the aromatic product comes from experiments with 2,4-dihydroxybenzoate as the substrate.…”

“…[50] The final steps in the catalytic cycle are the elimination of water from the flavin C(4a)-hydroxide and product release. Studies with 2-fluoro-4-hydroxybenzoate [51] and with the related phenol hydroxylase [47,52] have indicated that in this step a dead-end complex can be formed between the flavin C(4a)-hydroxide form of the enzyme and the substrate leading to substrate inhibition.…”