Flavin oxidation in flavin-dependent N -monooxygenases: Flavin Oxidation in NMO
More Like this
-
Cryptochromes are highly conserved blue light-absorbing flavoproteins which function as photoreceptors during plant development and in the entrainment of the circadian clock in animals. They have been linked to perception of electromagnetic fields in many organisms including plants, flies, and humans. The mechanism of magnetic field perception by cryptochromes is suggested to occur by the so-called radical pair mechanism, whereby the electron spins of radical pairs formed in the course of cryptochrome activation can be manipulated by external magnetic fields. However, the identity of the magnetosensitive step and of the magnetically sensitive radical pairs remains a matter of debate. Here we investigate the effect of a static magnetic field of 500 μT (10× earth's magnetic field) which was applied in the course of a series of iterated 5 min blue light/10 min dark pulses. Under the identical pulsed light conditions, cryptochrome responses were enhanced by a magnetic field even when exposure was provided exclusively in the 10 min dark intervals. However, when the magnetic stimulus was given exclusively during the 5 min light interval, no magnetic sensitivity could be detected. This result eliminates the possibility that magnetic field sensitivity could occur during forward electron transfer to the flavin in themore »
-
Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19 F NMR in conjunction with fluorinated substrate analogs to directly measure p K a values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the p K a of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19 F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the p K a of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C 2 component of HPAH. 19 F NMR also revealed a p K a of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that bindingmore »
