Abstract
Liver microsomal flavin-containing monooxygenase (MFMO) has been shown to exhibit a stable 4a-flavin hydroperoxide intermediate in the absence of oxygenatable substrate (Poulsen, L. L., and Ziegler, D. M. (1979) J. Biol. Chem. 254, 6449-6455; Beaty, N. B., and Ballou, D. P. (1981) J. Biol. Chem. 256, 4619-4625). The reaction of this intermediate with an assortment of substrates was studied by stopped flow techniques. The first observed spectral change is a small blue shift in the absorbance peak of the 4a-flavin intermediate. The rate of this spectral change is dependent on the concentration of the substrate. This small spectral change is succeeded by a large increase in the absorbance at 450 nm. The rate of appearance of oxidized flavin is independent of substrate concentration but does increase at higher pH. Steady state turnover rates also greater at higher pH, consistent with earlier observations that the formation of oxidized flavin is rate determining in catalysis. Upon oxygenation by MFMO, thiobenzamide and iodide each undergo a spectral change which is dependent on substrate concentration. The spectral changes corresponding to oxygenation of these substrates occur at the same rates as do the initial small spectral changes contributed by the flavin chromophore as observed with all substrates. However, no substrate tested to date shows any effect on the rate of formation of oxidized flavin. Previous work has shown MFMO to catalyze the oxygenation of a variety of nitrogen- and sulfur-containing hydrophobic compounds. Two new classes of compounds are shown here to be substrates for this enzyme. The nucleophilic anions, iodide and thiocyanate, catalyze the decomposition of the 4a-flavin hydroperoxide. Organic boronic acids (e.g. phenylboronic acid and butylboronic acid) also appear to be oxygenated with no striking differences in kinetic characteristics from those of nucleophilic substrates. These organic boronic acids are classic electrophiles and suggest that like peracids, the 4a-flavin hydroperoxide is capable of oxygenating both nucleophiles and electrophiles (Lee, J. B., and Uff, B. C. (1967) Quart. Rev. 21, 429-457).
Highlights
(MFMO) has been shown to exhibit a stable 4a-flavin catalyzes the oxygenation of a wide variety of compounds
The reaction of this intermediate with an as- using a combination of steady state (5) and single-turnover sortment of substrates was studied by stopped flow (6, 7) kinetic techniques
Stability of the 4aFlOOH"Poulsen and Ziegler (5) noted that a high steady state level of the4aF100H couldbe maintained by using a NADPH-regenerating system consisting of catalytic quantities of glucose 6-phosphate andglucose6-phosphate dehydrogenase and an amount of NADP+ approximately equimolar with MFMO
Summary
Muteriu&"FMO was isolated from pig livers as previously described (7). Prior to all spectral experiments, the enzyme was chromatographed on a G-25 gel filtration column (Pharmacia) to remove all free FAD and possible contaminating substrates. Phenylthiocarbamide (Sigma)was recrystallized 2 times from water. The rapid kinetic measurements were carried out on a stopped flow spectrophotometer described previously (7). Substrates were stored in the other syringe of the stopped flow prior to reaction. The typical reaction buffer was 50 mM potassium phosphate, pH 7.2. Studies at various conditions of pH were performed by mixing a solution of enzyme in buffer containing 2 mM potassium phosphate and 50 mM KCl, pH 6.8, with an equal volume of substrate in 100 mM buffer a t the desired pH, HEPES, Tris, Tricine, glycine and CAPS have been used as buffers for pH > 7.5. Steady state assays were carried out by measuring the rate of NADPH consumption at 340 nminairsaturated buffer in the presence of substrate and typically one p M MFMO. Steady state oxidase rates were measured in air saturated buffers with enzyme in the absence of substrate
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