Methionine S-oxidation in human and rabbit liver microsomes: Evidence for a high-affinity methionine S-oxidase activity that is distinct from flavin-containing monooxygenase 3

Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3 , which has a K-m value for methionine S-oxidation of approximately 4 mM, a nd exhibits high selectivity for formation of the D-diastereoisomer of meth ionine sulfoxide. The current studies provide evidence for an additional me thionine S-oxidase activity in liver microsomes. Human and rabbit Liver mic rosomes exhibited a biphasic response to methionine at concentrations rangi ng from 0.05 to 10 mM, as indicated by both Eadie-Hofstee plots and nonline ar regression. The low-affinity component of the biphasic response had II, values of approximately 3 and 5 nM for humans and rabbits, respectively, as web as high diastereoselectivity for methionine sulfoxide formation. The l ow-affinity activity in rabbit Liver microsomes was inhibited by methimazol e, S-allyl-L-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had K-m value s of approximately 0.07 and 0.01 mM for humans and rabbits, respectively, a s well as lower diastereoselectivity for methionine sulfoxide formation. Fu rther characterization of the high-affinity activity in rabbit liver micros omes indicated lack of involvement of cytochrome P450s or reactive oxygen s pecies. The high-affinity activity was inhibited 25% by potassium cyanide a nd greater than 50% by methimazole and S-allyl-L-cysteine. Mild heat treatm ent produced 85% inhibition of the low-affinity activity, but only 30% inhi bition of the high-affinity activity. Both high- and low-affinity activitie s were decreased by 85% in flavin-depleted microsomes. Because these result s suggested the additional S-oxidase activity has characteristics of an FMO , recombinant human FMO4 was evaluated as a potential catalyst of this acti vity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl -L-cysteine, with similar diastereoselectivity to the high-affinity microso mal S-oxidase; however, the K(m)values for both reactions appeared to be gr eater than 10 mM. In summary, these studies provide evidence for two micros omal methionine S-oxidase activities. FMO3 is the predominant catalyst at m illimolar concentrations of methionine. However, at micromolar methionine c oncentrations, there is an additional S-oxidase activity that is distinct f rom FMO3. (C) 1999 Academic Press.