Mammalian flavin-containing monooxygenase (FMO) as a source of hydrogen peroxide

Abstract

Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30-50% of O₂ consumed as H₂O₂ upon addition of NADPH. Addition of substrate had little effect on H₂O₂ production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H₂O₂. FMO2 S195L exhibited higher "leakage", producing much greater amounts of H₂O₂, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H₂O₂ generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H₂O₂ release. Catalase did not reduce levels of H₂O₂ with either FMO2.1 or FMO3 but inhibited H₂O₂ generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP(+) binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H₂O₂.

Keywords: Flavin-containing monooxygenase; Genetic polymorphism; Hydrogen peroxide; Oxidative stress; Pulmonary FMO2.

Figure 1. Structure of FAD during the Catalytic Cycle of Flavin-Containing Monooxygenases

Mammalian FMO and prokaryotic analogs have a catalytic cycle which first involves a rapid reduction by NADPH followed by binding of molecular oxygen and formation of the stable C4a-hydroperoxyflavin intermediate. This activated FAD was originally likened to a “cocked gun” by the late Dr. Henry Kamin, capable of reacting with any soft nucleophile (S) gaining proximity to this site. A nucleophilic attack by the substrate yielded an oxygenated product (S-O). The second atom of oxygen is then released as H₂O and the final step in the cycle is the release of NADP⁺. The breakdown of the hydroxy-FAD pseudo base and release of NADP⁺ are the slowest steps in the catalytic cycle and determine FMO turnover rate. Uncoupling (dashed line) with release of H₂O₂ is promoted if NADP⁺ binding is compromised. This figure was taken from Alfieri et al., [37] with permission. Alfieri et al., [34] “Revealing the Moonlighting Role of NADP in the Structure of a Flavin-Containing Monooxygenase”, Proceedings of the National Academy of Sciences, U.S.A. 105(18):6572-77. Reprinted with permission.

Figure 2

Figure 2A. H₂O₂ generation (closed symbols) and O₂ uptake (open symbols) monitored with an Apollo 4000 Radical Ion Analyzer in Sf9 microsomes from baculovirus expressed hFMO1 (diamonds), hFMO2.1(triangles), hFMO3 (squares) with ETU substrate (50 μM), and hFMO2.1 without substrate (circles) at pH 7.4, 37°C. Substrate was added after a 5 minute pre-incubation with 1 mM NADPH. Figure 2B. H₂O₂ yield as a percent of O₂ consumption by human FMO1 (diamonds), FMO2.1 (triangles) and FMO3 (squares) over an incubation time of 60 minutes.

Figure 3

A comparison of the time-dependent production of H₂O₂ using two methods, Amplex Red assay (top) and the Apollo dual-electrode instrument (bottom). The incubations were performed as described in Materials and Methods at pH 7.4, 37°C in the absence of substrate.

Figure 4

Top panel: O₂ consumption with time by FMO2.1, (open squares) and the N413K allelic variant (open triangles) and H₂O₂ production (closed symbols). ETU (50 μM) was added following a 5 minute pre-incubation with 1 mM NADPH and enzyme as described in Materials and Methods. Bottom panel: the percent yield of H₂O₂ as a function of O₂ consumption over time is depicted for FMO2.1 (closed squares) and for the N413K allelic variant (closed triangles).

Figure 5

H₂O₂ production by human FMO2.1, the N413K and S195L FMO2 allelic variants and FMO3. Enzymes were incubated for 30 minutes at pH 7.4, 37°C, in the absence (open bars) or presence (solid gray bars) of 75 μM ethionamide. Catalase was added to enzymes in the absence (hatched bars) or presence (solid black bars) of ethionamide. The yield of H₂O₂ was determined by the Amplex Red assay. Note: hFMO3 was measured only in the presence of substrate ± catalase.

Figure 6

A. The yield of H₂O₂ with FMO2 S195L over a 20 minute incubation (pH 7.4, 37°C) was determined with the Apollo 4000 H₂O₂ electrode in the absence (squares) or presence of 100 μM ETU (circles) or 100 μM MTS (diamonds). The results were compared to H₂O₂ production by ancestral FMO2.1 in the absence of substrate (triangles). The assay was performed with 0.1 mM NADP⁺ and an NADPH-generating system as described in Materials and Methods. B. The yield of H₂O₂ by the FMO2 S195L allelic variant measured with an Apollo electrode over a 20 minute incubation with 1 mM NADPH (pH 7.4, 37°C and no NADPH-generating system). The greatest rate of H₂O₂ production was seen in the absence of substrate and with 0.2 μM SOD (filled triangles) followed by conditions with no substrate or SOD (open triangles). The addition of 1 mM glutathione in the absence of substrate (circles) markedly reduced the yield of H₂O₂. The yield of H₂O₂ over time in the presence of 50 μM ETU (inset) was equivalent in the presence (dotted line) or absence (solid line) of SOD.

Figure 6

A. The yield of H₂O₂ with FMO2 S195L over a 20 minute incubation (pH 7.4, 37°C) was determined with the Apollo 4000 H₂O₂ electrode in the absence (squares) or presence of 100 μM ETU (circles) or 100 μM MTS (diamonds). The results were compared to H₂O₂ production by ancestral FMO2.1 in the absence of substrate (triangles). The assay was performed with 0.1 mM NADP⁺ and an NADPH-generating system as described in Materials and Methods. B. The yield of H₂O₂ by the FMO2 S195L allelic variant measured with an Apollo electrode over a 20 minute incubation with 1 mM NADPH (pH 7.4, 37°C and no NADPH-generating system). The greatest rate of H₂O₂ production was seen in the absence of substrate and with 0.2 μM SOD (filled triangles) followed by conditions with no substrate or SOD (open triangles). The addition of 1 mM glutathione in the absence of substrate (circles) markedly reduced the yield of H₂O₂. The yield of H₂O₂ over time in the presence of 50 μM ETU (inset) was equivalent in the presence (dotted line) or absence (solid line) of SOD.

Figure 7

H₂O₂ production in rat CYP3A2 supersomes (0.1 nmol containing cDNA expressed rat NADPH CYP oxidoreductase (NOR) 2.8 μmol/(min × mg protein)) incubated for 60 min with benzphetamine (200 μM). Enzyme and NADPH (1 mM) were pre-incubated in 100 mM PBS, pH 7.4 at 37°C for 2 minutes, substrate added at T = 0.

Figure 8

Baseline H₂O₂ generated with no substrate in control Sf9 microsomes (open circles) compared to CYP3A2 (closed circles), FMO2.1 (closed triangles), FMO1 (closed diamonds) and FMO3 (closed squares) Sf9 expressed microsomes.

Figure 9

Comparison of H₂O₂ generated in two separate batches of Sf9 expressed microsomes using the Apollo 4000 Radical Ion Analyzer, FMO2.1 (squares) and N413K variant (triangles), incubated with ETU (50 μM) at pH 7.4, 37°C and S195L variant (circles) with no substrate at pH 7.4, 37°C (inset).