Simultaneous detection of NADPH consumption and H2O2 production using the Ampliflu™ Red assay for screening of P450 activities and uncoupling.

Cytochrome P450s belong to a large and diverse group of heme-containing enzymes. These monooxygenases catalyze the incorporation of a single atom of molecular oxygen into their substrate. In contrast to most other enzymes, the activity of P450 enzymes is not only dependent on substrate and cofactor availability and reaction conditions, but also depends on the coupling efficiency of the catalytic cycle itself. Through the electron transfer from NAD(P)H to the heme-center of the P450, the enzyme becomes activated and binds oxygen. The thereby generated iron-oxygen complex undergoes multiple reductive steps forming different activated oxygen species. These intermediates can decay easily, releasing the reactive oxygen species superoxide anion and hydrogen peroxide (H2O2), which can also be further reduced to water. This so-called uncoupling of the reaction cycle drains electrons from the system, which consequently does not lead to the desired product, but merely H2O2 formation with stoichiometric consumption of NAD(P)H. Hence, measuring NAD(P)H consumption only can lead to an overestimation of substrate conversion. To measure this uncoupling, we herein report a microtiter plate-based assay for the simultaneous quantification of hydrogen peroxide formation and NAD(P)H consumption using Ampliflu™ Red as reporter. This was exemplified for the P450 monooxygenase from Bacillus megaterium (P450 BM3) and five mutants, using different substrates. We demonstrate the applicability of the assay, which provides a versatile basis for a high-throughput preliminary screening of P450 enzyme libraries without the need for GC or HPLC analysis and clear indication of the extent of hydrogen peroxide uncoupling.