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 (HO), 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 HO 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.

The CYP171 enzyme is known to catalyse a key step in the steroidogenesis of mammals. The substrates progesterone and pregnenolone are first hydroxylated at the C17 position, and this is followed by cleavage of the C17-C20 bond to yield important precursors for glucosteroids and androgens. In this study, we focused on the reaction of the bovine CYP17A1 enzyme with progesterone as a substrate. On the basis of a created homology model, active-site residues were identified and systematically mutated to alanine. In whole-cell biotransformations, the importance of the N202, R239, G297 and E305 residues for substrate conversion was confirmed. Additionally, mutation of the L206, V366 and V483 residues enhanced the formation of the 16α-hydroxyprogesterone side product up to 40 % of the total product formation. Furthermore, residue L105 was found not to be involved in this side activity, which contradicts a previous study with the human enzyme.