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NADPH oxidases (NOXs) are membrane enzymes whose sole function is the generation of reactive oxygen species. Humans have seven NOX isoenzymes that feature distinct functions in immune response and cell signaling but share the same catalytic core comprising a FAD-binding dehydrogenase domain and a heme-binding transmembrane domain. We previously described a mutation that stabilizes the dehydrogenase domain of a prokaryotic homolog of human NOX5. The thermostable mutant exhibited a large 19 C increase in the apparent melting temperature (app T m) and a much tighter binding of the FAD cofactor, which allowed the crystallization and structure determination of the domain holo-form. Here, we analyze the transferability of this mutation onto prokaryotic and eukaryotic full-length NOX enzymes. We found that the mutation exerts a significative stabilizing effect on the full-length NOX5 from both Cylindrospermum stagnale (app T m increase of 8 C) and Homo sapiens (app DT m of 2 C). Enhanced thermal stability resulted in more homogeneous preparations of the bacterial NOX5 with less aggregation problems. Moreover, we also found that the mutation increases the overall expression of recombinant human NOX4 and NOX5 in mammalian cells. Such a 2-5-fold increase is mainly due to the lowered cell toxicity, which leads to higher biomasses. Because of the high sequence identity of the catalytic core within this family of enzymes, this strategy can be a general tool to boost the production of all NOXs.

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Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron transfers represents an arduous task. Using extensive stopped-flow spectroscopy and kinetic simulations, Sucharitakul et al. now explore the bifurcation mechanism of the electron transfer flavoprotein EtfAB from the anaerobic gut bacterium Acidaminococcus fermentans . Strikingly, they illustrated that catalysis is orchestrated by a negatively charged radical, α-FAD, that inhibits further reductions and features an atypical inverted kinetic isotope effect. These results provide additional insight behind electron transfers that are prevalent within multienzyme governed reactions.