Binding Interface and Electron Transfer Between Nicotine Oxidoreductase and Its Cytochrome c Electron Acceptor

Abstract

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. 2022, 298 (8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein–protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2’s surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2’s flavin to CycN’s heme occurs without the assistance of a protein-derived wire.

Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme

“…Alternatively, NMM could create a suitable hydrophobic interface near the flavin for the reaction of O 2 , as has been demonstrated to be important for some other flavoenzymes 30 – 32 . Because NMM remains bound during CycN-dependent catalysis 9 and CycN appears to inhabit a binding site distinct from the location of our mutations 28 , 33 , it is not surprising that the oxidation rate with CycN is unimpaired in our variants.…”

FhlA is a Formate Binding Protein

“…ΔfhlA::KanProtein purificationPurification of FhlA derivatives was similar toSchlensog et al (1994) with modifications[32]. Overnight cultures were subcultured in 1.5 L of protein expression media (12 g/L tryptone, 24 g/L yeast extract, 40 ml/L glycerol, 2.13 g/L K 2 HPO 4 , and 12.54 g/L KH 2 PO 4 , pH 7.2)[33] and grown at 37°C with shaking (aerobic) to an optical density (OD ) of 0.6 to 0.8 (mid-log). Cultures were then induced with 100 μM IPTG and grown overnight at 20°C.…”