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A cytochrome c is the natural electron acceptor for nicotine oxidoreductase

An Author Correction to this article was published on 04 February 2021

This article has been updated


Nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, is of medical relevance as it shows potential as a therapeutic to aid cessation of smoking due to its ability to oxidize nicotine into a non-psychoactive metabolite. However, the use of NicA2 in this capacity is stymied by its dismal O2-dependent activity. Unlike other enzymes in the amine oxidase family, NicA2 reacts very slowly with O2, severely limiting its nicotine-degrading activity. Instead of using O2 as an oxidant, we discovered that NicA2 donates electrons to a cytochrome c, which means that NicA2 is actually a dehydrogenase. This is surprising, as enzymes of the flavin-containing amine oxidase family were invariably thought to use O2 as an electron acceptor. Our findings establish new perspectives for engineering this potentially useful therapeutic and prompt a reconsideration of the term ‘oxidase’ in referring to members of the flavin-containing amine ‘oxidase’ family.

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Fig. 1: NicA2 is rapidly reduced by nicotine, but slowly re-oxidizes with O2.
Fig. 2: A cycN knockout is unable to grow on or degrade nicotine.
Fig. 3: CycN is reduced by NicA2.
Fig. 4: NicA2 is rapidly oxidized by CycN.
Fig. 5: Proposed kinetic mechanism for the catalytic cycle of NicA2.

Data availability

All data used in this study are included in this published Article and its Supplementary Information files and are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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We thank B.A. Meinen for performing the analytical ultracentrifugation experiments. This work was supported, in part, by a Western Michigan University Faculty Research and Creative Activities Award to F.S. J.C.A.B. is an HHMI investigator.

Author information

Authors and Affiliations



M.D. performed the aerobic in vitro experiments and the in vivo experiments. C.T.C. performed all the stopped-flow experiments. All authors analyzed the data. J.C.A.B. and F.S. supervised the study. M.D., J.C.A.B. and F.S. wrote the manuscript.

Corresponding authors

Correspondence to James C. A. Bardwell or Frederick Stull.

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Competing interests

M.D., J.C.A.B. and F.S. are named on a provisional patent application partially based on the results of this work.

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Extended data

Extended Data Fig. 1 Reduction of NicA2 by nicotine, absorbance traces and intermediates.

a, Oxidized NicA2 was rapidly mixed with 0.2 mM nicotine and absorption of its flavin cofactor monitored using a CCD detector. The two intermediates detectable in reaction traces at 450 nm were maximally populated at 3.2 and 300 ms, and the absorbance spectra at these two points is shown. b, Absorbance trace overlay at 450 nm from stopped-flow experiments where NicA2 was reduced by rapid mixing with various concentrations of nicotine. Traces at all nicotine concentrations extrapolated back to the absorbance of NicA2-Flox at time zero, indicating that no observable kinetic events were missed within the dead time of the stopped-flow instrument. Note the logarithmic timescale. c, Partial reduction of oxidized NicA2 with sodium dithionite produced a species with an increased absorbance from 525–650 nm. The spectrum of the titration point with the highest absorbance in this region is most consistent with a mixed population of oxidized flavin, flavin hydroquinone, and neutral flavin semiquinone19. Further titration with sodium dithionite resulted in complete reduction to the hydroquinone (FADH2) state. NicA2-Flox, NicA2 containing oxidized FAD; NicA2-Flred, NicA2 containing FADH2; NicA2-FlSQ, NicA2 containing flavin semiquinone. d, NicA2-Flox was reduced by titration of one molar equivalent of nicotine, resulting in reduction of the flavin cofactor as monitored by absorbance. Additionally, a charge transfer band developed in the region of 500–700nm over the course of the titration, likely indicating that at least some amount of N-methylmyosmine product remains bound to NicA2 after reduction of its flavin.

Extended Data Fig. 2 NicA2 is a dimer in solution.

Previous work has described NicA2 as a monomer using size exclusion chromatography14,15. To determine the quaternary structure of NicA2 in our buffer conditions, a solution of NicA2 at 20 μM (monomer concentration) was subjected to analysis by sedimentation velocity AUC at a rotor speed of 44,000 rpm while monitoring 450 nm. Data were analyzed using UltraScan 4, version 4.0. One species predominated in solution, with a sedimentation coefficient of ~5.45 and an apparent molecular weight of ~115 kDa. The expected molecular weight for the NicA2 monomer is 53.13 kDa, indicating that NicA2 is a homodimer in solution under these conditions. This experiment was independently repeated twice with similar results.

Extended Data Fig. 3 NicA2 rapidly forms a complex when titrated with the non-catalytic nicotine analog myosmine.

a, Tryptophan fluorescence was used to quantify binding of myosmine as previously performed9. Traces from a stopped-flow experiment where oxidized NicA2 was mixed with varying concentrations of myosmine demonstrate a rapid binding event, occurring within the dead time (1 ms) of the instrument. b, Averaged fluorescence values of 5 traces per myosmine concentration were fit to determine the Kd for myosmine binding at 268 ± 3 μM (s.e.m.).

Extended Data Fig. 4 NicA2 is slowly re-oxidized by O2 in the presence and absence of N-methyl-myosmine.

a, Absorbance traces from stopped-flow experiments where NicA2, first reduced with dithionite, was then rapidly mixed with variable concentrations of O2. b, NicA2 was reduced with an equimolar amount of nicotine, and then rapidly mixed with O2 in a stopped-flow experiment which was monitored via the CCD detector. Inset: following the absorbance at 450 nm over time in this experiment, re-oxidation was very slow, similar to the behavior of dithionite reduced NicA2. NMM-NicA2-Flox, N-methylmyosmine bound NicA2 containing oxidized flavin; NMM-NicA2-Flred, N-methylmyosmine bound NicA2 containing reduced flavin. c, Absorbance traces from stopped-flow experiments where NicA2, first reduced with nicotine, resulting in NMM-NicA2-Flred, was then rapidly mixed with variable concentrations of O2. d, kobs values for the re-oxidation of NMM-bound NicA2 were plotted against the concentration of O2, demonstrating linear dependence. e, The absorbance spectrum of NicA2-Flox (yellow) was compared in two conditions. In one case (red dashed line), nicotine was added to 40 μM end concentration, and the reaction allowed to proceed for 30 minutes until complete re-oxidation of the flavin. In the other case (black solid line) pseudooxynicotine was added to an end concentration of 40 μM and the spectrum taken immediately. NicA2-Flox, NicA2 containing oxidized flavin; PON, pseudooxynicotine. These experiments were independently repeated twice with similar results. Values reported are the mean ± s.e.m. of the fit.

Extended Data Fig. 5 CycN is most closely related to cytochromes from other nicotine degrading species.

CycN (highlighted in red) was used as the template for an NCBI BLAST homology search. The sequences with highest homology were collected, and identical sequences removed. A tree was generated using the NGPhylogeny web server with default settings47, then formatted into a figure using the Interactive Tree of Life (iTOL)48. Notably, the cytochrome c from Pseudomonas sp. HZN649 (highlighted in red) is not included in the NCBI database, but was added to the sequence set after manual review of that organisms genome. It appears that other known nicotine degrading organisms also contain cytochromes c similar to CycN, suggesting that they use a similar electron transfer pathway49,50. Sequence analyses of CycN related sequences was complicated by the fact that there is relatively poor annotation of these proteins in nicotine degrading organisms. For example, manual review of the pyrrolidine-pathway nicotine degrading bacteria Pseudomonas sp. HZN6 revealed an unannotated cytochrome c homologous protein just downstream of nicotine oxidoreductase. This is the same genomic architecture as for P. putida S16. This poor annotation led us to manually review other nicotine degrading organism’s genomes, in which we identify a consistent pattern. In organisms that use the pyrrolidine pathway, like P. putida S16, there are nicotine oxidoreductase enzymes similar to NicA2 with neighboring cytochrome-c proteins. In those that metabolize nicotine via the pyridine pathway, there do not appear to be protein-based electron acceptors in the region of their nicotine degrading enzymes. For variant pyrrolidine/pyridine pathway (VPP) organisms, these do not appear to have cytochromes c, but often have pseudoazurin proteins in their nicotine degrading genomic islands. Pseudoazurins are able to participate in a range of electron transport reactions in the periplasm of bacteria51, though it is unclear if they could serve this role for flavin dependent amine oxidases in these organisms.

Extended Data Fig. 6 NicA2 and CycN’s interaction.

a, Oxidized NicA2 was incubated with nicotine under aerobic conditions in the presence (black filled circles) and absence (red filled circles) of oxidized CycN, and the amount of H2O2 produced by the reaction was monitored using the Amplex Red assay. Also included were conditions of NicA2 without nicotine (black empty circles) and with CycN but without nicotine (red empty circles). Only in the condition where NicA2 was incubated with nicotine in the absence of CycN was a significant amount of H2O2 produced. Three independent replicates were obtained and plotted. b, Oxidized CycN and reduced NicA2 were combined in an anaerobic stopped-flow spectrophotometer and observed for change in absorbance at 542 nm. When mixed in equimolar amounts (black points), absorbance rose and was maintained at an increased value indicating transition to the flavin semiquinone state. When mixed with excess CycN (blue points), NicA2 first reaches the semiquinone state (observable as an increase in absorbance) before becoming fully oxidized (observable as a subsequent decrease in absorbance). c, 40 µM CycN alone (black dashed line) or 40 µM CycN with 200 µM NicA2 (blue line) were run over a HiLoad Superdex 75 pg size exclusion column. CycN in the presence of excess NicA2 eluted with the same retention time as CycN alone and was well-resolved from the NicA2 peak. The insets show the absorbance spectrum of the two peaks in the chromatogram. Fractions 26 and 39 have spectra consistent with clean NicA2 and CycN, respectively, indicating that the two proteins do not bind with high affinity.

Extended Data Fig. 7 Reduction of CycN by dithionite.

UV-VIS spectra were recorded as sodium dithionite was serially titrated into a solution of oxidized CycN until it became fully reduced. Arrows represent the directionality of change during the titration. Inset: zooming in on just a small section of this titration, an isosbestic point is visible at 542 nm marked with an arrow. This wavelength was used to monitor the changes in absorbance for NicA2’s FAD in the experiments in Fig. 4.

Extended Data Fig. 8 Kinetic model for NicA2 oxidation by CycN.

In a two-step mechanism where rate-limiting CycNox binding to NicA2-Flred is followed by rapid electron transfer between the two redox centers, kobs should be linearly dependent on [CycNox] at low CycNox concentrations and should saturate at the sum of the rate constants for electron transfer (ke–forward + ke–reverse) at high CycNox concentrations52. Our data indicate that we have used CycNox concentrations at the low end of this concentration regime, where kobs increases linearly with CycNox concentration with a slope equal to kon for CycNox binding to NicA2-Flred. We presume that kobs would eventually reach a saturating value at high CycNox concentrations; however, we are not able to produce enough CycNox to explore the mM concentrations of CycNox that would be needed to achieve saturation. Notably, the rate constant for CycNox dissociation from NicA2-Flred (koff) should not contribute to the y-intercept of the kobs plot for the mechanism shown in this figure52. The kobs for the second phase also increased linearly with CycNox concentration, indicating that the above logic also applies for the reaction of the second CycNox with NicA2-FlSQ. This finding also indicates that CycNred resulting from the first one-electron transfer must dissociate from NicA2-FlSQ fast enough such that the second one-electron transfer event is also rate-limited by CycNox binding. In the kinetic scheme, the labels NicA2-Flred/SQ and NicA2-FlSQ/ox indicate that NicA2-Flred conversion to NicA2-FlSQ and NicA2-FlSQ conversion to NicA2-Flox are observed in the first and second phases, respectively.

Extended Data Fig. 9 CycN stopped-flow data.

a, Absorbance traces for the stopped flow reaction between ligand-free NicA2-Flred with variable concentrations of CycNox. In this case, the traces were able to capture formation and depletion of NicA2-FlSQ that occurred as the reaction proceeded. CycN contributes a substantial amount of absorbance at 542 nm. Accordingly, the traces were adjusted so that they all end at the same absorbance value to facilitate comparison. Note the logarithmic timescale. b, Signal change for the stopped-flow reaction was also monitored at 552 nm, a wavelength suitable for observing reduction of CycN. The trace required two exponentials (red curve) with similar amplitudes to fit properly, as one exponential (blue curve) was insufficient. Signal change occurred at the same time as NicA2 oxidation monitored at 542 nm, indicating that the processes occurred simultaneously. Note the logarithmic timescale. c, Signal change at 542 nm for the reaction of NMM-NicA2-Flred with CycNox. Curiously, traces at 542 nm, where NicA2-FlSQ is detectable, did not show the increase in absorbance that we observed with ligand-free NicA2-Flred; traces at this wavelength showed a decrease in absorbance that occurred in two phases, with the first phase contributing 80–90% of the total absorbance. This observation suggests that N-methylmyosmine in the active site inhibits NicA2’s FAD from populating a semiquinone state during the reaction with CycN. The decrease in absorbance at 542 nm may simply be due the small decrease in charge-transfer absorbance of N-methylmyosmine bound NicA2 that occurs when the flavin gets oxidized (Extended Data Fig. 4b). Reaction traces at 552 nm still showed the two kinetic phases with increasing absorbance (Extended Data Fig. 9b). Note the logarithmic timescale. d, Absorbance traces at 552 nm for the stopped flow reaction between NMM-NicA2-Flred and variable concentrations of CycNox. Traces fit best to two exponentials and the kobs values are reported in Fig. 4c of the main text. The traces were adjusted so that they all begin at the same absorbance value for comparison. Note the logarithmic timescale.

Extended Data Fig. 10 Bovine cytochrome c is not reduced by NicA2 and has different surface charge distribution than CycN.

a, Bovine cytochrome c combined with nicotine and NicA2 did not result in any reduction of the cytochrome c over 15 min of incubation, unlike the assay performed with CycN (Fig. 3). b, CycN was modeled onto the structure of bovine cytochrome c (PDB ID: 2B4Z) using the SWISS-MODEL online server53. Surface charge distribution of CycN and bovine cytochrome c was calculated using the APBS electrostatics plugin for PyMOL54. Bovine cytochrome c (top) is enriched for positive charge in the region where the heme is surface-exposed, whereas CycN (bottom) is closer to neutral/hydrophobic. Red color indicates negative charge density; blue color indicates positive charge density; heme cofactor is colored in magenta for both structures.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Table 1.

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Source Data Fig. 2

Unprocessed western blot for Fig. 2c.

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Dulchavsky, M., Clark, C.T., Bardwell, J.C.A. et al. A cytochrome c is the natural electron acceptor for nicotine oxidoreductase. Nat Chem Biol 17, 344–350 (2021).

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