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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

Abstract

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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References

  1. Massey, V. The chemical and biological versatility of riboflavin. Biochem. Soc. Trans. 28, 283–296 (2000).

    CAS  PubMed  Google Scholar 

  2. El-Gebali, S. et al. The Pfam protein families database in 2019. Nucleic Acids Res. 47, D427–D432 (2019).

    CAS  PubMed  Google Scholar 

  3. Tararina, M. A. & Allen, K. N. Bioinformatic analysis of the flavin-dependent amine oxidase superfamily: adaptations for substrate specificity and catalytic diversity. J. Mol. Biol. 432, 3269–3288 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Fitzpatrick, P. F. Oxidation of amines by flavoproteins. Arch. Biochem. Biophys. 493, 13–25 (2010).

    CAS  PubMed  Google Scholar 

  5. Binda, C., Mattevi, A. & Edmondson, D. E. Structure–function relationships in flavoenzyme-dependent amine oxidations: a comparison of polyamine oxidase and monoamine oxidase. J. Biol. Chem. 277, 23973–23976 (2002).

    CAS  PubMed  Google Scholar 

  6. Tang, H. et al. Novel nicotine oxidoreductase-encoding gene involved in nicotine degradation by Pseudomonas putida strain S16. Appl. Environ. Microbiol. 75, 772–778 (2009).

    CAS  PubMed  Google Scholar 

  7. Tang, H. et al. Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas. PLoS Genet. 9, e1003923 (2013).

    PubMed  PubMed Central  Google Scholar 

  8. Fitzpatrick, P. F. The enzymes of microbial nicotine metabolism. Beilstein J. Org. Chem. 14, 2295–2307 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Tararina, M. A. et al. Crystallography coupled with kinetic analysis provides mechanistic underpinnings of a nicotine-degrading enzyme. Biochemistry 57, 3741–3751 (2018).

    CAS  PubMed  Google Scholar 

  10. Xue, S., Schlosburg, J. E. & Janda, K. D. A new strategy for smoking cessation: characterization of a bacterial enzyme for the degradation of nicotine. J. Am. Chem. Soc. 137, 10136–10139 (2015).

    CAS  PubMed  Google Scholar 

  11. Pentel, P. R. et al. The nicotine-degrading enzyme NicA2 reduces nicotine levels in blood, nicotine distribution to brain, and nicotine discrimination and reinforcement in rats. BMC Biotechnol. 18, 1–14 (2018).

    Google Scholar 

  12. Thisted, T. et al. Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction. BMC Biotechnol. 19, 1–16 (2019).

    CAS  Google Scholar 

  13. Kallupi, M., Xue, S., Zhou, B., Janda, K. D. & George, O. An enzymatic approach reverses nicotine dependence, decreases compulsive-like intake, and prevents relapse. Sci. Adv. 4, eaat4751 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Tararina, M. A., Janda, K. D. & Allen, K. N. Structural analysis provides mechanistic insight into nicotine oxidoreductase from Pseudomonas putida. Biochemistry 55, 6595–6598 (2016).

    CAS  PubMed  Google Scholar 

  15. Xue, S. et al. An enzymatic advance in nicotine cessation therapy. Chem. Commun. 54, 1686–1689 (2018).

    CAS  Google Scholar 

  16. Kopacz, M. M., Heuts, D. P. H. M. & Fraaije, M. W. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis. FEBS J. 281, 4384–4393 (2014).

    CAS  PubMed  Google Scholar 

  17. Vintém, A. P. B., Price, N. T., Silverman, R. B. & Ramsay, R. R. Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines. Bioorg. Med. Chem. 13, 3487–3495 (2005).

    PubMed  Google Scholar 

  18. Fitzpatrick, P. F., Chadegani, F., Zhang, S., Roberts, K. M. & Hinck, C. S. Mechanism of the flavoprotein l-hydroxynicotine oxidase: kinetic mechanism, substrate specificity, reaction product, and roles of active-site residues. Biochemistry 55, 697–703 (2016).

    CAS  PubMed  Google Scholar 

  19. Su, D., Kabir, M. P., Orozco-Gonzalez, Y., Gozem, S. & Gadda, G. Fluorescence properties of flavin semiquinone radicals in nitronate monooxygenase. ChemBioChem 20, 1646–1652 (2019).

    CAS  PubMed  Google Scholar 

  20. Mattevi, A. To be or not to be an oxidase: challenging the oxygen reactivity of flavoenzymes. Trends Biochem. Sci. 31, 276–283 (2006).

    CAS  PubMed  Google Scholar 

  21. Yu, H. et al. Complete genome sequence of the nicotine-degrading Pseudomonas putida strain S16. J. Bacteriol. 193, 5541–5542 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Almagro Armenteros, J. J. et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 37, 420–423 (2019).

    CAS  PubMed  Google Scholar 

  23. Lehninger, A. L., Nelson, D. L. & Cox, M. M. Lehninger Principles of Biochemistry (W. H. Freeman, 2005).

  24. Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat. Protoc. 10, 1820–1841 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Leferink, N. G. H., Van Den Berg, W. A. M. & Van Berkel, W. J. H. l-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis. FEBS J. 275, 713–726 (2008).

    CAS  PubMed  Google Scholar 

  26. Kuwahara, T., White, R. A. & Agosin, M. A cytosolic flavin-containing enzyme catalyzing reduction of cytochrome c in Trypanosoma cruzi: kinetic studies with cytochrome c as substrate. Arch. Biochem. Biophys. 241, 45–49 (1985).

    CAS  PubMed  Google Scholar 

  27. Butt, W. D. & Keilen, D. Absorption spectra and some other properties of cytochrome c and of its compounds with ligands. Proc. R. Soc. Lond. B. Biol. Sci. 156, 429–458 (1962).

    CAS  PubMed  Google Scholar 

  28. Döpner, S. et al. The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase. Eur. J. Biochem. 261, 379–391 (1999).

    PubMed  Google Scholar 

  29. Kachalova, G. S. et al. Crystal structure analysis of free and substrate-bound 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans. J. Mol. Biol. 396, 785–799 (2010).

    CAS  PubMed  Google Scholar 

  30. Fitzpatrick, P. F., Chadegani, F., Zhang, S. & Dougherty, V. Mechanism of flavoprotein l-6-hydroxynicotine oxidase: pH and solvent isotope effects and identification of key active site residues. Biochemistry 56, 869–875 (2017).

    CAS  PubMed  Google Scholar 

  31. Tang, H. et al. Molecular deceleration regulates toxicant release to prevent cell damage in Pseudomonas putida S16 (DSM 28022). MBio 11, 1–12 (2020).

    Google Scholar 

  32. Arai, H. Regulation and function of versatile aerobic and anaerobic respiratory metabolism in Pseudomonas aeruginosa. Front. Microbiol. 2, 103 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Tribelli, P. M. et al. Core regulon of the global anaerobic regulator Anr targets central metabolism functions in Pseudomonas species. Sci. Rep. 9, 9065 (2019).

    PubMed  PubMed Central  Google Scholar 

  34. Chaiyen, P., Fraaije, M. W. & Mattevi, A. The enigmatic reaction of flavins with oxygen. Trends Biochem. Sci. 37, 373–380 (2012).

    CAS  PubMed  Google Scholar 

  35. Graves, S. M. et al. Dopamine metabolism by a monoamine oxidase mitochondrial shuttle activates the electron transport chain. Nat. Neurosci. 23, 15–20 (2020).

    CAS  PubMed  Google Scholar 

  36. Wang, J. & Edmondson, D. E. Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis. Biochemistry 50, 2499–2505 (2011).

    CAS  PubMed  Google Scholar 

  37. Zhuang, Z., Marks, B. & McCauley, R. B. The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria. J. Biol. Chem. 267, 591–596 (1992).

    CAS  PubMed  Google Scholar 

  38. Russell, S. M., Davey, J. & Mayer, R. J. The vectorial orientation of human monoamine oxidase in the mitochondrial outer membrane. Biochem. J. 181, 7–14 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Gaweska, H. & Fitzpatrick, P. F. Structures and mechanism of the monoamine oxidase family. Biomol. Concepts 2, 365–377 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Londer, Y. Y. Expression of recombinant cytochromes c in E. coli. Methods Mol. Biol. 705, 123–150 (2011).

    CAS  PubMed  Google Scholar 

  41. Demeler, B. & Gorbet, G. E. in Analytical Ultracentrifugation: Instrumentation, Software and Applications (eds Uchiyama, S. et al.) 119–143 (Springer, 2016).

  42. Demeler, B., Brookes, E. & Nagel-Steger, L. Analysis of heterogeneity in molecular weight and shape by analytical ultracentrifugation using parallel distributed computing. Methods Enzymol. 454, 87–113 (2009).

    CAS  PubMed  Google Scholar 

  43. Brookes, E., Cao, W. & Demeler, B. A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur. Biophys. J. 39, 405–414 (2010).

    PubMed  Google Scholar 

  44. Brookes, E. & Demeler, B. in Analytical Ultracentrifugation VIII. Progress in Colloid and Polymer Science Vol.131 (eds Wandrey, C. & Cölfen, H.) 33–40 (Springer, 2006).

  45. Moran, G. R. Anaerobic methods for the transient-state study of flavoproteins: the use of specialized glassware to define the concentration of dioxygen. Methods Enzymol. 620, 27–49 (2019).

    CAS  PubMed  Google Scholar 

  46. Zabinski-Snopko, R. M. & Czerlinski, G. H. Spectrophotometric titrations of ferricytochrome c with ferrohexacyanide in the pH range 5 to 7. J. Biol. Phys. 9, 155–167 (1981).

    CAS  Google Scholar 

  47. Lemoine, F. et al. NGPhylogeny.fr: new generation phylogenetic services for non-specialists. Nucleic Acids Res. 47, W260–W265 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Letunic, I. & Bork, P. Interactive Tree of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 47, 256–259 (2019).

    Google Scholar 

  49. Qiu, J. et al. Functional identification of two novel genes from Pseudomonas sp. strain HZN6 involved in the catabolism of nicotine. Appl. Environ. Microbiol. 78, 2154–2160 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Li, J. et al. Comparative genomics reveals specific genetic architectures in nicotine metabolism of Pseudomonas sp. JY-Q. Front. Microbiol. 8, 2085 (2017).

    PubMed  PubMed Central  Google Scholar 

  51. De Rienzo, F., Gabdoulline, R. R., Menziani, M. C. & Wade, R. C. Blue copper proteins: a comparative analysis of their molecular interaction properties. Protein Sci. 9, 1439–1454 (2000).

    PubMed  PubMed Central  Google Scholar 

  52. Bagshaw, C. in Biomolecular Kinetics Ch 2.7, 41–45 (CRC Press, 2017).

  53. Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Jurrus, E. et al. Improvements to the APBS biomolecular solvation software suite. Protein Sci. 27, 112–128 (2018).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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

Authors

Contributions

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.

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Supplementary Figs. 1–3 and Table 1.

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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). https://doi.org/10.1038/s41589-020-00712-3

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