Paracoccus denitrificans ArsH is encoded by two identical genes located in two distinct putative arsenic resistance (ars) operons. Escherichia coli-produced recombinant N-His6-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH2) and formation of phenylarsonous acid were observed. Growth experiments with P. denitrificans and E. coli revealed increased toxicity of phenylarsonic acid to cells expressing arsH, which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of ars operon.

Department of Biochemistry Faculty of Science Masaryk University Kotlářská 2 611 37 Brno Czech Republic

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Baker S.C., Ferguson S.J., Ludwig B., Page M.D., Richter O.M., van Spanning R.J. Molecular genetics of the genus Paracoccus: Metabolically versatile bacteria with bioenergetic flexibility. Microbiol. Mol. Biol. Rev. 1998;62:1046–1078. doi: 10.1128/MMBR.62.4.1046-1078.1998. PubMed DOI PMC

Sedlacek V., Klumpler T., Marek J., Kucera I. The structural and functional basis of catalysis mediated by NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans. PLoS ONE. 2014;9:e96262. doi: 10.1371/journal.pone.0096262. PubMed DOI PMC

Mazoch J., Tesarik R., Sedlacek V., Kucera I., Turanek J. Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans. Eur. J. Biochem. 2004;271:553–562. doi: 10.1046/j.1432-1033.2003.03957.x. PubMed DOI

Sedlacek V., van Spanning R.J.M., Kucera I. Characterization of the quinone reductase activity of the ferric reductase B protein from Paracoccus denitrificans. Arch. Biochem. Biophys. 2009;483:29–36. doi: 10.1016/j.abb.2008.12.016. PubMed DOI

Sedlacek V., Ptackova N., Rejmontova P., Kucera I. The flavoprotein FerB of Paracoccus denitrificans binds to membranes, reduces ubiquinone and superoxide, and acts as an in vivo antioxidant. FEBS J. 2015;282:283–296. doi: 10.1111/febs.13126. PubMed DOI

Sedlacek V., Kucera I. Chromate reductase activity of the Paracoccus denitrificans ferric reductase B (FerB) protein and its physiological relevance. Arch. Microbiol. 2010;192:919–926. doi: 10.1007/s00203-010-0622-4. PubMed DOI

Sedlacek V., Kucera I. Arginine-95 is important for recruiting superoxide to the active site of the FerB flavoenzyme of Paracoccus denitrificans. FEBS Lett. 2019;593:697–702. doi: 10.1002/1873-3468.13359. PubMed DOI

Pernikarova V., Sedlacek V., Potesil D., Prochazkova I., Zdrahal Z., Bouchal P., Kucera I. Proteomic responses to a methyl viologen-induced oxidative stress in the wild type and FerB mutant strains of Paracoccus denitrificans. J. Proteom. 2015;125:68–75. doi: 10.1016/j.jprot.2015.05.002. PubMed DOI

Sedlacek V., Kucera I. Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans. Mol. Microbiol. 2019;112:166–183. doi: 10.1111/mmi.14260. PubMed DOI

Paez-Espino D., Tamames J., de Lorenzo V., Canovas D. Microbial responses to environmental arsenic. Biometals. 2009;22:117–130. doi: 10.1007/s10534-008-9195-y. PubMed DOI

Mo H., Chen Q., Du J., Tang L., Qin F., Miao B., Wu X., Zeng J. Ferric reductase activity of the ArsH protein from Acidithiobacillus ferrooxidans. J. Microbiol. Biotechnol. 2011;21:464–469. doi: 10.4014/jmb.1101.01020. PubMed DOI

Xue X.M., Yan Y., Xu H.J., Wang N., Zhang X., Ye J. ArsH from Synechocystis sp. PCC 6803 reduces chromate and ferric iron. FEMS Microbiol. Lett. 2014;356:105–112. doi: 10.1111/1574-6968.12481. PubMed DOI

Hervas M., Lopez-Maury L., Leon P., Sanchez-Riego A.M., Florencio F.J., Navarro J.A. ArsH from the cyanobacterium Synechocystis sp. PCC 6803 is an efficient NADPH-dependent quinone reductase. Biochemistry. 2012;51:1178–1187. doi: 10.1021/bi201904p. PubMed DOI

Vorontsov I.I., Minasov G., Brunzelle J.S., Shuvalova L., Kiryukhina O., Collart F.R., Anderson W.F. Crystal structure of an apo form of Shigella flexneri ArsH protein with an NADPH-dependent FMN reductase activity. Protein Sci. 2007;16:2483–2490. doi: 10.1110/ps.073029607. PubMed DOI PMC

Ye J., Yang H.C., Rosen B.P., Bhattacharjee H. Crystal structure of the flavoprotein ArsH from Sinorhizobium meliloti. FEBS Lett. 2007;581:3996–4000. doi: 10.1016/j.febslet.2007.07.039. PubMed DOI PMC

Crescente V., Holland S.M., Kashyap S., Polycarpou E., Sim E., Ryan A. Identification of novel members of the bacterial azoreductase family in Pseudomonas aeruginosa. Biochem. J. 2016;473:549–558. doi: 10.1042/BJ20150856. PubMed DOI

Chen J., Bhattacharjee H., Rosen B.P. ArsH is an organoarsenical oxidase that confers resistance to trivalent forms of the herbicide monosodium methylarsenate and the poultry growth promoter roxarsone. Mol. Microbiol. 2015;96:1042–1052. doi: 10.1111/mmi.12988. PubMed DOI PMC

Paez-Espino A.D., Nikel P.I., Chavarria M., de Lorenzo V. ArsH protects Pseudomonas putida from oxidative damage caused by exposure to arsenic. Environ. Microbiol. 2020;22:2230–2242. doi: 10.1111/1462-2920.14991. PubMed DOI

Wijtzes T., de Wit J.C., In H., Van’t R., Zwietering M.H. Modelling bacterial growth of Lactobacillus curvatus as a function of acidity and temperature. Appl. Environ. Microbiol. 1995;61:2533–2539. doi: 10.1128/aem.61.7.2533-2539.1995. PubMed DOI PMC

Tesarik R., Sedlacek V., Plockova J., Wimmerova M., Turanek J., Kucera I. Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans. Protein Expres. Purif. 2009;68:233–238. doi: 10.1016/j.pep.2009.07.014. PubMed DOI

Battye T.G., Kontogiannis L., Johnson O., Powell H.R., Leslie A.G. iMOSFLM: A new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 2011;67:271–281. doi: 10.1107/S0907444910048675. PubMed DOI PMC

Krissinel E. Ccp4 software suite: History, evolution, content, challenges and future developments. Arbor. 2015;191:a220. doi: 10.3989/arbor.2015.772n2006. DOI

Long F., Vagin A.A., Young P., Murshudov G.N. BALBES: A molecular-replacement pipeline. Acta Crystallogr. D Biol. Crystallogr. 2008;64:125–132. doi: 10.1107/S0907444907050172. PubMed DOI PMC

Adams P.D., Afonine P.V., Bunkoczi G., Chen V.B., Davis I.W., Echols N., Headd J.J., Hung L.W., Kapral G.J., Grosse-Kunstleve R.W., et al. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 2010;66:213–221. doi: 10.1107/S0907444909052925. PubMed DOI PMC

Vagin A.A., Steiner R.A., Lebedev A.A., Potterton L., McNicholas S., Long F., Murshudov G.N. REFMAC5 dictionary: Organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 2004;60:2184–2195. doi: 10.1107/S0907444904023510. PubMed DOI

Emsley P., Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. PubMed DOI

Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. PubMed DOI

Horecker B.L., Kornberg A. The extinction coefficients of the reduced band of pyridine nucleotides. J. Biol. Chem. 1948;175:385–390. doi: 10.1016/S0021-9258(18)57268-9. PubMed DOI

Cleland W.W. Kinetics of enzyme-catalyzed reactions with 2 or more substrates or products. I. Nomenclature and rate equations. Biochim. Biophys. Acta. 1963;67:104–137. doi: 10.1016/0926-6569(63)90211-6. PubMed DOI

Whitby L.G. A new nethod for preparing flavin-adenine dinucleotide. Biochem. J. 1953;54:437–442. doi: 10.1042/bj0540437. PubMed DOI PMC

Heyduk T., Lee J.C. Application of fluorescence energy-transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction. Proc. Natl. Acad. Sci. USA. 1990;87:1744–1748. doi: 10.1073/pnas.87.5.1744. PubMed DOI PMC

Krissinel E., Henrick K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 2007;372:774–797. doi: 10.1016/j.jmb.2007.05.022. PubMed DOI

Agarwal R., Bonanno J.B., Burley S.K., Swaminathan S. Structure determination of an FMN reductase from Pseudomonas aeruginosa PA01 using sulfur anomalous signal. Acta Crystallogr. D. 2006;62:383–391. doi: 10.1107/S0907444906001600. PubMed DOI PMC

Hua Y.H., Wu C.Y., Sargsyan K., Lim C. Sequence-motif detection of NAD(P)-binding proteins: Discovery of a unique antibacterial drug target. Sci. Rep. 2014;4:6471. doi: 10.1038/srep06471. PubMed DOI PMC

Wu J.H., Rosen B.P. Metalloregulated expression of the ars operon. J. Biol. Chem. 1993;268:52–58. doi: 10.1016/S0021-9258(18)54113-2. PubMed DOI

Prabaharan C., Kandavelu P., Packianathan C., Rosen B.P., Thiyagarajana S. Structures of two ArsR As(III)-responsive transcriptional repressors: Implications for the mechanism of derepression. J. Struct. Biol. 2019;207:209–217. doi: 10.1016/j.jsb.2019.05.009. PubMed DOI PMC

Wang L.P., Jeon B.W., Sahin O., Zhang Q.J. Identification of an arsenic resistance and arsenic-sensing system in Campylobacter jejuni. Appl. Environ. Microb. 2009;75:5064–5073. doi: 10.1128/AEM.00149-09. PubMed DOI PMC

Xu C., Rosen B.P. Dimerization is essential for DNA binding and repression by the ArsR metalloregulatory protein of Escherichia coli. J. Biol. Chem. 1997;272:15734–15738. doi: 10.1074/jbc.272.25.15734. PubMed DOI

Kretzschmar J., Brendler E., Wagler J., Schmidt A.C. Kinetics and activation parameters of the reaction of organoarsenic(V) compounds with glutathione. J. Hazard. Mater. 2014;280:734–740. doi: 10.1016/j.jhazmat.2014.08.036. PubMed DOI

Nuallain C.O., Cinneide S.O. Thermodynamic ionization constants of aromatic arsonic acids. J. Inorg. Nucl. Chem. 1973;35:2871–2881. doi: 10.1016/0022-1902(73)80519-6. DOI

Millis K.K., Weaver K.H., Rabenstein D.L. Oxidation/reduction potential of glutathione. J. Org. Chem. 1993;58:4144–4146. doi: 10.1021/jo00067a060. DOI

Knowles F.C. Reactions of lipoamide dehydrogenase and glutathione reductase with arsonic acids and arsonous acids. Arch. Biochem. Biophys. 1985;242:1–10. doi: 10.1016/0003-9861(85)90472-2. PubMed DOI

Walsh C.T., Wencewicz T.A. Flavoenzymes: Versatile catalysts in biosynthetic pathways. Nat. Prod. Rep. 2013;30:175–200. doi: 10.1039/C2NP20069D. PubMed DOI PMC

Mayhew S.G. The effects of pH and semiquinone formation on the oxidation-reduction potentials of flavin mononucleotide. A reappraisal. Eur. J. Biochem. 1999;265:698–702. doi: 10.1046/j.1432-1327.1999.00767.x. PubMed DOI

Pi K.F., Markelova E., Zhang P., van Cappellen P. Arsenic oxidation by flavin-derived reactive species under oxic and anoxic conditions: Oxidant Formation and pH Dependence. Environ. Sci. Technol. 2019;53:10897–10905. doi: 10.1021/acs.est.9b03188. PubMed DOI

Hirose K., Ezaki B., Liu T., Nakashima S. Diamide stress induces a metallothionein BmtA through a repressor BxmR and is modulated by Zn-inducible BmtA in the cyanobacterium Oscillatoria brevis. Toxicol. Lett. 2006;163:250–256. doi: 10.1016/j.toxlet.2005.11.008. PubMed DOI

Ehira S., Ohmori M. The redox-sensing transcriptional regulator RexT controls expression of thioredoxin A2 in the Cyanobacterium anabaena sp. strain PCC 7120. J. Biol. Chem. 2012;287:40433–40440. doi: 10.1074/jbc.M112.384206. PubMed DOI PMC

Palm G.J., Chi B.K., Waack P., Gronau K., Becher D., Albrecht D., Hinrichs W., Read R.J., Antelmann H. Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR. Nucleic Acids Res. 2012;40:4178–4192. doi: 10.1093/nar/gkr1316. PubMed DOI PMC

Guimaraes B.G., Barbosa R.L., Soprano A.S., Campos B.M., de Souza T.A., Tonoli C.C.C., Leme A.F.P., Murakami M.T., Benedetti C.E. Plant pathogenic bacteria utilize biofilm growth-associated repressor (BigR), a novel winged-helix redox switch, to control hydrogen sulfide detoxification under hypoxia. J. Biol. Chem. 2011;286:26148–26157. doi: 10.1074/jbc.M111.234039. PubMed DOI PMC

Madeira F., Park Y.M., Lee J., Buso N., Gur T., Madhusoodanan N., Basutkar P., Tivey A.R.N., Potter S.C., Finn R.D., et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019;47:W636–W641. doi: 10.1093/nar/gkz268. PubMed DOI PMC

Kelley L.A., Mezulis S., Yates C.M., Wass M.N., Sternberg M.J.E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 2015;10:845–858. doi: 10.1038/nprot.2015.053. PubMed DOI PMC

Structural Insight into Catalysis by the Flavin-Dependent NADH Oxidase (Pden_5119) of Paracoccus denitrificans