{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T19:54:04Z","timestamp":1772826844453,"version":"3.50.1"},"reference-count":24,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2024,9,10]],"date-time":"2024-09-10T00:00:00Z","timestamp":1725926400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"DGAPA-PAPIIT, UNAM","award":["IN217520"],"award-info":[{"award-number":["IN217520"]}]},{"name":"DGAPA-PAPIIT, UNAM","award":["IN211923"],"award-info":[{"award-number":["IN211923"]}]},{"name":"DGAPA-PAPIIT, UNAM","award":["IN201021"],"award-info":[{"award-number":["IN201021"]}]},{"name":"DGAPA-PAPIIT, UNAM","award":["IN214724"],"award-info":[{"award-number":["IN214724"]}]},{"name":"DGAPA-PAPIIT, UNAM","award":["IN211224"],"award-info":[{"award-number":["IN211224"]}]},{"name":"DGAPA-PAPIIT, UNAM","award":["58540"],"award-info":[{"award-number":["58540"]}]},{"name":"DGAPA-PAPIIT, UNAM","award":["782527"],"award-info":[{"award-number":["782527"]}]},{"name":"DGAPA-PAPIIT, UNAM","award":["CF2023-I-199"],"award-info":[{"award-number":["CF2023-I-199"]}]},{"name":"CONAHCyT Ciencia de Frontera 2019","award":["IN217520"],"award-info":[{"award-number":["IN217520"]}]},{"name":"CONAHCyT Ciencia de Frontera 2019","award":["IN211923"],"award-info":[{"award-number":["IN211923"]}]},{"name":"CONAHCyT Ciencia de Frontera 2019","award":["IN201021"],"award-info":[{"award-number":["IN201021"]}]},{"name":"CONAHCyT Ciencia de Frontera 2019","award":["IN214724"],"award-info":[{"award-number":["IN214724"]}]},{"name":"CONAHCyT Ciencia de Frontera 2019","award":["IN211224"],"award-info":[{"award-number":["IN211224"]}]},{"name":"CONAHCyT Ciencia de Frontera 2019","award":["58540"],"award-info":[{"award-number":["58540"]}]},{"name":"CONAHCyT Ciencia de Frontera 2019","award":["782527"],"award-info":[{"award-number":["782527"]}]},{"name":"CONAHCyT Ciencia de Frontera 2019","award":["CF2023-I-199"],"award-info":[{"award-number":["CF2023-I-199"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The transmembrane nitrate reductase (Nar) is the first enzyme in the dissimilatory alternate anaerobic nitrate respiratory chain in denitrifying bacteria. To date, there has been no real-time method to determine its specific activity embedded in its native membrane; here, we describe such a new method, which is useful with the inside-out membranes of Paracoccus denitrificans and other denitrifying bacteria. This new method takes advantage of the native coupling of the endogenous NADH dehydrogenase or Complex I with the reduction of nitrate by Nar through the quinone pool of the inner membranes of P. denitrificans. This is achieved under previously reached anaerobic conditions. Inner controls confirming the specific Nar activity determined by this new method were made by the total inhibition of the Nar enzyme by sodium azide and cyanide, well-known Nar inhibitors. The estimation of the Michaelis\u2013Menten affinity of Nar for NO3\u2212 using this so-called Nar-JJ assay gave a Km of 70.4 \u03bcM, similar to previously determined values. This new Nar-JJ assay is a suitable, low-cost, and reproducible method to determine in real-time the endogenous Nar activity not only in P. denitrificans, but in other denitrifying bacteria such as Brucella canis, and potentially in other entero-pathogenic bacteria.<\/jats:p>","DOI":"10.3390\/ijms25189770","type":"journal-article","created":{"date-parts":[[2024,9,10]],"date-time":"2024-09-10T03:16:51Z","timestamp":1725938211000},"page":"9770","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A New Real-Time Simple Method to Measure the Endogenous Nitrate Reductase Activity (Nar) in Paracoccus denitrificans and Other Denitrifying Bacteria"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2597-9457","authenticated-orcid":false,"given":"Jos\u00e9 J.","family":"Garc\u00eda-Trejo","sequence":"first","affiliation":[{"name":"Departamento de Biolog\u00eda, Facultad de Qu\u00edmica, Universidad Nacional Aut\u00f3noma de M\u00e9xico (U.N.A.M.), Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sharon","family":"Rojas-Alcantar","sequence":"additional","affiliation":[{"name":"Departamento de Biolog\u00eda, Facultad de Qu\u00edmica, Universidad Nacional Aut\u00f3noma de M\u00e9xico (U.N.A.M.), Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4103-3162","authenticated-orcid":false,"given":"Monserrat","family":"Alonso-Vargas","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioprocesos Ambientales, Universidad Polit\u00e9cnica de Pachuca (U.P.P.), Zempoala, Pachuca 43830, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raquel","family":"Ortega","sequence":"additional","affiliation":[{"name":"Departamento de Biolog\u00eda, Facultad de Qu\u00edmica, Universidad Nacional Aut\u00f3noma de M\u00e9xico (U.N.A.M.), Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alejandro","family":"Ben\u00edtez-Guzm\u00e1n","sequence":"additional","affiliation":[{"name":"Departamento de Microbiolog\u00eda e Inmunolog\u00eda, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut\u00f3noma de M\u00e9xico (U.N.A.M.), Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0488-6385","authenticated-orcid":false,"given":"Leticia","family":"Ram\u00edrez-Silva","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Facultad de Medicina, Universidad Nacional Aut\u00f3noma de M\u00e9xico (U.N.A.M.), Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Natalia","family":"Pav\u00f3n","sequence":"additional","affiliation":[{"name":"Departamento de Farmacolog\u00eda, Instituto Nacional de Cardiolog\u00eda \u201cIgnacio Ch\u00e1vez\u201d, Ciudad de M\u00e9xico 14080, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudia","family":"Pe\u00f1a-Segura","sequence":"additional","affiliation":[{"name":"Departamento de Bioterio, Instituto de Fisiolog\u00eda Celular, Universidad Nacional Aut\u00f3noma de M\u00e9xico (U.N.A.M.), Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ofelia","family":"M\u00e9ndez-Romero","sequence":"additional","affiliation":[{"name":"Departamento de Gen\u00e9tica Molecular, Instituto de Fisiolog\u00eda Celular, Universidad Nacional Aut\u00f3noma de M\u00e9xico (U.N.A.M.), Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Salvador","family":"Uribe-Carvajal","sequence":"additional","affiliation":[{"name":"Departamento de Gen\u00e9tica Molecular, Instituto de Fisiolog\u00eda Celular, Universidad Nacional Aut\u00f3noma de M\u00e9xico (U.N.A.M.), Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arturo","family":"Cadena-Ram\u00edrez","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioprocesos Ambientales, Universidad Polit\u00e9cnica de Pachuca (U.P.P.), Zempoala, Pachuca 43830, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,9,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1016\/0005-2728(70)90225-2","article-title":"Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans","volume":"216","author":"John","year":"1970","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1111\/j.1432-1033.1986.tb09771.x","article-title":"The respiratory nitrate reductase from Paracoccus denitrificans. Molecular characterisation and kinetic properties","volume":"158","author":"Craske","year":"1986","journal-title":"Eur. J. Biochem."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Craske, A.L., and Ferguson, S.J. (1986). The nitrate reductase of Paracoccus denitrificans. Biochem. Soc. Trans., 14.","DOI":"10.1042\/bst0141204"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Spees, A.M., Wangdi, T., Lopez, C.A., Kingsbury, D.D., Xavier, M.N., Winter, S.E., Tsolis, R.M., and B\u00e4umler, A.J. (2013). Streptomycin-induced inflammation enhances Escherichia coli gut colonization through nitrate respiration. mBio, 4.","DOI":"10.1128\/mBio.00430-13"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3470","DOI":"10.1128\/IAI.00351-15","article-title":"The Periplasmic Nitrate Reductase NapABC Supports Luminal Growth of Salmonella enterica Serovar Typhimurium during Colitis","volume":"83","author":"Lopez","year":"2015","journal-title":"Infect. Immun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4953","DOI":"10.1111\/1462-2920.13974","article-title":"Tuning the modular Paracoccus denitrificans respirome to adapt from aerobic respiration to anaerobic denitrification","volume":"19","author":"Giannopoulos","year":"2017","journal-title":"Environ. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Olaya-Abril, A., Hidalgo-Carrillo, J., Luque-Almagro, V.M., Fuentes-Almagro, C., Urbano, F.J., Moreno-Vivi\u00e1n, C., Richardson, D.J., and Rold\u00e1n, M.D. (2018). Exploring the Denitrification Proteome of Paracoccus denitrificans PD1222. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.01137"},{"key":"ref_8","unstructured":"Nicholls, D.G., and Ferguson, S.J. (2013). Bioenergetics 4, Academic Press."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"681","DOI":"10.1038\/nsb969","article-title":"Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A","volume":"10","author":"Bertero","year":"2003","journal-title":"Nat. Struct. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1080\/00103627509366547","article-title":"Rapid colorimetric determination of nitrate in plant tissue bi nitration of salicylic acid","volume":"6","author":"Cataldo","year":"1975","journal-title":"Soil. Sci. Plant Anal."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1042\/BJ20101920","article-title":"A composite biochemical system for bacterial nitrate and nitrite assimilation as exemplified by Paracoccus denitrificans","volume":"435","author":"Gates","year":"2011","journal-title":"Biochem. J."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Parsonage, D., Greenfield, A.J., and Ferguson, S.J. (1985). The high affinity of Paracoccus denitrificans cells for nitrate as an electronacceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells. Biochim. Biophys. Acta, 807.","DOI":"10.1016\/0005-2728(85)90055-6"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1096\/fj.09-137356","article-title":"A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related alpha-proteobacteria","volume":"24","author":"Celis","year":"2010","journal-title":"FASEB J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"10503","DOI":"10.1021\/bi00498a013","article-title":"Kinetics of oxidative phosphorylation in Paracoccus denitrificans. 1. Mechanism of ATP synthesis at the active site(s) of F0F1-ATPase","volume":"29","author":"Perez","year":"1990","journal-title":"Biochemistry"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10518","DOI":"10.1021\/bi00498a014","article-title":"Kinetics of oxidative phosphorylation in Paracoccus denitrificans. 2. Evidence for a kinetic and thermodynamic modulation of F0F1-ATPase by the activity of the respiratory chain","volume":"29","author":"Perez","year":"1990","journal-title":"Biochemistry"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1111\/j.1432-1033.1974.tb03343.x","article-title":"The bacterial nitrate reductases. Solubilization, purification and properties of the enzyme A of Escherichia coli K 12","volume":"42","author":"Forget","year":"1974","journal-title":"Eur. J. Biochem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4987","DOI":"10.1074\/jbc.M116.771899","article-title":"Respiratory Complex I in Bos taurus and Paracoccus denitrificans Pumps Four Protons across the Membrane for Every NADH Oxidized","volume":"292","author":"Jones","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1021\/bi00322a007","article-title":"Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12","volume":"24","author":"Morpeth","year":"1985","journal-title":"Biochemistry"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4082","DOI":"10.1038\/s41467-022-31718-1","article-title":"Cryo-EM structures of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae","volume":"13","author":"Kishikawa","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Mann, A., Nicholls, P., Sharpe, M., and Cooper, C.E. (2002). Inhibition of cytochrome c oxidase by nitrite and nitric oxide:reversible and irreversible. Biochem. Soc. Trans., 30.","DOI":"10.1042\/bst030a074b"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2146","DOI":"10.1096\/fj.13-241430","article-title":"The zeta subunit of the F1FO-ATP synthase of alpha-proteobacteria controls rotation of the nanomotor with a different structure","volume":"28","year":"2014","journal-title":"FASEB J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1007\/BF02110340","article-title":"Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: Competition with H2PO4","volume":"27","author":"Garcia","year":"1995","journal-title":"J. Bioenerg. Biomembr."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0021-9258(19)52451-6","article-title":"Protein measurement with the Folin phenol reagent","volume":"193","author":"Lowry","year":"1951","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1016\/0003-2697(77)90043-4","article-title":"A simplification of the protein assay method of Lowry et al. which is more generally applicable","volume":"83","author":"Peterson","year":"1977","journal-title":"Anal. Biochem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/18\/9770\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:52:39Z","timestamp":1760111559000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/18\/9770"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,10]]},"references-count":24,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2024,9]]}},"alternative-id":["ijms25189770"],"URL":"https:\/\/doi.org\/10.3390\/ijms25189770","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,9,10]]}}}