title: Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase.
abstract: Quercetin dioxygenase (QDO) catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Fe, Mn, and Co. Previous comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme. We herein report the unprecedented substitution of nitrosyl hydride (HNO) for dioxygen in the activity of Mn-QDO, resulting in the incorporation of both N and O atoms into the product. Turnover is demonstrated by consumption of quercetin and other related substrates under anaerobic conditions in the presence of HNO-releasing compounds and the enzyme. As with dioxygenase activity, a nonenzymatic base-catalyzed reaction of quercetin with HNO is observed above pH 7, but no enhancement of this basal reactivity is found upon addition of divalent metal salts. Unique and regioselective N-containing products ((14)N/(15)N) have been characterized by MS analysis for both the enzymatic and nonenzymatic reactions. Of the several metallo-QDO enzymes examined for nitroxygenase activity under anaerobic condition, only the Mn(II) is active; the Fe(II) and Co(II) substituted enzymes show little or no activity. This result represents an enzymatic catalysis which we denote nitroxygenase activity; the unique reactivity of the Mn-QDO suggests a metal-mediated electron transfer mechanism rather than metal activation of the substrate's inherent base-catalyzed reactivity.
authors.affiliation: Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76706, USA.
authors.forename: Murugaeson R
authors.initials: MR
authors.lastname: Kumar
authors.forename: Adrian
authors.initials: A
authors.lastname: Zapata
authors.forename: Alejandro J
authors.initials: AJ
authors.lastname: Ramirez
authors.forename: Sara K
authors.initials: SK
authors.lastname: Bowen
authors.forename: Wilson A
authors.initials: WA
authors.lastname: Francisco
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Farmer
mesh_terms: D000693:Anaerobiosis; D001412:Bacillus subtilis / Q000201:enzymology*; D002384:Catalysis; D002853:Chromatography, Liquid; D049308:Dioxygenases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D015394:Molecular Structure; D009584:Nitrogen / Q000378:metabolism; D009589:Nitrogen Oxides / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011794:Quercetin / Q000378:metabolism
chemical_list: D009589:Nitrogen Oxides; D008345:Manganese; D011794:Quercetin; D049308:Dioxygenases; C039900:nitroxyl; D009584:Nitrogen; D010100:Oxygen
references.citation: Biochem Pharmacol. 1998 Jun 15;55(12):2007-15
references.pmid: 9714321
references.citation: Eur J Biochem. 1996 Mar 15;236(3):871-6
references.pmid: 8665908
references.citation: Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5537-42
references.pmid: 12704230
references.citation: Antioxid Redox Signal. 2011 May 1;14(9):1649-57
references.pmid: 21235348
references.citation: Biochem J. 1982 Jul 1;205(1):239-44
references.pmid: 7126180
references.citation: Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12705-10
references.pmid: 9356514
references.citation: Free Radic Biol Med. 2008 Sep 1;45(5):578-84
references.pmid: 18503778
references.citation: Methods Enzymol. 1999;301:279-87
references.pmid: 9919577
references.citation: Biochemistry. 2008 Nov 18;47(46):12185-96
references.pmid: 18950192
references.citation: Biochemistry. 2006 Jan 24;45(3):1009-16
references.pmid: 16411777
references.citation: Biochem Biophys Res Commun. 1971 Apr 2;43(1):1-5
references.pmid: 5579942
references.citation: Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2194-8
references.pmid: 11226215
references.citation: J Am Chem Soc. 2001 Jun 20;123(24):5680-3
references.pmid: 11403599
references.citation: Science. 2007 Apr 20;316(5823):453-7
references.pmid: 17446402
references.citation: Free Radic Biol Med. 2001 Oct 1;31(7):869-81
references.pmid: 11585705
references.citation: J Org Chem. 2001 Nov 30;66(24):7974-8
references.pmid: 11722193
references.citation: Biochemistry. 1998 Nov 3;37(44):15503-12
references.pmid: 9799513
references.citation: Biochemistry. 2005 Jan 11;44(1):193-201
references.pmid: 15628860
references.citation: Inorg Chem. 2010 Jul 19;49(14):6283-92
references.pmid: 20666387
references.citation: Protein Expr Purif. 2004 May;35(1):131-41
references.pmid: 15039076
references.citation: Org Biomol Chem. 2009 May 7;7(9):1954-62
references.pmid: 19590793
references.citation: J Am Chem Soc. 2007 Aug 8;129(31):9590-1
references.pmid: 17630747
references.citation: Annu Rev Pharmacol Toxicol. 2005;45:335-55
references.pmid: 15822180
references.citation: Inorg Chem. 2005 May 30;44(11):4024-8
references.pmid: 15907131
references.citation: J Phys Chem A. 2006 Jan 12;110(1):192-7
references.pmid: 16392855
references.citation: J Inorg Biochem. 2002 Feb;88(3-4):343-52
references.pmid: 11897349
references.citation: Circ Res. 2007 Jan 5;100(1):96-104
references.pmid: 17138943
references.citation: Chem Res Toxicol. 2005 May;18(5):790-801
references.pmid: 15892572
references.citation: FEBS Lett. 2004 Jan 16;557(1-3):45-8
references.pmid: 14741339
references.citation: J Physiol. 2007 May 1;580(Pt.3):951-60
references.pmid: 17331988
references.citation: Inorg Chem. 2005 Nov 14;44(23):8259-64
references.pmid: 16270963
references.citation: J Am Chem Soc. 2003 May 28;125(21):6547-52
references.pmid: 12785794
references.citation: Int J Cancer. 2008 Apr 15;122(8):1905-10
references.pmid: 18076071
references.citation: J Inorg Biochem. 2005 Jan;99(1):166-84
references.pmid: 15598500
references.citation: Proc Natl Acad Sci U S A. 2010 Sep 28;107(39):16788-93
references.pmid: 20837547
references.citation: Nat Chem Biol. 2008 Mar;4(3):186-93
references.pmid: 18277980
references.citation: J Am Chem Soc. 2008 Nov 5;130(44):14465-7
references.pmid: 18839948
references.citation: Eur J Biochem. 1999 Aug;263(3):871-8
references.pmid: 10469153
references.citation: Structure. 2002 Feb;10(2):259-68
references.pmid: 11839311
references.citation: Chem Rev. 2004 Feb;104(2):939-86
references.pmid: 14871146
references.citation: J Biol Inorg Chem. 2003 Feb;8(3):348-52
references.pmid: 12589571
references.citation: J Am Chem Soc. 2004 Feb 4;126(4):1096-101
references.pmid: 14746478
references.citation: Chem Rev. 1996 Nov 7;96(7):2951-2964
references.pmid: 11848847
references.citation: Biochemistry. 2009 Jun 9;48(22):5018-25
references.pmid: 19368336
references.citation: J Am Chem Soc. 2002 Oct 2;124(39):11737-45
references.pmid: 12296741
references.citation: Methods Enzymol. 1999;301:211-20
references.pmid: 9919569
references.citation: Inorg Chem. 2005 Jul 25;44(15):5212-21
references.pmid: 16022518
references.citation: Free Radic Biol Med. 2003 Dec 1;35(11):1515-26
references.pmid: 14642399