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 metalmediated electron transfer mechanism rather than metal activation of the substrate's inherent base-catalyzed reactivity.nitroxyl | quercetinase D ioxygenases are enzymes that incorporate both atoms of dioxygen into a targeted substrate, e.g., the enzyme catechol 1,2-dioxygenase cleaves the C-C bond between catechol's two hydroxyl groups forming the dicarboxylate cis,cis-muconic acid (1). Although highly divergent, there are two broadly defined classes of nonheme dioxygenases: those that directly bind dioxygen at the metal center or those in which dioxygen binds to the substrate that has been activated by interaction with the metal center (2). Such questions also apply to quercetin dioxygenase (QDO), which catalyzes the oxidation of the flavonol quercetin (1) with dioxygen ( Fig. 1). During turnover, QDO cleaves the central heterocyclic ring of quercetin, releasing CO, and yielding the corresponding depside. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Cu, Fe, Mn, and Co. Comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme (3).The simple molecule nitrosyl hydride (HNO) is the singly reduced and protonated form of nitric oxide, NO. HNO has garnered much interest because it demonstrates a physiological reactivity distinctly different from its congener nitric oxide (4, 5). In a series of papers, we have reported the preparation and characterization of the HNO adduct of myoglobin (HNO-Mb) by reduction of 7) and by trapping of free HNO by deoxymyoglobin (Mb-Fe II ) (8)...

Recent advances in the chemical biology of nitroxyl (HNO) detection and generation

“…On-going studies outside the scope of this review continue to explore HNO’s role in basic cardiac physiology or define HNO’s reactivity with oxygen or new targets, such as cobalt-containing proteins or manganese quercetin dioxygenase. 110,156–158 Reactions of HNO with the proteins that control hydrogen peroxide degradation reveals a “cross-talk” between HNO and hydrogen peroxide that may define new redox based signaling or gene expression pathways. 159 The combination of the advances in HNO detection and donation with these emerging areas highlights the enormous potential of these new chemical biology tools to understanding HNO’s actions in biology, physiology and medicine.…”

A singular value decomposition approach for kinetic analysis of reactions of HNO with myoglobin

“…As HNO is isoelectronic with O 2 and forms stable diamagnetic adducts with ferrous hemoglobins from various species, we have suggested its use as an O 2 analog [18]. Most recently, we have shown that HNO may replace O 2 during turnover of a non-heme Mn dioxygenase enzyme, quercetin dioxygenase; this nitroxygenase activity results in the regioselective incorporation of the N atom derived from HNO within the enzymatic product [20].…”

Computational investigations of HNO in biology

“…These observations strongly suggest that HNO has significant roles in biology and medicine. It is interesting to note that quite a number of the HNO biological interactions occur with metalloproteins, such as heme proteins [1–3,15–18], Cu,Zn-SOD (superoxide dismutase) [19], and manganese quercetin dioxygenase [20]. However, the atomic level structural and functional details of HNO interactions with biological systems are largely unknown.…”