Quinone- and nitroreductase reactions of Thermotoga maritima peroxiredoxin–nitroreductase hybrid enzyme Z ˇ ilvinas Anusevic ˇius a , Lina Misevic ˇiene ˙ a , Jonas Šarlauskas a , Nicolas Rouhier b , Jean-Pierre Jacquot b , Narimantas C ˇ e ˙ nas a,⇑ a Institute of Biochemistry of Vilnius University, Mokslininku z 12, LT-08662 Vilnius, Lithuania b Unité Mixte de Recherches 1136, Universite de Lorraine-INRA, IFR 110 Faculté des Sciences, BP 239, 54506 Vandoeuvre Cedex, France article info Article history: Received 6 August 2012 and in revised form 29 August 2012 Available online 12 September 2012 Keywords: Flavoenzymes Nitroreductase Peroxiredoxin Thermotoga maritima Quinones Nitroaromatic compounds abstract Thermotoga maritima peroxiredoxin–nitroreductase hybrid enzyme (Prx–NR) consists of a FMN-contain- ing nitroreductase (NR) domain fused to a peroxiredoxin (Prx) domain. These domains seem to function independently as no electron transfer occurs between them. The reduction of quinones and nitroaromat- ics by NR proceeded in a two-electron manner, and follows a ‘ping-pong’ scheme with sometimes pro- nounced inhibition by quinone substrate. The comparison of steady- and presteady-state kinetic data shows that in most cases, the oxidative half-reaction may be rate-limiting in the catalytic cycle of NR. The enzyme was inhibited by dicumarol, a classical inhibitor of oxygen-insensitive nitroreductases. The reduction of quinones and nitroaromatic compounds by Prx–NR was characterized by the linear dependence of their reactivity (log k cat /K m ) on their single-electron reduction potentials E 1 7 , while the reactivity of quinones markedly exceeded the one with nitroaromatics. It shows that NR lacks the spec- ificity for the particular structure of these oxidants, except their single-electron accepting potency and the rate of electron self-exchange. It points to the possibility of a single-electron transfer step in a net two-electron reduction of quinones and nitroaromatics by T. maritima Prx–NR, and to a significant diver- sity of the structures of flavoenzymes which may perform the two-electron reduction of quinones and nitroaromatics. Ó 2012 Elsevier Inc. All rights reserved. Introduction Bacterial oxygen-insensitive NAD(P)H:nitroreductases (NRs) contain flavin mononucleotide (FMN) 1 in their active center, and perform a net two-electron reduction of nitroaromatic compounds to nitroso, and, subsequently, to hydroxylamine products ([1–11], and references therein). There are two groups of NRs with low se- quence homology, which are similar with Escherichia coli NADPH- dependent major nitroreductase-A [3], e.g., Vibrio harveyi FMN- reductase [4], and Salmonella typhimurium nitroreductase [5], or with E. coli NAD(P)H-dependent minor nitroreductase-B [6], e.g., Enterobacter cloacae nitroreductase [2,7–9], Vibrio fisheri FMN- reductase [10], and Thermus thermophilus NADH-oxidase [11]. The physiological role(s) of NRs are unclear except their possible antioxidant functions, because they are induced by oxidative stress and other environmental hazards [12]. On the other hand, bacterial NRs are of considerable interest due to their participation in the biodegradation of explosives and other polynitroaromatic environ- mental pollutants ([12,13], and references therein), and their pos- sible utility in the antibody-, gene-, or virus- directed prodrug therapies ([14,15], and references therein). It is generally accepted that a net two-electron (hydride) reduction of nitroaromatics by NRs is linked to the instability of their FMN sem- iquinone state, which makes the single-electron transfer thermody- namically unfavorable [8,10]. However, the other aspects of the catalytic mechanisms of bacterial oxygen-insensitive NRs, e.g., their substrate specificity, and the general mechanism of two-electron nitroreduction remain vaguely understood. Except for the more de- tailed examination of E. cloacae NR [7,16], most kinetic studies of other NRs were confined to a limited number of oxidants with some- times poorly characterized reduction thermodynamics ([3,6,17], and references therein). The few available crystal structures of E. cloacae NR and E. coli NR-B with bound nitroaromatic compounds and other 0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.08.014 ⇑ Corresponding author. E-mail address: narimantas.cenas@bchi.vu.lt (N. C ˇ e ˙ nas). 1 Abbreviations used: Ar-NO 2 , nitroaromatic compound; BCP, bacterioferritin comi- gratory protein; CB-1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide; DNBF, 4,6-dini- trobenzofuroxan; DZQ, diaziridinyl-1,4-benzoquinone; EE 1 7 , single-electron reduction potential at pH 7.0; FMN, flavin mononucleotide; Grx, glutaredoxin; k cat , catalytic constant; k cat /K m , second-order rate reaction rate constant; K iq , quinone substrate inhibition constant; MeDZQ, 2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone; NQO1, NAD(P)H:quinone oxidoreductase; NR, nitroreductase; Prx, peroxiredoxin; Prx-NR, peroxiredoxin–nitroreductase hybrid enzyme; Q, quinone; SN-23862, 5-(bis(2,2’- chloroethyl-amino))-2,4-dinitrobenzamide; TNC, 1,3,6,8-tetranitrocarbazole; TNT, 2,4,6-trinitrotoluene; VdWvol, van der Waals volume. Archives of Biochemistry and Biophysics 528 (2012) 50–56 Contents lists available at SciVerse ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi