Quinone-and nitroreductase reactions of Thermotoga maritima thioredoxin reductase

The Thermotoga maritima NADH:thioredoxin reductase (TmTR) contains FAD and a catalytic disulfide in the active center, and uses a relatively poorly studied physiological oxidant Grx-1-type glutaredoxin. In order to further assess the redox properties of TmTR, we used series of quinoidal and nitroaromatic oxidants with a wide range of single-electron reduction potentials (E7, –0.49–0.09 V). We found that TmTR catalyzed the mixed singleand two-electron reduction of quinones and nitroaromatic compounds, which was much faster than the reduction of Grx-1. The reactivity of both groups of oxidants increased with an increase in their E7, thus pointing to the absence of their structural specificity. The maximal rates of quinone reduction in the steady-state reactions were lower than the maximal rates of reduction of FAD by NADH, obtained in presteady-state experiments. The mixed-type reaction inhibition by NAD+ was consistent with its competition for a NADH binding site in the oxidized enzyme form, and also with the reoxidation of the reduced enzyme form. The inhibition data yielded a value of the standard potential for TmTR of –0.31±0.03 V at pH 7.0, which may correspond to the FAD/FADH2 redox couple. Overall, the mechanism of quinoneand nitroreductase reactions of T. maritima TR was similar to the previously described mechanism of Arabidopsis thaliana TR, and points to their prooxidant and possibly cytotoxic role.


INTRODUCTION
NAD(P)H:thioredoxin reductase (TR) and thioredoxin (TRx) systems are ubiquitous in both prokaryotes and eukaryotes, where they mainly perform antioxidant functions (Williams, 1990).TRs are members of the family of flavoenzyme pyridine nucleotide-disulfide reductases, which include glutathione reductase, trypanothione reductase, lipoamide dehydrogenase, and some other related enzymes (Williams, 1990;Arner et al., 2000;Nordberg et al., 2001).These NAD(P)H-dependent enzymes contain a flavin adenine dinucleotide (FAD) and a redoxactive disulfide in their active center.In the catalysis, the FAD accepts two electrons (hydride) from NAD(P) H and transfers it to the catalytic disulfide which subsequently reduces the disulfide oxidant.Importantly, there exist two groups of TRs, namely the low M.W. TR (~35 kD, L-TR) of plants, prokarya and many eukarya, which contain FAD and the catalytic disulfide (Williams, 1990;Lennon et al., 1999), and high m.w.TR (~55 kD, H-TR), which contain an additional redox-active -Se-Smoiety (mammalian TR), or an additional redox-active disulfide (Plasmodium spp.) (Arner et al., 2000;Nordberg et al., 2001;Kanzok et al., 2000).These additional redox groups of H-TR accept two redox equivalents from the reduced catalytic disulfide adjacent to FAD, and subsequently reduce the corresponding physiological disulfide oxidants.
Like other flavoenzymes-disulfide reductases, both L-and H-TRs reduce quinones and nitroaromatic compounds (Vienožinskis et al., 1990;Čėnas et al., 1994;2004;2006;Bironaitė et al., 1997;Miškinienė et al., 1998;and references therein).These reactions are of some biomedical importance, because the antioxidant disulfide reductases are considered potential targets of quinones and nitroaromatic compounds with respect to their potential anticancer, antimicrobial, and antiparasitic activities (Čėnas et al., 1994; 2004; 2006; and the references therein).The above compounds may act as "subversive substrates" for TRs, converting antioxidant functions into prooxidant ones due to redox-cycling reactions.In these cases, both H-TRs and L-TRs, e.g., L-TR of Trichomonas vaginalis, may be considered as potential redox-drug targets (Čėnas et al., 2004;2006;McMillan et al., 2009;and references therein).However, the reactions of L-TRs with quinones and nitroaromatic compounds have not been thoroughly studied so far, except for the Arabidopsis thaliana TR (Bironaitė et al., 1997;Miškinienė et al., 1998).
Thermotoga maritima TR (TmTR, M.W. 34.3 kD) was the first TR isolated from a hyperthermophylic organism (Yang et al., 2009;Couturier et al., 2013).As the other L-TRs, it contains the NAD(P)H binding motif, FAD, and the pair of active-site cysteines (McMillan et al., 2009).It participates in the antioxidant defence of T. maritima, providing electrons for peroxiredoxins via glutaredoxin-like proteins (Yang et al., 2009;Couturier et al., 2013).The catalytic mechanism of TmTR is not characterized so far.
In this paper, we provide a detailed analysis of the catalytic mechanism of T. maritima TR using a series of quinoidal and nitroaromatic electron acceptors.Apart from the quantitative structure-activity relationships in these reactions, our data enabled us to characterize the thermodynamic properties of TmTR involving the redox equilibrium with the NAD + /NADH couple.
Steady-state kinetic studies.Steady-state kinetic measurements were performed spectrophotometrically by monitoring the oxidation of NADH (Δε 340 =6.2 mM -1 cm -1 ) in the presence of TmTR and quinones or nitroaromatic compounds in 0.1 M K-phosphate (pH 7.0) containing 1 mM EDTA, at 25°C using Perkin Elmer Lambda 25 spectrophotometer.The initial rates of reaction were corrected for the intrinsic NADH oxidase activity of the enzyme, and for the nonenzymatic oxidation of NADH by 1,4-benzoquinone and 2-methyl-1,4benzoquinone, and for the spectral changes at 340 nm corresponding to reduction of nitrobenzenes (Nivinskas et al., 2001).The steady-state parameters of reactions, the catalytic constants (k cat ) and the bimolecular rate constants (k cat /K m ) of the oxidants at fixed concentration of NADH correspond to the reciprocal intercepts and slopes of Lineweaver-Burk plots, [E]/V vs. 1/[Q], where V is the reaction rate, [E] is the enzyme concentration, and [Q] is the concentration of quinone or nitroaromatic oxidant.k cat represents the number of molecules of NADH oxidized by a single active center of the enzyme per second.These parameters were obtained by the fitting of kinetic data to the parabolic expression using SigmaPlot 2000 (version 11.0, SPSS Inc.).Single-electron flux in the reduction of quinones was determined by monitoring 1,4-benzoquinone (50 µM)-mediated reduc-tion of cytochrome c (50 µM) by TmTR in the presence of 100 µM NADH assuming ∆ε 550 =20 mM -1 cm -1 .The same conditions were used in the monitoring of TmTRmediated reduction of cytochrome c by 50 µM tetryl or p-dinitrobenzene in the presence of 100 µM NADH and in the absence or presence of 100 U/ml SOD.Reduction rate of Grx-1 was monitored in the presence of 0.1 mM DTNB assuming Δε 412 =27.2 mM -1 cm -1 .
Presteady-state kinetic measurements.The presteady-state kinetic measurement of reduction of TR by excess NADH was carried out in 0.1 M K-phosphate buffer containing 1 mM EDTA at pH 7.0 and 25°C using a stopped-flow SX.17 MV spectrophotometer (Applied Photophysics).The final concentration of the enzyme was 5.0 µM.Because of the low NADH oxidase activity of TmTr, 0.05 s 1 , which could marginally affect the data of the measurements, the experiments were performed under aerobic conditions.The kinetics of absorbance decrease at 460 nm was analyzed according to the single exponent fit using SigmaPlot software.
Amino acid sequence alignment.Amino acid sequence alignment of TmTR and related enzymes was performed using Clustal Omega software (www.ebi.ac.uk/Tools/msa/clustalo/).

Steady-state reactions of T. maritima TR
The physiological electron acceptors of T. maritima TR are poorly characterized except for glutaredoxin-1 (Grx-1) (McMillan et al., 2009;Yang et al., 2009), which also possesses a relatively low activity.The maximal rate of Grx-1 reduction at pH 7.0 (0.1 M K-phosphate, 100 µM NADH, 3-10 µM Grx-1) using the DTNB assay was 0.2-0.3s -1 , which is close to the intrinsic NADH oxidase activity of the enzyme, 0.05 s -1 .In agreement with the previous data (Yang et al., 2009), an increase in pH to 8.0 (0.03 M Tris-HCl) increased the reactivity of Grx-1.Under these conditions, k cat was equal to 2.1±0.6 s -1 , and the k cat /K m for Grx-1 was 6.3±0.6 × 10 5 M -1 s -1 .Relationship between log k cat /K m of quinones (solid circles) and nitroaromatic compounds (blank circles) in TmTR catalyzed reaction and their single-electron reduction potentials at pH 7.0 (E 1 7 ) is shown.Numbers refer to quinones and nitroaromatic compounds whose redox potentials and reduction rate constants are given in Table 1.The catalytic mechanism of Thermotoga maritima TR.
In contrast, the TmTR-catalyzed oxidation of NADH by a number of quinones and nitroaromatic compounds at pH 7.0 was much faster (Table 1).Using varied concentrations of 5,8-dihydroxy-1,4-naphthoquinone (naphthazarin) as an electron acceptor at several fixed concentrations of NADH, the reaction was characterized by a series of parallel Lineweaver-Burk plots (Fig. 1).It corresponds to a "ping-pong" scheme, which was also characteristic for the quinone-and nitroreductase reactions of A. thaliana TR (Bironaitė et al., 1997;Miškinienė et al., 1998).In our case, k cat /K m for naphtazarin was 3.3±0.3× 10 5 M -1 s -1 , the k cat /K m for NADH was 1.7±0.2× 10 6 M -1 s -1 , and k cat , obtained by extrapolation to infinite concentrations of NADH and napthhazarin, was equal to 39.1±3.5 s -1 .
The studies of quinone and nitroaromatic (ArNO 2 ) electron acceptors revealed that the reactivity of compounds (log k cat /K m ) generally increased with an increase in their single-electron reduction potential (E 1 7 ), i.e., the potential of the quinone/semiquinone or ArNO 2 / ArNO 2 -• redox couples (Fig. 2).The reactivity of nitroaromatics was systematically lower than that of quinones with similar E 1 7 values (Fig. 2).Next we aimed at characterizing the possible singleelectron transfer events in the reduction of quinones and nitroaromatics by T. maritima TR.The percentage of the single-electron flux in the reduction of quinones by NAD(P)H-oxidizing flavoenzymes can be determined using 1,4-benzoquinone-mediated reduction of added cytochrome c (Iyanagi et al., 1990, Table 1.Kinetic parameters of reduction of quinones and nitroaromatic compounds by T. maritima thioredoxin reductase.The catalytic constants (k cat ) and bimolecular rate constants (k cat /K m ) of electron acceptors in TmTR catalyzed reactions at pH 7.0 and 25 o C were obtained in the presence of 100 µM NADH.Their single-electron reduction potentials at pH 7.0 (E 1 7 ) are taken from Wardman (1989), and Šarlauskas et al., (2006).

No
Compound and references therein), and it is equal to the ratio between the cytochrome c reduction rate and a doubled rate of NAD(P)H oxidation.This is based on the fact that at pH < 7.2 the rate of reduction of cytochrome c by 1,4-hydroquinone is negligible, whereas 1,4-benzosemiquinone, formed during the single-electron reduction, reduces cytochrome c at a high rate (k≈10 6 M -1 s - 1 ) (Iyanagi, 1990) redox couples are under equilibrium, and only the O 2 -• -mediated, but not the nitroradical-mediated, cytochrome c reduction is inhibited by SOD.Nevertheless, it points to an at least 20% share of singleelectron flux in the reduction of nitroaromatic compounds by TmTR.
It is commonly accepted that quinoidal compouds are reduced by the reduced FAD of flavoenzymes containing a catalytic disulfide (Vienožinskis et al., 1990;Čėnas et al., 1994;2004;2006;Bironaitė et al., 1997;Miškinienė et al., 1998;and references therein).This may also be true for TmTR, because a 40-50 min-incubation of the reduced enzyme with the thiolalkylating agent p-chloromercury benzoate (p-CMB) (200 µM NADH and 100 µM p-CMB) resulted in a 95% inhibition of the reduction of Grx-1 (4.0 µM).In contrast, the reduction of 100 µM naphthazarin was inhibited by 40-50%.In control experiments, incubation of TrxR with 200 µM NADH resulted in the inhibition of Grx-1 and naphthazarin reduction activity by 10-15%.It shows that reduced thiol groups do not participate in the reduction of quinones.Because the reduction of quinones and nitroaromatics involves a single-electron transfer step, this selectivity may be caused by the high endothermicity of a single-electron transfer from the dithiol moiety, i.e., the high redox potential of the thiyl radical/thiol redox couple, ~1.0 V (Wardman, 1990).

Presteady-state reactions of T. maritima TR
The preliminary prestady-state measurements of reduction of TR by excess NADH monitored at 460 nm show that the kinetics of the reaction are best described by a single-exponent fit.The reduction of FAD is characterized by a maximal rate of 82±3.0 s -1 , and the apparent bimolecular rate constant of TmTR reduction by NADH, 1.6±0.15× 10 6 M -1 s -1 (Fig. 3).The latter value is close to the k cat /K m for NADH, obtained in the steady-state experiments (Fig. 1).Typically, the absorbance of FAD at 460 nm decreased by 70-80% at the end of the reaction, as in the case of E. coli TR (Lennon et al., 1997).

Inhibition of quinone reductase reaction of TmTR by NAD +
The inhibition of NAD(P)H-oxidizing flavoenzymes by the reaction product, NAD(P) + , gives some information on the binding of NAD(P)H/NAD(P) + to the different redox states of the enzyme, and, in certain cases, on its redox equilibrium with the NAD(P) + /NAD(P)H couple (Vienožinskis et al., Bironaitė et al., 1997;Čėnas et al., 2004).In the case of T. maritima TR, NAD + acted as a mixed-type inhibitor towards NADH (Fig. 4A), i.e., it decreased both the maximal rate of the reaction and the k cat /K m for NADH.This means that NAD + not only competes with NADH for the binding to the oxidized enzyme form, but also competes with the quinoidal electron acceptor for the binding to the reduced enzyme form, or even reoxidizes the reduced enzyme (Rudolph et al., 1979).In agreement with this, NAD + acts as a mixed-type inhibitor towards naphthazarin (Fig. 4B), decreasing both its k cat /K m and the maximal reaction rate.Importantly, the inhibitory effect of NAD + towards the k cat /K m of naphthazarin decreased with an increase in NADH concentration from 30 µM to 100 µM (Fig. 5A).Besides, in the presence of 600 µM NAD + , varied naphthazarin concentrations at several fixed concentrations of NADH give a series of converging Lineweaver-Burk plots (Fig. 5B).This is in contrast with the series of parallel double-reciprocal plots obtained in the absence of NAD + (Fig. 1).Thus, based on the general effects of the reaction products on the reaction rates (Rudolph & Fromm, 1979;Rudolph, 1979), the data of Fig. 5A, B indicate that NAD + not only competes with naphthazarin for the binding to the reduced enzyme, but also reoxidizes the reduced enzyme at a rate comeasurable with the forward reaction, i.e., the reduction of the oxidized enzyme by NADH (Rudolph et al., 1979;Rudolph, 1979).Taken together, the data of Fig. 1, 4A, B, and 5A, B enable us to present a general scheme of quinone reduction by T. maritima TR (Scheme 1).Assuming a rapid equilibrium between the oxidized (E o ) and reduced (E r ) enzyme forms and NADH (S) and NAD + (P), Scheme 1 gives the following steady-state rate equation (Eqn.1), where Q is the electron acceptor: In the absence of NAD + ([P]=0), Eqn. 1 is reduced to Eqn. 2, which corresponds to a simple "ping-pong" scheme: Importantly, Eqn. 1 shows that the k cat /K m of reduction of quinone (k 3 k 4 /(k -3 + k 4 )) should decrease with an increase in the [P]/[S] ratio (i.e., the [NAD + ]/[NADH] ratio) which, in the case of the rapid redox equilibrium, corresponds to the redox potential of the medium, E h .(1) (2) We summarized the data from Fig. 4A, B, and 5A, B in Fig. 6, expressing k cat /K m for naphthazarin as a function of E h , and taking the standard redox potential for [NAD + ]/[NADH] couple as -0.32 V.The data of Fig. 6 show that 50% inhibition of quinone reductase activity expressed as k cat /K m takes place at E h = -0.31±0.03V.

DISCUSSION
In comparison with other L-TRs, the redox properties of TmTR are incompletely characterized so far, although some data point to the similarity of its properties to those of previously studied homologues.The sequence of TmTR (Genbank accession number TM_0869 or AAD35951) shows that its catalytic disulfide motif  which likely participates in the reduction of glutaredoxin-1 is similar to that of E. coli TR, which reduces thioredoxins (Lennon et al., 1999).The interdomain sequence motifs of E. coli TR (Gly-244, Pro-247) and A. thaliana TR (Gly-298, Pro-301) correspond to Gly-251 and Pro-254 in T. maritima TR.These data indirectly point to a possible similarity in the catalysis of those TRs, involving a transition between the flavin-to-disulfide-and flavin-to-NAD(P)H-binding site conformations (Dai et al., 1996;Lennon et al., 1997Lennon et al., , 1999)).
In this study, we aimed to characterize the redox properties of T. maritima TR, using quinones and nitroaromatic compounds as nopnphysiological oxidants.The comparison of presteady-and steady-state kinetic data of the enzyme (Fig. 1, 3, Table 1) shows that the oxidative half-reaction is rate-limiting in the catalysis, with a possible exception of 2,3-dichloro-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 9,10-phenanthrene quinone, where the reductive reaction may be partly rate-limiting.
There are also other similarities in the action of TmTR and A. thaliana TR, namely the mixed singleand two-electron reduction of quinones and nitroaromatics, which follow parabolic dependences of log k cat / K m on their E 1 7 (Table 1, Fig. 2) (Bironaitė et al., 1997;Miškinienė et al., 1998) This is in line with an "outersphere" electron transfer pathway (Marcus and Sutin,1985), or with a mixed one-and two-electron transfer (e -, H + , e -) with a rate limiting first electron transfer (Anusevičius et al., 2005).In this case, the systematic lower reactivity of nitroaromatic compounds may be attributed to their lower single-electron self-exchange rate constant (~10 5 M -1 s -1 ) (Meotner et al., 1986), in comparison to that of quinones (~10 8 M -1 s -1 ) (Grampp et al., 1987).These reactions contrast with the physiological reactions of L-TRs, the obligatory net two-electron (hydride) transfer from NAD(P)H to the disulfide oxidant.Nevertheless, the data on quinone-and nitroreductase reactions of T. maritima and A. thaliana L-TRs complement each other (Miškinienė et al., 1998) and show that the single-electron transfer is feasible in this group of pyridine nucleotide-disulfide reductases.It is indirectly supported by the stabilization of the neutral flavin semiquinone of E. coli TrxR under photoirradiation conditions (Zanetti et al., 1968).We suppose that these reactions may contribute to the prooxidant cytotoxicity of these compounds.
However, there are also some differences between the reactions of T. maritima and A. thaliana TRs.First, the quinone reductase reaction of A. thaliana TR is characterized by a competitive inhibition of NADP + vs. NADPH, and by an uncompetitive inhibition of NADP + vs. the quinone oxidant (Bironaitė et al., 1997).Besides, in the presence of NADP + , the "ping-pong" scheme of quinone reduction was unchanged, i.e., it was not converted into a series of converging double-reciprocal plots, like in Fig. 5B.This is in line with the low oxidation rate of reduced A. thaliana TR by NADP + , and the previously established E 0 7 of FAD of A. thaliana TR, -0.244 V (Bironaitė et al., 1997).In contrast, the inhibition of TmTR by NAD + (Fig. 4A, B, 5A, B) corresponds to the rapid establishment of the redox equilibrium between TR and NAD + /NADH (Scheme 1, Eqn. 1), thus pointing to a lower redox potential of FAD.The dependence of k cat /K m of naphthazarin on the [NAD + ]/ [NADH] ratio (Fig. 6) shows that the E 0 7 value of the FAD cofactor of T. maritima TR may be equal to -0.31±0.03V.This value may be taken as realistic, because it is close to the redox potential of the catalytic disulfide, -0.295 V (Couturier et al., 2013).Similar small differences between the E 0 7 values of FAD and the catalytic disulfide, about 0.02 V, were observed in potentiometric studies of E. coli L-TR (Williams, 1990).
In conclusion, our study preliminarily assessed the previously uncharacterized redox and kinetic properties of T. maritima L-TR.This enzyme, like the previously studied A. thaliana TR, may be considered as a potential target for redox active herbicides like 2,3-dichloro-1,4-naphthoquinone, the air pollution agent 9,10-phenanthrene quinone, and the explosives tetryl and TNT (Table 1), which are important soil and groundwater pollutants at their disposal sites (Bironaitė et al., 1997;Miškinienė et al., 1998;and references therein).These compounds may be considered as the "subversive substrates" for T. maritima TR because they convert the antioxidant functions of this enzyme into prooxidant ones due to their redox cycling.These reactions may be important in their action against T. maritima which, although anaerobic, can proliferate under low O 2 tension as well, and suffer from oxidative stress (Lefourn et al., 2008).These features may be even more important in view of the recently emerged interest in the use of T. maritima spp. in food, paper, and biofuel-related applications (Frock et al., 2010).

Figure 2 .
Figure 2. Reactivity of quinones and nitroaromatic compouns in T. maritima TR-catalyzed reactions.Relationship between log k cat /K m of quinones (solid circles) and nitroaromatic compounds (blank circles) in TmTR catalyzed reaction and their single-electron reduction potentials at pH 7.0 (E 1 7 ) is shown.Numbers refer to quinones and nitroaromatic compounds whose redox potentials and reduction rate constants are given in Table1.
. During the TmTR-catalyzed reduction of 50 µM 1,4-benzoquinone by 150 µM NADH, the rate of reduction of added 50 µM cytochrome c was equal to 50% of NADH oxidation rate.This shows that single-electron flux makes up 25% of the overall electron flux in this case.The quantitation of single-electron flux in the nitroreductase reactions of flavoenzymes is less straightforward, because both nitroaromatic anion-radicals and the products of their two(four)-electron reduction (hydroxylamines) reduce cytochrome c(Šarlauskas et al., 2004).During the TmTR-catalyzed oxidation of 100 µM NADH by 100 µM p-dinitrobenzene, the rate of reduction of added 50 µM cytochrome c was 160% of NADH oxidation rate.The cytochrome c reduction was inhibited by 100 U/mg superoxide dismutase (SOD) by 25%.In this case, the ArNO 2 /ArNO 2 and O

Figure 3 .
Figure 3. Presteady-state reduction of TmTR by NADH monitored at 460 nm.The dependence of pseudofirst-order presteady-state reduction rate constant (k) of reduction of TmTR on NADH concentration is shown.