Reactions of Plasmodium Falciparum Ferredoxin:NADP Oxidoreductase

International Journal of Molecular Sciences

Article Reactions of Plasmodium falciparum Ferredoxin:NADP+ Oxidoreductase with Redox Cycling Xenobiotics: A Mechanistic Study

Mindaugas Lesanaviˇcius 1, Alessandro Aliverti 2 , Jonas Šarlauskas 1 and Narimantas Cˇ enas˙ 1,* 1 Department of Xenobiotics Biochemistry, Institute of Biochemistry of Vilnius University, Sauletekio˙ 7, LT-10257 Vilnius, Lithuania; [email protected] (M.L.); [email protected] (J.Š.) 2 Department of Biosciences, Università degli Studi di Milano, via Celoria 26, I-20133 Milano, Italy; [email protected] * Correspondence: [email protected]; Tel.: +37-223-4392

Received: 6 April 2020; Accepted: 30 April 2020; Published: 2 May 2020

Abstract: Ferredoxin:NADP+ oxidoreductase from Plasmodium falciparum (Pf FNR) catalyzes the NADPH-dependent reduction of ferredoxin (Pf Fd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other ﬂavin-dependent electrontransferases, Pf FNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic N-oxides by Pf FNR. We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction 1 midpoint potential (E 7). The reactivity of nitroaromatics is lower than that of quinones and aromatic N-oxides, which is in line with the diﬀerences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH. to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of Pf FNR with various electron acceptors are similar to those of Anabaena FNR, thus demonstrating their similar “intrinsic” reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of Pf FNR, evidently providing an additional reduction pathway via reduced Pf Fd. Based on the available data, Pf FNR and possibly Pf Fd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.

Keywords: ferredoxin:NADP+ oxidoreductase; Plasmodium falciparum; quinones; nitroaromatic compounds; aromatic N-oxides; oxidative stress

1. Introduction The emergence of a malarial parasite Plasmodium falciparum resistance to available drugs, e.g., chloroquine or artemisinin ([1] and references therein), results in both a demand for new antimalarial agents and a better understanding of their mechanisms of action. P. falciparum is particularly vulnerable to oxidative stress, which might be caused by its lack of the antioxidant enzymes catalase and glutathione peroxidase [2]. For this reason, redox cycling compounds such as quinones, aromatic nitrocompounds and aromatic N-oxides, which frequently exhibit antiplasmodial in vitro activity at micromolar or lower concentrations, were a subject of great interest for a number of years ([3–8] and references therein). However, only fragmental data are available on their reactions with P. falciparum redox enzymes [6,9–11]. It is commonly accepted that the single-electron reduction of quinones and other classes of prooxidant compounds is performed by ﬂavin-dependent dehydrogenases-electrontransferases

Int. J. Mol. Sci. 2020, 21, 3234; doi:10.3390/ijms21093234 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3234 2 of 15

It is commonly accepted that the single-electron reduction of quinones and other classes of Int.prooxidant J. Mol. Sci. compounds2020, 21, 3234 is performed by flavin-dependent dehydrogenases-electrontransferases2 such of 15 as NADPH:cytochrome P-450 reductase (P-450R), ferredoxin:NADP+ oxidoreductase (FNR) or suchNO-synthase as NADPH:cytochrome (NOS) ([12–14] and P-450 references reductase therein) (P-450R),. These ferredoxin:NADP enzymes, working+ oxidoreductase in conjunction (FNR) with orphysiological NO-synthase single-electron (NOS) ([12–14 acceptors,] and references transform therein). a two-electron These enzymes, (hydride) working transfer in into conjunction a single- withelectron physiological one by stabilizing single-electron the neutral acceptors, (blue) semiquinone transform form a two-electron of the flavin (hydride) nucleotide transfer as the reaction into a single-electronintermediates [15–17]. one by stabilizing the neutral (blue) semiquinone form of the flavin nucleotide as the + reactionIn P. intermediates falciparum, an [15 FAD-containing–17]. ferredoxin:NADP oxidoreductase (PfFNR, EC 1.18.1.2) is localizedIn P. in falciparum a nonphotosynthetic, an FAD-containing plastid ferredoxin:NADPorganelle called +apicoplastoxidoreductase [18,19], (PfwhichFNR, performs EC 1.18.1.2) the isbiosynthesis localized in of a isoprenoids nonphotosynthetic and fatty plastid acids an organelled is essential called for apicoplast the parasite’s [18, survival.19], which PfFNR performs supplies the biosynthesisredox equivalents of isoprenoids to the apic andoplast fatty redox acids system and via is essential a Fe2S2-protein for the ferredoxin parasite’s ( survival.PfFd) [18]. PfPfFNRFd is characterized by a standard redox potential (E07.5) of -0.26 V and possesses about 50% amino acid supplies redox equivalents to the apicoplast redox system via a Fe2S2-protein ferredoxin (Pf Fd) [18]. sequence homology with plant ferredoxins [18]. PfFNR0 is characterized by E07 = −0.28 V [19]; it Pf Fd is characterized by a standard redox potential (E 7.5) of -0.26 V and possesses about 50% aminopossesses acid low sequence homology homology (20–30%) with with plant plant ferredoxins FNRs, displaying [18]. Pf uniqueFNR is characterizedlarge insertions by andE0 deletions= 0.28 7 − V[[20].19 ];The it possesses protein complex low homology formation (20–30%) is attributed with plant to the FNRs, electrostatic displaying interaction unique large between insertions the basic and deletionsresidues of [20 Pf].FNR The proteinand acidic complex residues formation of PfFd isand attributed is sensitive to the to electrostaticionic strength interaction [18,19,21]. between the basicPf residuesFNR reduces of Pf FNR quinones and acidic and nitroaromatic residues of Pf Fdcompounds and is sensitive in a single-electron to ionic strength way [18 and,,19, 21based]. on currentlyPf FNR available reduces data, quinones may be and considered nitroaromatic as an compounds important insource a single-electron of their radicals way in and, P. falciparum based on currently[6,11]. In availablethis work, data, we may extended be considered the studies as an of important PfFNR using source a oflarge their number radicals inof P.falciparumnonphysiological[6,11]. Inelectron this work, acceptors we extended with different the studies structures, of Pf FNR redu usingction a largepotentials number and of nonphysiologicalelectrostatic charges. electron Our acceptorsresults provide with di aff generalerent structures, insight into reduction their reduction potentials mechanisms and electrostatic and highlight charges. the Our specific results providefeatures aof general PfFNR insightrelevant into to theirthese reduction processes. mechanisms and highlight the specific features of Pf FNR relevant to these processes. 2. Results 2. Results 2.1. Steady-State Kinetics and Substrate Specificity Studies of PfFNR 2.1. Steady-State Kinetics and Substrate Specificity Studies of PfFNR In a previous study, juglone (5-hydroxy-1,4-naphthoquinone) was identified as one of the most activeIn nonphysiological a previous study, electron juglone (5-hydroxy-1,4-naphthoquinone)acceptors of PfFNR [6]. In this work was a identified series of asparallel one of lines the most was activeobtained nonphysiological in double-reciprocal electron plots acceptors at varied of PfconcFNRentrations [6]. In thisof juglone work a and series fixed of parallelconcentrations lines was of obtainedNADPH (Figure in double-reciprocal 1). This indicates plots that at variedthe quinone-reductase concentrations ofreaction juglone catalyzed and fixed by concentrations PfFNR follows of a NADPH“ping-pong” (Figure mechanism.1). This indicates As deduced that from the quinone-reductase Equation (A1) (Appendix reaction A), catalyzed the kcat value by Pf forFNR the followsjuglone −1 areduction “ping-pong” at an mechanism. infinite NADPH As deduced concentration from Equation is equal (A1) to 63.2 (Appendix ± 4.1 sA,), and the thekcat valuesvalue forof thethe 1 juglonebimolecular reduction rate constants at an infinite (or ca NADPHtalytic efficiency concentration constants, is equal kcat/ toKm 63.2) for NADPH4.1 s− , and and juglone the values are ofequal the 5 −1 −1 6 −1 −1 ± bimolecularto 6.0 ± 0.4 × rate 10 constantsM s and (or 1.1 catalytic ± 0.1 × 10 effi Mciencys , respectively. constants, kcat /Km) for NADPH and juglone are equal to 6.0 0.4 105 M 1s 1 and 1.1 0.1 106 M 1s 1, respectively. ± × − − ± × − − 0.16 6 0.14

0.12 5 4 0.10 3 2 0.08 1

[E]/V (s) 0.06

0.04

0.02

0.00 0.01 0.02 0.03 0.04 0.05 0.06 μ -1 1/[juglone] ( M ) FigureFigure 1.1. Steady-stateSteady-state kinetics of aa reductionreduction ofof juglonejuglone byby NADPHNADPH catalyzedcatalyzed byby PfPfFNR.FNR. NADPHNADPH concentrations:concentrations: 200200 µµMM (1),(1), 150150 µµMM (2),(2), 100100 µµMM (3),(3), 7575 µµMM (4),(4), 5050µ µMM(5) (5)and and25 25µ µMM (6). (6).

In order to assess the substrate speciﬁcity of Pf FNR, we examined the reduction of three series of electron acceptors, namely quinones (Q), nitroaromatic compounds (ArNO2) and aromatic N-oxides Int. J. Mol. Sci. 2020, 21, 3234 3 of 15

(ArN O), whose single-electron reduction midpoint potentials (E1 ) vary from 0.01 V to 0.575 V. → 7 − In addition, several single-electron acceptors, ferricyanide, Fe(EDTA)− and benzylviologen have been studied. The apparent reduction maximal rate constants, kcat(app), of electron acceptors at 100 µM NADPH, and their respective kcat/Km, are given in Table1. The kcat values for a number of less-active oxidants were not determined because of a nearly linear dependence of the reaction rate on their concentrations.

Table 1. Steady-state rate constants of the reduction of nonphysiological electron acceptors by NADPH

catalyzed by Pf FNR. (NADPH) = 100 µM, 0.1-M K-phosphate + 1.0-mM EDTA, pH 7.0, 25 ◦C.

1 1 1 1 No. Compound E 7 (V) [22–25] kcat(app) (s− ) kcat/Km (M− s− ) Quinones 1 2-Methyl-1,4-benzoquinone 0.01 32.4 4.1 2.8 0.2 105 ± ± × 2 2,5-Dimethyl-1,4-benzoquinone 0.07 26.0 2.3 1.8 0.2 105 − ± ± × 3 5-Hydroxy-1,4-naphthoquinone 0.09 35.7 5.1 1.1 0.1 106 − ± ± × 4 5,8-Dihydroxy-1,4-naphthoquinone 0.11 25.0 3.8 4.1 0.5 105 − ± ± × 5 9,10-Phenanthrene quinone 0.12 20.3 3.3 2.0 0.3 105 − ± ± × 6 1,4-Naphthoquinone 0.15 16.9 2.1 3.1 0.3 105 − ± ± × 7 2-Methyl-1,4-naphthoquinone 0.20 26.5 3.2 2.6 0.3 105 − ± ± × 8 Tetramethyl-1,4-benzoquinone 0.26 33.1 4.3 7.2 0.8 104 − ± ± × 9 Benzylviologen 0.354 4.8 0.6 2.1 0.1 104 − ± ± × 10 9,10-Anthraquinone-2-sulphonate 0.38 27.0 3.2 1.6 0.2 105 − ± ± × 11 2-Hydroxy-1,4-naphthoquinone 0.41 2.6 0.3 9.8 0.2 103 − ± ± × 12 2-Methyl-3-hydroxy-1,4-naphthoquinone 0.46 14.0 1.2 1.1 0.1 104 − ± ± × Nitroaromatic Compounds 13 Tetryl 0.191 40.0 5.1 2.1 0.4 105 − ± ± × 14 2,4,6-Trinitrotoluene a 0.253 1.3 0.3 104 − ± × 15 Nifuroxime a 0.255 3.3 0.2 104 − ± × 16 Nitrofurantoin a 0.255 6.8 0.5 104 − ± × 17 1,4-Dinitrobenzene 0.257 9.1 0.8 104 − ± × 18 1,2-Dinitrobenzene a 0.287 1.1 0.2 104 − ± × 19 5-Nitrothiophene-2-carbonic acid morpholide 0.305 2.0 0.2 104 − ± × 20 4-Nitrobenzaldehyde a 0.325 4.0 0.3 103 − ± × 21 3,5-Dinitrobenzoic acid a 0.344 3.9 0.5 103 − ± × 22 1,3-Dinitrobenzene a 0.348 2.7 0.2 103 − ± × 23 4-Nitroacetophenone a 0.355 3.2 0.4 103 − ± × 24 2-Nitrothiophene 0.390 2.2 0.2 103 − ± × 25 4-Nitrobenzoic acid a 0.425 4.5 0.4 102 − ± × 26 4-Nitrobenzyl alcohol 0.475 3.9 0.2 102 − ± × 27 Nitrobenzene a 0.485 5.5 0.6 101 − ± × Aromatic N-Oxides 28 7-CF -tirapazamine 0.345 11.5 2.0 5.2 0.4 104 3 − ± ± × 29 7-Cl-tirapazamine 0.400 14.8 1.3 3.7 0.4 104 − ± ± × 30 7-F-tirapazamine 0.400 2.7 0.2 104 − ± × 31 3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine) 0.456 4.4 0.5 103 − ± × 32 7-CH -tirapazamine 0.474 5.0 0.6 103 3 − ± × 33 7-C H O-tirapazamine 0.494 4.5 0.5 103 2 5 − ± × 34 3-Amino-1,2,4-benzotriazine-1-oxide 0.568 3.2 0.2 103 − ± × 35 Quinoxaline-1,4-dioxide 0.575 8.2 0.9 102 − ± × Inorganic Complexes 36 Ferricyanide b 0.41 47.9 4.0 3.0 0.4 106 ± ± × 4 37 Fe (EDTA)− 0.12 4.3 0.2 10 ± × a b Taken from Reference [11]. Calculated on a single-electron base. Catalytic eﬃciency constants, kcat/Km; reduction 1 maximal rate constants, kcat(app) and single-electron reduction midpoint potentials, E 7.

1 The log kcat/Km of ArNO2 exhibits a linear dependence on their E 7 (Table1 and Figure2). In general, the log kcat/Km values of quinones and aromatic N-oxides are higher than those of nitroaromatics and 1 are characterized by a parabolic dependence on their E 7 values (Figure3). It is important to note Int. J. Mol. Sci. 2020, 21, 3234 4 of 15

a Taken from Reference [11]. b Calculated on a single-electron base. Catalytic efficiency constants,

kcat/Km; reduction maximal rate constants, kcat(app) and single-electron reduction midpoint potentials, E17.

The log kcat/Km of ArNO2 exhibits a linear dependence on their E17 (Table 1 and Figure 2). In general, the log kcat/Km values of quinones and aromatic N-oxides are higher than those of Int. J. Mol. Sci. 2020, 21, 3234 4 of 15 nitroaromatics and are characterized by a parabolic dependence on their E17 values (Figure 3). It is important to note that the reactivity of the single-electron acceptor benzylviologen matches the thatreactivity the reactivity of quinones of the (Figure single-electron 2). acceptor benzylviologen matches the reactivity of quinones (Figure2).

3 6 4 7 6 10 17 1 13 5 28 16 5 2 29 8 m 19 K 15 / 12 30 9 cat 4 14 k 11 21 18 34 23 33 32 31 20 24 22 Log 3 35 25 26 2

-0.6 -0.4 -0.2 0.0 E1 (V) 7 FigureFigure 2. 2.Dependence Dependence ofof thethe reactivityreactivity ofof quinones,quinones, nitroaromaticnitroaromatic compounds compounds and and aromatic aromaticN N--oxidesoxides

onon their their single-electron single-electron reduction reduction midpoint midpoint potentials. potentials. Relationship Relationship between between the log k catthe/K mlogof quinoneskcat/Km of (solidquinones circles), (solid nitroaromatics circles), nitroaromatics (blank triangles) (blank triangles) and N-oxides and (blankN-oxides circles) (blank and circles) their and single-electron their single- Int. J. Mol. Sci. 2020, 21, 3234 1 5 of 15 reductionelectron reduction midpoint potentialsmidpoint atpotentials pH 7.0 (E at7). pH Numbers 7.0 (E and17). Numbers reduction potentialsand reduction of compounds potentials are of givencompounds in Table are1. given in Table 1. 5 Previously, we found that PfFNR reduces quinones and nitroaromatic4 compounds in a single- electron way [6,11]. Here, we found5 that the PfFNR-catalyzed oxidation3 of NADPH by 100–200 µM tirapazamine was accompanied 4by O2 consumption at a rate close to2 that of NADPH oxidation. The addition of 50-µM cytochrome c to the reaction mixture resulted in its reduction at a rate representing 3 1 180%–190% of that of NADPH oxidation. Superoxide dismutase (100 U/mL) inhibited the reduction [E]/V (s) of cytochrome c by 40%–65%. 2This shows that the single-electron flux in the PfFNR-catalyzed reduction of ArN→O is equal to1 90%–95% and that cytochrome c is reduced by their radicals, which are under a steady state with the O2/O2−. couple. Pyridine nucleotide analogues of NAD(P)0.005 0.010+ are frequently 0.015 0.020 used in the analysis of mechanisms of NAD(P)H-oxidizing flavoenzymes. The kcat of1/[AcPyP the transhydrogenase+] (μM-1) reaction of PfFNR, the formation of reduced 3-acetylpyridineadenine dinucleotide phosphate (AcPyPH) from AcPyP+ at the expense FigureFigure 3. Steady-state kinetics of the reduction of 3-ac 3-acetylpyridineadenineetylpyridineadenine dinucleotide phosphate by of NADPH, did not depend on the NADPH concentration, in the 25–200 µM range, and was equal to PfPfFNR. NADPH concentrations: 200 µMµM (1), 150 µµMM (2), 100 µµMM (3),(3), 5050 µµMM (4)(4) andand 2525 µµMM (5).(5). 3.2 ± 0.4 s−1 (Figure 3). However, in this case, the kcat/Km for the oxidant decreased with the increase in the Previously,NADPHThe affinity concentration of we Pf foundFNR for (Figure that its physiologicalPf FNR3). This reduces shows oxidant,quinones that Pf NADPHFd, decreases and acts nitroaromatic withas a competitivethe ionic compounds strength inhibitor of in the ato + single-electronmedium,AcPyP , occupyingdue to way the the [electrostatic6,11 pyridine]. Here, characternucleo we foundtide of binding their that theinteraction sitePf FNR-catalyzedof the [18,21]. reduced In enzymeorder oxidation to form.assess of NADPHAs the deduced role byof from Equation (A2) (Appendix A), the Kis of NADPH, describing the effect of NADPH on the slopes 100–200electrostaticµM interactions tirapazamine in wasthe reactions accompanied of PfFNR by O with2 consumption nonphysiological at a rate oxidants, close to we that examined of NADPH the + oxidation.effectsin the Lineweaver–Burkof Theionic addition strength of plots,on 50- µtheirM is cytochrome equal reduction to 140 crate to± 20 thes. µM, reactionIt was and reportedthe mixture kcat/K resultedmthat for AcPyPthe in NADPH-ferricyanide its reductionat (NADPH) at a = rate 0 is 3 −1 −1 representingreductaseequal to 9.3 reaction ± 180–190% 0.8 × of10 Pf M ofFNR thats .was of NADPH inhibited oxidation. by high concentratio Superoxidens dismutase of ferricyanide, (100 U/mL) which inhibited acted as the a reductioncompetitive of cytochromeinhibitor withc by respect 40–65%. to ThisNADPH shows (K thati = 230 the single-electronµM) [20]. However, flux in when the Pf FNR-catalyzedthe phosphate reductionbuffer was of used ArN insteadO is equal of 0.1-M to 90–95% Tris-HCl and [20], that the cytochrome substratec inhibitionis reduced by by ferricyanide their radicals, was which absent. are → . underThis enabled a steady us state to perform with the a Omore2/O2 thorough− couple. analysis of its reduction kinetics. + PyridineThe data nucleotideof Figure 4 analoguesshow a bell-shape of NAD(P) dependenceare frequently of log k usedcat/Km in for the ferricyanide, analysis of mechanismsFe(EDTA)- and of NAD(P)H-oxidizingbenzylviologen on the flavoenzymes. ionic strength The of kthecat ofsolution the transhydrogenase, irrespective of reactionthe opposite of Pf FNR,electrostatic the formation charge + ofof reducedthe latter 3-acetylpyridineadenine oxidant. In contrast, dinucleotide the phosphatekcat/Km for (AcPyPH) the fromuncharged AcPyP electron-acceptorat the expense of NADPH,tetramethyl-1,4-benzoquinone did not depend on the did NADPH not depend concentration, on the ionic in strength the 25–200 (FigureµM range,4). and was equal to 1 3.2 0.4 s (Figure3). However, in this case, the kcat/Km for the oxidant decreased with the increase ± − in the NADPH concentration (Figure3). This shows that NADPH acts as a competitive inhibitor to AcPyP+, occupying the pyridine nucleotide binding site of the reduced enzyme form. As deduced from Equation (A2) (AppendixA), the Kis of NADPH, describing the effect of NADPH on the slopes in 6 1 m K / cat

k 4 5 log

4 2 3

0.2 0.4 0.6 0.8 μ1/2 (M1/2) Figure 4. Effects of the ionic strength on the reactivity of PfFNR towards the electron acceptors. The

dependence of log kcat/Km for ferricyanide (1), Fe(EDTA)- (2), benzylviologen (3) and tetramethyl-1,4- benzoquinone (4) on the ionic strength of the phosphate buffer at pH 7.0 is shown.

2.2. Kinetics of PfFNR Oxidation under Multiple Turnover Conditions In order to get insight into the enzyme reoxidation mechanism, we investigated the spectral changes of PfFNR-bound FAD during its multiple turnover under aerobic conditions in the presence of NADPH and tetramethyl-1,4-benzoquinone. This electron acceptor does not absorb light at ≥460 nm, and its semiquinone form is rapidly reoxidized by oxygen [26]. In control experiments performed in the absence of quinone, the initial fast phase of FAD reduction by NADPH monitored at 460 nm is followed by its slower reoxidation by oxygen (Figure 5A). Importantly, this is accompanied by a transient increase in absorbance at 600 nm at the same time scale (Figure 5A). The addition of quinone accelerates the reoxidation of FADH- and the decay of the 600-nm absorbing

Int. J. Mol. Sci. 2020, 21, 3234 5 of 15

5 4 5 3

4 2

3 1 [E]/V (s) 2

0.005 0.010 0.015 0.020 1/[AcPyP+] (μM-1) Int. J. Mol. Sci. 2020, 21, 3234 5 of 15 Figure 3. Steady-state kinetics of the reduction of 3-acetylpyridineadenine dinucleotide phosphate by PfFNR. NADPH concentrations: 200 µM (1), 150 µM (2), 100 µM (3), 50 µM (4) and 25 µM (5). + the Lineweaver–Burk plots, is equal to 140 20 µM, and the kcat/Km for AcPyP at (NADPH) = 0 is ± equalThe to 9.3 affinity0.8 of10 Pf3FNRM 1 sfor1 .its physiological oxidant, PfFd, decreases with the ionic strength of the ± × − − medium,The a ffiduenity to ofthePf electrostaticFNR for its physiologicalcharacter of their oxidant, interactionPf Fd, decreases[18,21]. In withorder the to ionicassess strength the role ofof theelectrostatic medium, interactions due to the electrostatic in the reactions character of PfFNR of their with interaction nonphysiological [18,21]. oxidants, In order towe assess examined the role the ofeffects electrostatic of ionic interactions strength on in thetheir reactions reduction of PfrateFNRs. It with was nonphysiological reported that the oxidants, NADPH-ferricyanide we examined thereductase effects ofreaction ionic strength of PfFNR on was their inhibited reduction by rates.high concentratio It was reportedns of that ferricyanide, the NADPH-ferricyanide which acted as a reductasecompetitive reaction inhibitor of Pf withFNR respect was inhibited to NADPH by high (Ki = concentrations 230 µM) [20]. ofHowever, ferricyanide, when which the phosphate acted as abuffer competitive was used inhibitor instead with of 0.1-M respect Tris-HCl to NADPH [20], (theKi =substrate230 µM) inhibition [20]. However, by ferricyanide when the was phosphate absent. buThisffer enabled was used us insteadto perform of 0.1-M a more Tris-HCl thorough [20 analysis], the substrate of its reduction inhibition kinetics. by ferricyanide was absent. This enabledThe data us of to Figure perform 4 show a more a bell-shape thorough dependence analysis of its of reductionlog kcat/Kmkinetics. for ferricyanide, Fe(EDTA)- and benzylviologenThe data of Figureon the 4ionic show strength a bell-shape of the dependencesolution, irrespective of log kcat of/K them for opposite ferricyanide, electrostatic Fe(EDTA) charge− andof benzylviologenthe latter oxidant. on the ionicIn strengthcontrast, ofthe the solution,kcat/Km irrespectivefor the uncharged of the opposite electron-acceptor electrostatic chargetetramethyl-1,4-benzoquinone of the latter oxidant. did In not contrast, depend on the thekcat ionic/Km strengthfor the (Figure uncharged 4). electron-acceptor tetramethyl-1,4-benzoquinone did not depend on the ionic strength (Figure4).

6 1 m K / cat

k 4 5 log

4 2 3

0.2 0.4 0.6 0.8 μ1/2 (M1/2) FigureFigure 4.4. EffectsEﬀects of of the the ionic ionic strength strength on on the the reactivity reactivity of ofPfFNR PfFNR towards towards the theelectron electron acceptors. acceptors. The

Thedependence dependence of log of kcat log/Km kforcat /ferricyanideKm for ferricyanide (1), Fe(EDTA) (1),- Fe(EDTA)(2), benzylviologen− (2), benzylviologen (3) and tetramethyl-1,4- (3) and tetramethyl-1,4-benzoquinonebenzoquinone (4) on the ionic (4)strength on the of ionic the strengthphosphate of thebuffer phosphate at pH 7.0 bu isff showner at pH. 7.0 is shown. 2.2. Kinetics of PfFNR Oxidation under Multiple Turnover Conditions 2.2. Kinetics of PfFNR Oxidation under Multiple Turnover Conditions In order to get insight into the enzyme reoxidation mechanism, we investigated the spectral In order to get insight into the enzyme reoxidation mechanism, we investigated the spectral changes of Pf FNR-bound FAD during its multiple turnover under aerobic conditions in the presence of changes of PfFNR-bound FAD during its multiple turnover under aerobic conditions in the presence NADPH and tetramethyl-1,4-benzoquinone. This electron acceptor does not absorb light at 460 nm, of NADPH and tetramethyl-1,4-benzoquinone. This electron acceptor does not absorb≥ light at and its semiquinone form is rapidly reoxidized by oxygen [26]. In control experiments performed in the ≥460 nm, and its semiquinone form is rapidly reoxidized by oxygen [26]. In control experiments absence of quinone, the initial fast phase of FAD reduction by NADPH monitored at 460 nm is followed performed in the absence of quinone, the initial fast phase of FAD reduction by NADPH monitored by its slower reoxidation by oxygen (Figure5A). Importantly, this is accompanied by a transient at 460 nm is followed by its slower reoxidation by oxygen (Figure 5A). Importantly, this is increase in absorbance at 600 nm at the same time scale (Figure5A). The addition of quinone accelerates accompanied by a transient increase in absorbance at 600 nm at the same time scale (Figure 5A). The the reoxidation of FADH and the decay of the 600-nm absorbing species by about two orders of addition of quinone accelera− tes the reoxidation of FADH- and the decay of the 600-nm absorbing magnitude (Figure5B). This shows that, under our experimental conditions, O 2 plays a negligible role in the kinetics of enzyme reoxidation. This process is also accompanied by a transient increase in absorbance at 600 nm. The kinetics of reoxidation were analyzed by the method of chance [27] using Equation (1), where kox is the apparent first-order rate constant of enzyme reoxidation, (NADPH)0 is the initial NADPH concentration, (Ered)max is the maximal concentration of the reduced enzyme formed during the turnover and t1/2(off) is the time interval between the formation of the half-maximal amount of Ered and its decay to the half-maximal value:

[NADPH] kox = 0 (1) [E ] t red max· 1/2(off) Int. J. Mol. Sci. 2020, 21, 3234 6 of 15 species by about two orders of magnitude (Figure 5B). This shows that, under our experimental conditions, O2 plays a negligible role in the kinetics of enzyme reoxidation. This process is also accompanied by a transient increase in absorbance at 600 nm. The kinetics of reoxidation were analyzed by the method of chance [27] using Equation (1), where kox is the apparent first-order rate constant of enzyme reoxidation, (NADPH)0 is the initial NADPH concentration, (Ered)max is the maximal concentration of the reduced enzyme formed during the turnover and t1/2(off) is the time interval between the formation of the half-maximal amount of Ered and its decay to the half-maximal value:Int. J. Mol. Sci. 2020, 21, 3234 6 of 15 [NADPH] ox = (1) [E] ∙t/() It was assumed that complete FAD reduction corresponds to the maximal ∆A460 after the enzyme It was assumed that complete FAD reduction corresponds to the maximal ∆A460 after the enzyme mixingmixing with with NADPH NADPH in the in absence the absence of quinone of quinone (Figure (Figure 5A). This5A). 460-nm This 460-nm absorbance absorbance change change was was 1 1 close to that expected using the value of ∆ε = 7.8−1 mM−1 cm for the absorbance difference between close to that expected using the value of ∆ε460 = 4607.8 mM cm − for the− absorbance difference between thethe oxidized oxidized and and two-electron two-electron reduced reduced PfFNRPf FNR[19,28,29]. [19,28 For,29 ].the For reoxidation the reoxidation of PfFNR of PfwithFNR oxygen with oxygen 1 (Figure5A), we obtained a kox = 0.18 −0.021 s , which was close to the enzyme NADPH oxidase activity (Figure 5A), we obtained a kox = 0.18 ± 0.02± s , which− was close to the enzyme NADPH oxidase activity underunder a steady a steady state. state. The Thedependence dependence of kox of onkox theon tetramethyl-1,4-benzoquinone the tetramethyl-1,4-benzoquinone concentration concentration 5 1 1 (Figure(Figure 5C)5 C)gives gives an anapparent apparent bimolecular bimolecular rate rate constant constant of 1.34 of1.34 ± 0.37 ×0.37 105 M10−1s−1M, which− s− ,is which comparable is comparable ± × 1 withwith the the steady-state steady-state kcat/kKcatm /forKm thisfor oxidant this oxidant (Table (Table 1) and1) andkox(max)k = 155 ±= 32155 s−1. However,32 s . However, the later the later ox(max) ± − valuevalue may may lack lack sufficient sufficient accuracy, accuracy, because because it wa its impossible was impossible to obtain toobtain a saturating a saturating concentration concentration (sufficiently(sufficiently high high kox values)kox values) due to due the to limited the limited solubility solubility of the ofoxidant. the oxidant.

0.01 0.01 1 A 1 B C 0.00 0.06 0.00 -0.01 2 ) -1 A (s -0.02 A 0.04 Δ Δ

2 ox -0.01 -0.03 1/k 0.02 -0.04 -0.02 -0.05 0 102030405060 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.000 0.002 0.004 0.006 0.008 0.010 Time (s) Time (s) 1/[Q] (μM-1)

Figure 5. Turnover of PfFNR in the presence of NADPH and oxidants. (A,B) The kinetics of the absorbance changes at 600 nm (1) and 460 nm (2) during the reduction of Pf FNR (6.0 µM) by 50-µM NADPH and its subsequent reoxidation by oxygen (A) or 250-µM tetramethyl-1,4-benzoquinone (B) (concentrations after mixing). (C) The dependence of an apparent first-order reoxidation rate constant on the concentration of tetramethyl-1,4-benzoquinone. 2.3. NADP+ Inhibition Studies 2.3. NADP+ Inhibition Studies At a fixed juglone concentration, the reaction product NADP+ acted as a competitive inhibitor At a fixed juglone concentration, the reaction product NADP+ acted as a competitive inhibitor towards NADH (Figure 6A) with Kis = 1.42 ± 0.13 mM, as deduced from Equation (A2) (Appendix A). towards NADH (Figure6A) with K = 1.42 0.13 mM, as deduced from Equation (A2) (AppendixA). In turn, at a fixed concentration of 50 µMis NADH,± NADP+ acted as an uncompetitive inhibitor towards In turn, at a fixed concentration of 50 µM NADH, NADP+ acted as an uncompetitive inhibitor towards juglone (Figure 6B) with Kii = 1.83 ± 0.19 mM, as deduced from Equation (A3) (Appendix A), describingjuglone (Figurethe effects6B) of with NADPKii =+ on1.83 the intercepts0.19 mM, asin deducedthe Lineweaver–Burk from Equation plots. (A3) As (Appendix comparedA ),with describing + ± thethe previous effects data of NADP obtainedon in the 0.05-M intercepts Hepe ins, thethe Lineweaver–Burkuse of 0.1-M phosphate plots. Asdecreased compared the withkcat/K them for previous NADPHdataInt. J. Mol. obtained and Sci. increased 2020 in, 21 0.05-M, 3234 the Kis Hepes, of NADP the+ almost use of 0.1-Mby one phosphateorder of magnitude decreased [28,29]. the k cat/Km for NADPH7 andof 15 + increased the Kis of NADP almost by one order of magnitude [28,29].

1.0 A 0.10 5 B 5 0.8 4 0.08 4 3 3 0.6 0.06 2 1 2 [E]/V (s)

[E]/V (s) [E]/V 0.4 0.04 1 0.2 0.02

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.00 0.01 0.02 0.03 0.04 0.05 0.06 -1 1/[NADPH] (μM ) 1/[juglone] (μM-1) Figure 6.6. InhibitionInhibition ofof thethe juglonejuglone reductasereductase reactionreaction ofofPf PfFNRFNR byby NADPNADP++.(. (A) InhibitionInhibition atat variedvaried NADPH concentrations in the presence of 100-100-µMµM juglone,juglone, and ((B)) inhibitioninhibition atat variedvaried juglonejuglone concentrationsconcentrations in thethe presencepresence ofof 100-100-µMµM NADPH,NADPH, NADPNADP++ concentrations: 0 0 mM mM (1), (1), 1.0 mM (2), 2.0 mMmM (3),(3), 3.03.0 mMmM (4)(4) andand 5.05.0 mMmM (5).(5).

2.4. Stimulation of Quinone- and Nitroreductase Activity of PfFNR by Ferredoxin

The complex of PfFNR with PfFd is characterized by micromolar Kd values [19,21]. Therefore, it is important to characterize the reduction of nonphysiological electron acceptors in the presence of both redox proteins. PfFd stimulated the reduction of quinones and nitroaromatics by PfFNR, concomitantly causing a biphasicity of the corresponding Lineweaver-Burk plots (Figure 7A). The maximal rates of their “slower” phase (low concentrations and higher kcat/Km of the oxidant) were close to the rates of cytochrome c reduction at corresponding PfFd concentrations (Figure 7B), which, in turn, are equal to the rate of PfFd reduction by PfFNR. In 0.1-M K-phosphate, pH 7.0, the maximal rate of this reaction at saturating the PfFd concentration is 16 ± 2.5 s−1, on the one-electron base, which is close to the previously determined values, 13–15 s−1 [19]. The Km(app) for PfFd is 5.2 ± 1.3 µM, a value significantly higher than that previously reported [19], which may be attributed to the higher ionic strength of the medium.

1 2 1.0 6 A B 5 0.8

4 4 0.6 3 3 3

[E]/v (s) [E]/v 0.4 [E]/V (s) 2 4 2 1 0.2 1

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 -1 μ -1 1/[Concentration] (μM ) 1/[PfFd] ( M ) Figure 7. Stimulation of quinone- and nitroreductase reactions of PfFNR by ferredoxin. (A) The dependence of the PfFNR-catalyzed NADPH oxidation rate on the concentration of p-nitroacetophenone (1,4) or 2-hydroxy-1,4-naphthoquinone (2,3) in the absence of PfFd (1,2), and in the presence of 1.7-µM (3) or 4.0-µM (4) PfFd; concentration of NADPH is 100 µM. (B) The dependence of the cytochrome c reduction rate by PfFNR on the concentration of PfFd (solid circles), concentration of NADPH, 100 µM and concentration of cytochrome c, 50 µM. Blank circles show the doubled maximal rates of the ”slower” phase of NADPH oxidation in the presence of tetramethyl- 1,4-benzoquinone (1), p-nitroacetophenone (2,4) and 2-hydroxy-1,4-naphthoquinone (3) at corresponding concentrations of PfFd. The maximal rates were obtained by the fitting of kinetic data of the “slower” phase (5–6 lower concentrations of oxidant) to the parabolic expression.

Int. J. Mol. Sci. 2020, 21, 3234 7 of 15

1.0 A 0.10 5 B 5 0.8 4 0.08 4 3 3 0.6 0.06 2 1 2 [E]/V (s)

[E]/V (s) [E]/V 0.4 0.04 1 0.2 0.02

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.00 0.01 0.02 0.03 0.04 0.05 0.06 -1 1/[NADPH] (μM ) 1/[juglone] (μM-1) Figure 6. Inhibition of the juglone reductase reaction of PfFNR by NADP+. (A) Inhibition at varied NADPH concentrations in the presence of 100-µM juglone, and (B) inhibition at varied juglone concentrations in the presence of 100-µM NADPH, NADP+ concentrations: 0 mM (1), 1.0 mM (2), Int. J. Mol.2.0 mM Sci. 2020(3), ,3.021, mM 3234 (4) and 5.0 mM (5). 7 of 15

2.4. Stimulation of Quinone- and Nitroreductase Activity of PfFNR by Ferredoxin 2.4. Stimulation of Quinone- and Nitroreductase Activity of PfFNR by Ferredoxin The complex of PfFNR with PfFd is characterized by micromolar Kd values [19,21]. Therefore, it is importantThe complex to characterize of Pf FNR with the PfreductionFd is characterized of nonphy bysiological micromolar electronKd values acceptors [19, 21in]. the Therefore, presence it isof importantboth redox to proteins. characterize PfFd the stimulated reduction ofthe nonphysiological reduction of quinones electron acceptorsand nitroaromatics in the presence by ofPfFNR, both redoxconcomitantly proteins. PfcausingFd stimulated a biphasicity the reduction of the corresp of quinonesonding and Lineweaver-Burk nitroaromatics by plotsPf FNR, (Figure concomitantly 7A). The causing a biphasicity of the corresponding Lineweaver-Burk plots (Figure7A). The maximal rates of maximal rates of their “slower” phase (low concentrations and higher kcat/Km of the oxidant) were theirclose “slower”to the rates phase of cytochrome (low concentrations c reduction and at highercorrespondingkcat/Km of Pf theFd concentrations oxidant) were close(Figure to the7B), rates which, of cytochromein turn, are equalc reduction to the atrate corresponding of PfFd reductionPf Fd concentrationsby PfFNR. In 0.1-M (Figure K-phosphate,7B), which, inpH turn, 7.0, arethe equalmaximal to therate rate of this of Pf reactionFd reduction at saturating by Pf FNR. the Pf InFd 0.1-M concentration K-phosphate, is 16 pH± 2.5 7.0, s−1 the, on maximalthe one-electron rate of this base, reaction which 1 at saturating the Pf Fd concentration is 16 2.5 s− ,−1 on the one-electron base, which is close to the is close to the previously determined values,± 13–15 s [19]. The Km(app) for PfFd is 5.2 ± 1.3 µM, a value previously determined values, 13–15 s 1 [19]. The K for Pf Fd is 5.2 1.3 µM, a value signiﬁcantly significantly higher than that previously− reportedm(app) [19], which may be ±attributed to the higher ionic higherstrength than of the that medium. previously reported [19], which may be attributed to the higher ionic strength of the medium.

1 2 1.0 6 A B 5 0.8

4 4 0.6 3 3 3

[E]/v (s) [E]/v 0.4 [E]/V (s) 2 4 2 1 0.2 1

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 -1 μ -1 1/[Concentration] (μM ) 1/[PfFd] ( M ) FigureFigure 7.7. Stimulation of quinone- and nitroreductasenitroreductase reactionsreactions ofof PfPfFNR by ferredoxin.ferredoxin. ((AA)) TheThe dependencedependence of theof PftheFNR-catalyzed PfFNR-catalyzed NADPH NADPH oxidation rateoxidation on the concentrationrate on the of p -nitroacetophenoneconcentration of (1,4)p-nitroacetophenone or 2-hydroxy-1,4-naphthoquinone (1,4) or 2-hydroxy-1,4- (2,3) innaphthoquinone the absence of Pf (2,3)Fd (1,2),in the and absence in the of presence PfFd (1,2), of 1.7- andµ Min (3)the or presence 4.0-µM (4)of Pf1.7-µMFd; concentration (3) or 4.0-µM of NADPH (4) PfFd; is 100concentrationµM. (B) The of dependence NADPH is of 100 the cytochromeµM. (B) Thec reductiondependence rate of by thePf cytochromeFNR on the concentrationc reduction rate of PfbyFd PfFNR (solid on circles), the concentration concentration of of Pf NADPH,Fd (solid 100circles),µM andconcentration concentration of NADPH, of cytochrome 100 µMc, 50 andµM. concentration Blank circles of show cytochrome the doubled c, 50 maximal µM. Blank rates circles of the show ”slower” the phasedoubled of NADPHmaximal oxidation rates of the in the”slower” presence phase of tetramethyl-1,4-benzoquinone of NADPH oxidation in the presence (1), p-nitroacetophenone of tetramethyl- (2,4)1,4-benzoquinone and 2-hydroxy-1,4-naphthoquinone (1), p-nitroacetophenone (3) at corresponding(2,4) and 2-hydroxy-1,4-naphthoquinone concentrations of Pf Fd. The maximal (3) at ratescorresponding were obtained concentrations by the ﬁtting of Pf ofFd. kinetic The maximal data of the rates “slower” were obtained phase (5–6 by the lower fitting concentrations of kinetic data of oxidant)of the “slower” to the parabolic phase (5–6 expression. lower concentrations of oxidant) to the parabolic expression. 3. Discussion

These results provide a general insight into the mechanism of reduction of several groups of nonphysiological oxidants by Pf FNR and complement the data on the mechanism of its interaction with the physiological electron-acceptor Pf Fd [18,19]. First, let us consider the possible rate-limiting step of the reaction. Since Pf FNR follows a ping-pong mechanism (Figure1), its reductive and oxidative half-reactions can proceed independently, with the kcat expressed as 1/kcat = 1/kred(max) + 1/kox(max), where kred(max) and kox(max) are the maximal rates of the reductive and oxidative half-reactions, respectively. Our data show that, in the reduction of tetramethyl-1,4-benzoquinone, the overall catalytic process should be partly limited by both oxidative and reductive half-reactions, because the maximal rate of Pf FNR reduction by NADPH at pH 7.0 is 125–148 s 1 [19,29], and a comparable value, k = 155 32 s 1, − ox(max) ± − was obtained for tetramethyl-1,4-benzoquinone under multiple turnover conditions (Figure5C). On the other hand, the rate of the enzyme reduction with a fixed concentration of NADPH should be the same, irrespectively of the oxidant used, and the observed kcat(app) differences (Table1) should be attributed to different maximal rates of reoxidation. Thus, for oxidants 1–4, 7, 10 and 13, whose kcat(app) values Int. J. Mol. Sci. 2020, 21, 3234 8 of 15 are close to that of tetramethyl-1,4-benzoquinone (Table1), the overall catalytic process should be partly limited by both oxidative and reductive reactions, as well. The lower values of kcat(app) for other compounds (Table1) imply that, in these cases, the process is limited by the oxidative half-reaction. The nature of these differences will be the object of our future studies. Since reduced PfFNR is reoxidized in a single-electron fashion, the oxidative half-reaction must proceed through the two steps of FADH FADH. and FADH. FAD. There are several arguments − → → supporting FAD semiquinone oxidation as a possible rate-limiting step of the overall process: (a) The 600-nm absorbing species is formed during the reoxidation of reduced Pf FNR by quinones, which may point to a transient FADH. accumulation [13,30] (Figure5B); (b) NADPH acts as a competitive inhibitor with respect to the oxidant in the reduction of AcPyP+ by Pf FNR (Figure3). In contrast, NADPH does not inhibit quinone reduction (Figure1). This shows that quinones and AcPyP + oxidize different redox forms of Pf FNR, which possess different affinities for NADPH. Since AcPyP+ is an obligatory two-electron (hydride) acceptor, it can be reduced only by a two-electron reduced FAD. This argues against its involvement as a rate-limiting step in quinone reduction, and (c) NADP+ acts as a competitive inhibitor with respect to NADPH (Figure6A) and as an uncompetitive inhibitor with respect to quinones (Figure6B). This may be attributed to a specific case of a ping-pong mechanism, where NADP + binds relatively tightly to the oxidized enzyme form but binds weakly or not at all to its reduced state [31]. Since NADP+ binds tightly to the two-electron reduced form of Pf FNR [28,29], this also argues against its involvement as a rate-limiting step in quinone reduction. Thus, Pf FNR may have properties in common with FNRs from Anabaena PCC7118. and from spinach, where the oxidation of FADH. is the rate-limiting step of reactions with various nonphysiological electron acceptors [13,30,32–34]. Moreover, our study discloses some specific properties of Pf FNR relevant to the reduction of nonphysiological redox agents. The experiments at varied ionic strengths may characterize the surface region of Pf FNR that interacts with charged oxidants. In this regard, the observed bell-shape dependences of the reactivity of oppositely charged oxidants on the ionic strength of the solution (Figure4) were unexpected. Similar, although less pronounced, dependences were observed in the reactions of FNR from Anabaena PCC7118 [13]. A possible explanation of this phenomenon is that the oxidants may interact with both the negatively charged Glu-314 (Glu-301 in Anabaena PCC7118 FNR [18]) and positively charged Lys-287 (Arg-274 in Anabaena PCC7118 FNR), the latter participating in the binding of Pf Fd, which are located close to the dimethylbenzene part of the isoalloxazine ring [20,21]. This is in line with the finding that ferricyanide competes with the nicotinamide mononucleotide part of NADP+ for binding close to the FAD isoalloxazine ring [20]. Other positively charged residues participating in the binding of Pf Fd, such as Arg-98, Arg-290 and Lys-308, are too distant from the isoalloxazine ring [21] and may not be likely to interact with low molecular weight oxidants. Our data point to an absence of a strict substrate specificity in the reduction of quinones, ArNO2, and ArN O by Pf FNR, with the exception of an increase in their log kcat/Km with an increase in 1 → E 7 (Figure3). The latter feature points to an “outer-sphere” electron transfer mechanism of their reduction, which is established for FNR from Anabaena PCC7119, P-450R and NOS [12–14]. According to this mechanism, the bimolecular rate constant of the electron transfer between the reactants (k12) is expressed as: k = (k k K f )1/2, (2) 12 11 × 22 × × where k11 and k22 are the electron self-exchange rate constants of the reactants, K is the equilibrium constant of the reaction (log K = ∆E1/0.059 V) and f is expressed as:

log f = (log K)2/4log (k k /Z2), (3) 11 × 22 11 1 1 where Z is the frequency factor, 10 M− s− [35]. According to Equations (2 and 3), in the reaction of the electron donor with a series of homologous electron acceptors (which display the same k22), log k will exhibit a parabolic (quadratic) dependence on ∆E1 with a slope 8.45 V 1 at ∆E1 = 0.15 V. 12 − ± In particular, the lower reactivity of ArNO as compared with quinones and ArN O, which possess 2 → Int. J. Mol. Sci. 2020, 21, 3234 9 of 15

1 6 1 1 similar E 7 values (Figure3), is explained by their k22 = ~10 M− s− [36], which are much lower than those of quinones and ArN O, ~108 M 1s 1 [36,37]. → − − According to the model of Mauk et al. [38], at an inﬁnite ionic strength, where electrostatic interactions are absent, the k11 value of metalloproteins for the reactions with the inorganic complexes is related to the distance of the electron transfer, Rp:

Rp (Å) = 6.3 0.35 ln k (4) − 11 We have applied this approach for the analysis of the single-electron oxidation of P-450R, FNR from Anabaena PCC7118 and NOS by quinones, nitroaromatics and inorganic complexes [12–14]. Their reactions with Q and ArNO2 are characterized by Rp ranging from 3.4 to 5.0 Å, whereas, for the reactions with hydrophilic ferricyanide and Fe(EDTA)−, which are incapable of entering the protein globule, the Rp values are much higher (Table2). However, it is possible that this procedure gives slightly overestimated distances in the case of flavoproteins, since the dimethylbenzene part of the flavin isoalloxazine ring in P-450R, FNR and NOS is partly exposed to the solvent [39–41]. Thus, these values may be useful only for an approximate assessment of the “intrinsic” flavoenzyme reactivity. 1 The estimation of k11 and Rp for the reactions of Pf FNR is complicated by the unknown E 7 value for the FAD/FADH. couple. Based on the available data [19], the FAD semiquinone state in Pf FNR is not . stabilized. Thus, the values of k11 were calculated tentatively assuming 15% and 5% FAD formations at the equilibrium (AppendixB). The resulting values of Rp for Pf FNR are given in Table2.

Table 2. Distances of the electron transfer (Rp) in reactions of ﬂavin-dependent electrontransferases with nonphysiological electron acceptors, calculated according to Equation (4).

R (Å) Flavoprotein Reaction p 3 Q ArNO2 Fe (CN) −6 Fe (EDTA)− FMNH e FMNH., P-450R, rat [12] − − − → 3.4 4.2 8.1 7.3 E1 = 0.270 V 7 − FMNH e FMNH., n-NOS, rat [14] − − − → 4.7 3.9 - - E1 = 0.274 V 7 − FNR, FADH. e H+ FAD, Anabaena − − − → 5.0 4.4 9.2 10.4–11.4 E1 = 0.280 V PCC7118 a 7 − FADH. e H+ FAD, − − − → 4.8 4.9 9.5 9.1 E1 = 0.308 V b Pf FNR, this 7 − . + work FADH e− H FAD, − − → 5.0 5.6 9.8 9.4 E1 = 0.337 V c 7 − b . b,c Calculated according to the data of Reference [13], using the value of E7 (FAD/FADH ) = 0.280 V [30]. The values 1 . − of E 7 are calculated assuming 15% and 5% FADH stabilizations at the equilibrium, respectively.

Typically, they are larger than those of P-450R but close to those of FNR from Anabaena PCC7118. This shows that low molecular weight oxidants may access the FAD isoalloxazine ring of both representatives of FNR with similar ease, in spite of some differences in their surroundings [20,21,41]. Finally, the stimulation of the nonphysiological acceptor reductase reactions of PfFNR by PfFd (Figure 7A,B) shows that PfFd provides an alternative pathway for their reduction via reduced PfFd. Since the redox potentials of both proteins are similar [18,19], this may be most easily explained by a better accessibility of the active center of PfFd. This phenomenon has been previously observed in reactions of bovine adrenodoxin reductase and adrenodoxin and Anabaena PCC7118 FNR and Fd [13,42]; thus, it may be a general feature of this group of redox proteins. In conclusion, our comprehensive study characterized the mechanism of reactions of PfFNR withInt. J. Mol.redox-cycling Sci. 2020, 21, 3234 xenobiotics, which may be instrumental in the further development10 ofof 15 redox-active antiplasmodial agents [3–8]. In terms of kcat/Km for the reduction of quinones and nitroaromatics (Table 1 and Figure 2), the reactivity of PfFNR is considerably higher as compared to other(Table P.1 and falciparum Figure2 ),flavoenzymes, the reactivity ofglutathionePf FNR is reductase, considerably thioredoxin higher as comparedreductase toand other typeP. falciparum2 NADH dehydrogenaseﬂavoenzymes, glutathione [9,10]. To the reductase, best of our thioredoxin knowledge, reductase the reactions and type of the 2 NADH above groups dehydrogenase of xenobiotics[9,10] . withTo the other best electrontransferases of our knowledge, of the the reactions P. falciparum of the mitochondrial above groups respiratory of xenobiotics chain, with namely other dihydroorotateelectrontransferases dehydrogenase, of the P. falciparum succinate mitochondrialdehydrogenase respiratory and malate:quinone chain, namely oxidoreductase, dihydroorotate have notdehydrogenase, been studied succinateso far. Thus, dehydrogenase based on the andavailable malate:quinone data, PfFNR oxidoreductase, and possibly Pf haveFd may not play been a studiedcentral roleso far. in the Thus, reductive basedon activation the available of pro-oxidant data, Pf FNR xenobiotics and possibly relevantPf Fd for may malaria play chemotherapy. a central role in This the alsoreductive points activation to a versatility of pro-oxidant of the properties xenobiotics of Pf relevantFNR, which for malaria may be chemotherapy. relevant for the This design also points of new to antiplasmodiala versatility of theagents, properties because of PfanotherFNR, which intriguing may be approach relevant forto this the designtask is of the new development antiplasmodial of compoundsagents, because that may another bind intriguing at the PfFNR- approachPfFd interface to this and task inhibit is the the development physiological of reduction compounds of Pf thatFd [43,44].may bind Thus, at the anPf attractiveFNR-Pf Fd possibility interface andcould inhibit be the the derivatization physiological reductionof these ligands, of Pf Fd [chalcones43,44]. Thus, or an attractive possibility could be the derivatization of these ligands, chalcones or alkaloids [43,44] alkaloids [43,44] by redox-active quinone, ArNO2 or ArN→O moieties. These hybrid molecules may by redox-active quinone, ArNO or ArN O moieties. These hybrid molecules may combine two combine two mechanisms of antiplasmodial2 → action: the inhibition of the electron supply to the apicoplastmechanisms redox of antiplasmodial system and redox action: cycling. the inhibition of the electron supply to the apicoplast redox system and redox cycling. 4. Materials and Methods 4. Materials and Methods

4.1.4.1. Enzymes Enzymes and and Reagents Reagents Recombinant P. falciparum ferredoxin:NADP+ oxidoreductase and ferredoxin were prepared as Recombinant P. falciparum ferredoxin:NADP+ oxidoreductase and ferredoxin were prepared previously described [19], and their concentrations were determined spectrophotometrically as previously described [19], and their concentrations were determined spectrophotometrically −1 −1 −1 −1 according to ε454 = 10.0 mM 1 cm 1 and ε424 = 9.68 mM 1 cm 1, respectively. Compounds according to ε454 = 10.0 mM− cm− and ε424 = 9.68 mM− cm− , respectively. Compounds 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl) were synthesized as 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl) were synthesized described [45]. The 5-Nitrothiophene-2-carbonic acid morpholide was synthesized as described [25]. as described [45]. The 5-Nitrothiophene-2-carbonic acid morpholide was synthesized as described [25]. The 7-substituted tirapazamines, quinoxaline 1,4-dioxide and 1-oxide of tirapazamine (Figure 8) were The 7-substituted tirapazamines, quinoxaline 1,4-dioxide and 1-oxide of tirapazamine (Figure8) were synthesized as described [23,46,47]. synthesized as described [23,46,47].

FigureFigure 8.8. StructuralStructural formulae formulae of aromaticof aromaticN-oxides N-oxides used inused this work.in this Derivatives work. Derivatives of 3-amino-1,2, of 3-amino-1,2,4-benzotriazine-1,4-dioxide4-benzotriazine-1,4-dioxide (tirapazamine) (tirapazamin (1), 3-amino-1,2,4-benzotriazinee) (1), 3-amino-1,2,4-benzotriazine 1-oxide (2) and quinoxaline-1 1-oxide (2) , 4-dioxide. Formulae were drawn using ISIS/Draw (v. 2002, MDL Information Systems, San Leandro,

CA, USA).

All compounds were characterized by determining their melting point, as well as their 1H-NMR, UV and IR spectra. The purity of the compounds, determined using a high-performance liquid chromatography system equipped with a mass spectrometer (LCMS-2020, Shimadzu, Kyoto, Japan), was >98%. Cytochrome c, NADPH, superoxide dismutase and other compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as received. Int. J. Mol. Sci. 2020, 21, 3234 11 of 15

4.2. Steady-State Kinetic Studies All kinetic experiments were carried out spectrophotometrically using a PerkinElmer Lambda 25 UV–VIS spectrophotometer (PerkinElmer, Waltham, MA, USA) in 0.1-M K-phosphate buffer (pH 7.0) containing 1-mM EDTA at 25 ◦C. The steady-state parameters of the reactions, the catalytic constants (kcat(app.)) and the bimolecular rate constants (or catalytic efficiency constants, kcat/Km) of the oxidants at fixed concentrations of NADPH were obtained by fitting the kinetic data to the parabolic expression using SigmaPlot 2000 (v. 11.0, SPSS Inc., Chicago, IL, USA). They correspond to the reciprocal intercepts and slopes of Lineweaver-Burk plots, (E)/V vs. 1/(oxidant) respectively, where V is the reaction rate, and (E) is the enzyme concentration. kcat represents the number of molecules of NADPH oxidized by a single active center of the enzyme per second. In the case of two-substrate reactions or inhibitions, the data were fitted to Equations (A1–3). The rates of Pf FNR-catalyzed NADPH oxidation in the presence of quinones, nitroaromatic compounds or tirapazamine derivatives were 1 1 determined using the value ∆ε340 = 6.2 mM− cm− . The rates were corrected for the intrinsic 1 NADPH-oxidase activity of the enzyme, determined as 0.12 s− . In separate experiments, in which 50-µM cytochrome c was included in the reaction mixture, its tirapazamine-mediated reduction was 1 1 measured using the value ∆ε550 = 20 mM− cm− . Ferricyanide reduction rate was determined using 1 1 + the value ∆ε420 = 1.03 mM− cm− . The reduction rate of AcPyP was determined using the value 1 1 ∆ε363 = 5.6 mM− cm− [48]. The rates of oxygen consumption during the reactions were monitored under identical conditions using a Clark electrode (Rank Brothers Ltd., Bottisham, UK).

4.3. Presteady-State Kinetic Studies Enzyme rapid kinetic studies were performed using a SX20 stopped-ﬂow spectrophotometer (Applied Photophysics, Leatherhead, UK) under aerobic conditions. The enzyme reduction by NADPH and its reoxidation was monitored at 460 and 600 nm, respectively. During turnover studies, the enzyme in the ﬁrst syringe (6.0–7.0 µM after mixing) was mixed with the contents of the second syringe (50-µM NADPH and 100–500-µM tetramethyl-1,4-benzoquinone after mixing).

Author Contributions: M.L. performed kinetic experiments, A.A. purified enzymes, J.Š. synthesized compounds and N.C.ˇ designed and supervised the experiments and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the European Social Fund (Measure No. 09.33-LMT-K-712, grant No. DOTSUT-34/09.3.3.-LMT-K712-01-0058/LSS-600000-58) (M.L., J.Š and N.C.)ˇ and by the Fund Linea-2-2016/2017 grant by the Universita degli Studi di Milano (A.A.). Acknowledgments: We thank Saulius Klimašauskas for access to the stopped-flow facility. Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Abbreviations

AcPyP+ 3-Acetylpyridineadenine dinucleotide phosphate ArNO2 aromatic nitrocompound ArN O aromatic N-oxide 1 → E 7 single-electron reduction midpoint potential at pH 7.0 0 E 7 two-electron (standard) reduction midpoint potential at pH 7.0 Fd ferredoxin FNR ferredoxin:NADP+ oxidoreductase kcat catalytic constant kcat/Km bimolecular rate constant Q quinone Int. J. Mol. Sci. 2020, 21, 3234 12 of 15

Appendix A Kinetic parameters of steady-state reactions according to a ping-pong mechanism were calculated according to Equation (A1): V kcat [S][Q] = (A1) [E] Km(S)[Q] + Km (Q)[S] + [S][Q] where S stands for NADPH, and Q stands for the electron acceptor. The competitive inhibition constant (Kis) of NADP+ (I) vs. NADPH (S) was calculated according to Equation (A2):

V kcat [S] = (A2) [ ] [I] E K 1 + + [S] m(S) Kis

For the transhydrogenase reaction, S stands for AcPyP+, and I stands for NADPH. The noncompetitive + inhibition constant (Kii) of NADP vs. the electron acceptor was calculated according to Equation (A3):

V kcat [Q] = (A3) [ ] [I] E K + [Q] 1 + m(Q) Kii

Appendix B

The k values of ferricyanide and Fe(EDTA) at inﬁnite ionic strengths are equal to 4.6 105 M 1s 1 22 − × − − and 6.9 104 M 1s 1, respectively [38]. At the ionic strength of the solution, 1.10 M, the k values of × − − 12 ferricyanide and Fe(EDTA) are equal to 1.0 106 M 1s 1 and 1.0 104 M 1s 1, respectively (Figure4). − × − − × − − Using E7(FAD/FADH−) = 0.28 V for Pf FNR [19], the calculations according to the Nernst equation give E (FAD/FADH.) = 0.308 V− (15% FADH. stabilization) and E (FAD/FADH.) = 0.337 V (5% FADH. stabilization). 7 − 7 − The electron self-exchange rate constants of Pf FNR, k11, are calculated after the rearrangement of Equation (3) [49]:

log k = log k log k 0.5 log K + log Z [(log Z log k )2 + log K (log Z log k )]1/2 (A4) 11 12 − 22 − − − 12 − 12 4 1 1 . For the reactions of Pf FNR with ferricyanide, Equation (A4) gives k11 = 10− M− s− (15% FADH stabilization) and k = 4.1 10 5 M 1s 1 (5% FADH. stabilization). Similarly, for the reactions with Fe(EDTA), it yields 11 × − − − k = 3.5 10 4 M 1s 1 (15% FADH. stabilization) and k = 1.3 10 4 M 1s 1 (5% FADH. stabilization). For the 11 × − − − 11 × − − − reactions of Pf FNR with Q and ArNO2, the approximate k11 values may be estimated from the data of Figure3 at 1 1/2 . 1 ∆E 7 = 0, where k12 = (k11 k22) . At a 15% FADH stabilization, i.e., at the E 7 of the oxidant being equal to × 1 1 1 1 . 0.308 V, the k11 values are equal to 79 M− s− (quinones) and to 63 M− s− (nitroaromatics). At a 5% FADH − 1 1 1 stabilization, i.e., at the E 7 of the oxidant being equal to 0.337 V, the k11 values are equal to 40 M− s− (quinones) 1 1 − and to 6.3 M− s− (nitroaromatics).

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