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Reactions of Plasmodium Falciparum Ferredoxin:NADP Oxidoreductase

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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
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