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

Abstract

Ferredoxin:NADP+ oxidoreductase from Plasmodium falciparum (PfFNR) catalyzes the NADPH-dependent reduction of ferredoxin (PfFd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, PfFNR 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 PfFNR. 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 midpoint potential (E17). 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 PfFNR 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 PfFNR, evidently providing an additional reduction pathway via reduced PfFd. Based on the available data, PfFNR and possibly PfFd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.

Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1)

“…In this study, we examined the relationship between enzymatic redox properties and aerobic cytotoxicity of aromatic N -oxides with a particular emphasis on the role of NQO1. The rate constants of single-electron reduction of ArN→O 1–9 by P-450R and Pf FNR ( Table 1 ) are in line with previous findings [ 22 , 27 ], showing that the reactivity is mostly determined by the electron-accepting potency of ArN→O, and hardly depend on their structural peculiarities. Their log k cat / K m (avge) proved to be a reasonable substitute for E 1 7 because of their good interrelationship (Equations (1) and (2)).…”

“…In other cases, the reaction rates were proportional to the concentration of compounds up to the limits of their solubility, 300–600 µM. Because Pf FNR does not reduce cytochrome c directly [ 27 ], it was possible to demonstrate the redox cycling of synthesized ArN→O monitoring the reduction of cytochrome c added to the reaction mixture. The rates of this reaction were equal to 175–190% NADPH oxidation rate.…”

“…Typically, the logarithms of k cat / K m of single-electron reduction of ArN→O by flavoenzymes dehydrogenases-electrontransferases such as P-450R or Pf FNR linearly increase with E 1 7 [ 22 , 27 ]. This is attributed to an “outer-sphere” electron transfer mechanism, where the reactivity of compounds is insignificantly influenced by their structural peculiarities [ 28 ].…”

“…Table 1 contains the logarithms of geometric averages of k cat / K m of ArN→O in P-450R- and Pf FNR-catalyzed reactions (log k cat / K m (avge) = 0.5 log k cat / K m (P-450R) + 0.5 log k cat / K m ( Pf FNR)). For comparison, the reduction rate constants of compounds 10 – 18 obtained in our previous studies [ 22 , 27 ] are also presented ( Table 1 ). The log k cat / K m (avge) of these compounds correlates well with their E 1 7 values: log k cat / K m (avge) = (7.23 ± 0.29) + (6.96 ± 0.65) E 1 7 , r 2 = 0.9430.…”

Genetic alteration of ferredoxin NADP+-reductase or cysteine desulfurase in piperaquine-resistant Plasmodium berghei restores susceptibility to lumefantrine and abolishes the impact of probenecid, verapamil, and cyproheptadine

“…We provide a clear link between LM activity, RA action, and overexpression of the FNR gene. Redox enzymes such as FNR convert inactive compounds into active molecules 45 , 46 . Two possible assumptions exist; First, FNR may be a target for LM.…”