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Neuronal nitric oxide synthase (nNOS) catalyzes single-electron reduction of quinones (Q), nitroaromatic compounds (ArNO 2 ) and aromatic N -oxides (ArN → O), and is partly responsible for their oxidative stress-type cytotoxicity. In order to expand a limited knowledge on the enzymatic mechanisms of these processes, we aimed to disclose the specific features of nNOS in the reduction of such xenobiotics. In the absence or presence of calmodulin (CAM), the reactivity of Q and ArN → O increases with their single-electron reduction midpoint potential ( E 1 7 ). ArNO 2 -NO form. This finding may help to design the heme-targeted bioreductively activated agents and contribute to the understanding of the role of P-450-type heme proteins in the bioreduction of quinones and other prooxidant xenobiotics.2 to quinone by 1-2 Å. The effects of ionic strength point to the interaction of oxidants with a negatively charged protein domain close to FMN, and to an increase in accessibility of the active center induced by high ionic strength. The multiple turnover experiments of nNOS show that, in parallel with reduced FAD-FMN, duroquinone reoxidizes the reduced heme, in particular its Fe 2+ -NO form. This finding may help to design the heme-targeted bioreductively activated agents and contribute to the understanding of the role of P-450-type heme proteins in the bioreduction of quinones and other prooxidant xenobiotics.

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 midpoint potential ( E 1 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., Competing Interests: 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.

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be −0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]