Ferredoxin:NADP+ oxidoreductases (FNRs, EC 1.18.1.2) are flavoenzymes classified as dehydrogenases – electrontransferases acting in electron transfer in plastids, mitochondria and bacteria. FNR catalyzed two electron transfer from two equivalents of reduced ferredoxin to NADP+ is the final step of electron transfer in photosynthesis, while electron transfer in the opposite direction is implicated in nitrogen fixation or isoprenoid biosynthesis. After the transfer of a single electron to the oxidant a FAD semiquinone forms. Rhodopseudomonas palustris is a Gram-negative purple nonsulfur bacterium exhibiting the ability to switch its metabolism between four different modes [1]. R. palustris ferredoxin:NADP+ oxidoreductase (RpFNR) belongs to novel subgroup of glutathione reductase-like FNRs resembling thioredoxin reductase. The key differences from other FNRs are a native homodimeric structure and NADP(H) binding domain being inserted between the two segments of an FAD binding domain.
By combining steady-state kinetics and photosensitization methods, here we present a study on the RpFNR catalyzed oxidation of NADPH in the presence of nonphysiological electron acceptors commonly considered to be xenobiotic compounds, often found as environmental pollutants or various drugs. These compounds exhibit redox-cycling behavior under aerobic conditions and this is considered to be the basis of their prooxidant cytotoxicity.
Based on the reactions of RpFNR with the 3-acetylpyridine adenine dinucleotide phosphate redox couple, we estimated the two-electron reduction midpoint potential of the FAD cofactor to be −0.285 V. 5-DeazaFMN-sensitized photoreduction revealed a −0.017 V separation of the redox potentials between the first and second electron transfer events. The mechanism of oxidation of RpFNR by several different groups of nonphysiological electron acceptors was examined. Quinone reductase and nitroreductase reactions proceed via a ping-pong mechanism. The kcat/Km values of quinones and aromatic N-oxides towards RpFNR increase with their single-electron reduction midpoint potential with a parabolic dependence. The same is true for nitroaromatic compounds, albeit their reactivity is lower and the increase is linear, owing to their electron self-exchange constants being lower by several orders of magnitude. This then illustrates that the reactivity is governed by the energetics of the compounds and not their structural features. A mixed single- and two-electron reduction was characteristic of quinones, whereas nitroaromatics were reduced entirely in a single-electron way. The calculated electron transfer distances in the reaction with quinones and nitroaromatics were close to the ones obtained in the studies of Anabaena and Plasmodium falciparum FNRs highlighting similar 'intrinsic' reactivities [2,3].