$ OXIDOREDUCTASE: POTENTIOMETRIC CHARACTERISTICS AND REACTIONS WITH PROOXIDANT XENOBIOTICS} {Chlorobaculum tepidum} ferredoxin:NADP\(^{+}\) oxidoreductase ({Ct}FNR) is a thioredoxin reductase (TrxR)-type FNR with scarcely characterized redox properties and reactivity with nonphysiological electron acceptors. The enzyme employs a noncovalently bound FAD and acts in transforming between one- and two-electron transfer with its native substrates NADP(H) and low molecular weight protein ferredoxin (Fd). The TrxR-type FNRs exhibit a homodimeric structure with their NADP(H)-binding domain being inserted between two segments of FAD-binding domains. The domains are mobile during catalysis, as the distance between the bound NADP(H) and FAD is ~15 Å, too great for efficient electron transfer [1,2].
In this study, we combined steady-state and presteady-state kinetic methods to examine the potentiometric characteristics of {Ct}FNR and its reactions with prooxidant xenobiotics. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of FAD cofactor was estimated to be −0.275 V, and photoreduction using 5-deazaFMN as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.042 V. The enzymatic reaction proceeds via the {ping-pong} mechanism and exhibits the expected inhibition patterns by the reaction product NADP\(^{+}\), namely competitive inhibition towards NADPH and uncompetitive inhibition towards a quinone. The reactivity of quinones and nitroaromatics towards {Ct}FNR increases with their increasing single-electron reduction midpoint potentials; however, the reactivity of nitroaromatics was lower due to their lower electron self-exchange rate. The reduction reaction of quinones proceeds in a mixed single- and two-electron way, and nitroaromatics are reduced via the single-electron reaction. The experiments with varied ionic strength of the buffer exhibit a trend similar to that seen in other TrxR-type FNRs from {Bacillus subtilis} and {Rhodopseudomonas palustris}: the {k}\(_{cat}\)/{K}\(_{m}\) of a noncharged electron acceptor remains constant, while it increases for the negatively charged FeEDTA\(^{-}\) and decreases for the positively charged benzyl viologen upon increasing the ionic strength, thus giving insight into the role of charged amino acid residues in the vicinity of enzyme-bound FAD [3,4].
The obtained results are generally similar to those obtained with {Rp}FNR and {Bs}FNR and further expand our understanding of the novel TrxR-type FNRs and their catalysis.