Functional analyses of plasmodium ferredoxin Asp97Tyr mutant related to artemisinin resistance of human malaria parasites.

Mutation of Asp97Tyr in the C-terminal region of ferredoxin (PfFd) in the apicoplast of malaria parasites was recently reported to be strongly related to the parasite's resistance to the frontline antimalarial drug, artemisinin. We previously showed that the aromatic amino acid in the C-terminal region of PfFd is important for the interaction with its electron transfer partner, Fd-NADP+ reductase (PfFNR). Here, the importance of the aromatic-aromatic interaction between PfFd and PfFNR was shown using the kinetic analysis of the electron transfer reaction of site-directed mutants of PfFNR with PfFd. Mutation of Asp97Tyr of PfFd was further shown to increase the affinity with PfFNR by the measurements of the dissociation constant (Kd) using tryptophan fluorescence titration and the Michaelis constant (Km) in the kinetic analysis with PfFNRs. Diaphorase activity of PfFNR was inhibited by D97Y PfFd at lower concentration as compared to wild-type PfFd. Ascorbate radical scavenging activity of PfFd and electron transfer activity to a heterogeneous Fd-dependent enzyme was lower with D97Y PfFd than that of wild-type PfFd. These results showed that D97Y mutant of PfFd binds to PfFNR tighter than wild-type PfFd, and thus may suppress the function of PfFNR which could be associated with the action of artemisinin.