Structure−Function Relationships in Anabaena Ferredoxin:  Correlations between X-ray Crystal Structures, Reduction Potentials, and Rate Constants of Electron Transfer to Ferredoxin:NADP⁺ Reductase for Site-Specific Ferredoxin Mutants^,

Abstract

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.

Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

“…The effects on PetF-dependent hydrogenase activity observed in this study indicate that most of the residues that are involved in PetF-ferredoxin-NADPH oxidoreductase interaction (21,30,33) also participate in complex formation between PetF and HydA1. Nevertheless, there are some significant exceptions.…”

“…Because the in vitro assays were conducted using sodium dithionite in excess to completely reduce the respective PetF variants (32), a dominating influence of a possible change in the PetF redox potential is unlikely (33). Exchanging the residues Glu 94 and Phe 65 in PetF of Anabaena (corresponding to Glu 122 and Phe 93 in PetF of C. reinhardtii) had a similar impact on electron transfer between PetF and ferredoxin-NADPH oxidoreductase as described here for the PetF-HydA1 complex (33,34).…”

“…Because the in vitro assays were conducted using sodium dithionite in excess to completely reduce the respective PetF variants (32), a dominating influence of a possible change in the PetF redox potential is unlikely (33). Exchanging the residues Glu 94 and Phe 65 in PetF of Anabaena (corresponding to Glu 122 and Phe 93 in PetF of C. reinhardtii) had a similar impact on electron transfer between PetF and ferredoxin-NADPH oxidoreductase as described here for the PetF-HydA1 complex (33,34). Accordingly, in the crystal structure of the PetF-ferredoxin-NADPH oxidoreductase complex from Anabaena, residue Glu 94 has been located as part of an important intermolecular salt bridge, whereas Phe 65 turned out to be involved in hydrophobic interactions that stabilize the final complex orientation (14).…”

“…Furthermore, the homologous positions of Glu 122 and the C-terminal tyrosine group (Tyr 126 ) in PetF of Anabaena have been described to take part in a hydrogen bond network originating from the loop that surrounds the 2Fe2S cluster (33,36 (Fig. 6).…”

Complex Formation between Azotobacter vinelandiiFerredoxin I and Its Physiological Electron Donor NADPH-Ferredoxin Reductase

“…Electron transfer via exposed methyl groups of the flavin ring has been proposed in other systems in which protein-protein interactions were known (46,47,26). Mutagenesis studies have implicated the complementary interaction of basic residues on Anabaena ferredoxin-NADP ϩ reductase with acidic residues on the Anabaena [2Fe-2S] Fd (48,49). Fig.…”