STRUCTURE-FUNCTION-RELATIONSHIPS IN ANABAENA FERREDOXIN - CORRELATIONS BETWEEN X-RAY CRYSTAL-STRUCTURES, REDUCTION POTENTIALS, AND RATE CONSTANTS OF ELECTRON-TRANSFER TO FERREDOXIN-NADP(-SPECIFIC FERREDOXIN MUTANTS() REDUCTASE FOR SITE)

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 le ading to electron transfer from reduced ferredoxin to oxidized ferredo xin:NADP(+) reductase (FNR). We have determined the reduction potentia ls of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 s ite-specific mutants at seven surface residues of vegetative ferredoxi n, as well as the one-and two-electron reduction potentials of FNR, bo th 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 signifi cant structural changes in the immediate vicinity of the [2Fe-2S] clus ter, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94 K and 47 mV for S47A). Furthermore, the relatively small changes in C- alpha 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 S 47A mutant, implicating the importance of the hydrogen bond which exis ts between the side chain hydroxyl group of S47 and the side chain car boxyl oxygen of E94. Other ferredoxin mutations that alter both reduct ion potential and electron-transfer reactivity are E94Q, F65A, and F65 I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that an similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredo xin, induced large (approximately 40 mV) positive shifts in the reduct ion potential of the flavoprotein, thereby making electron transfer th ermodynamically feasible. On the basis of these observations, we concl ude that nonconservative mutations of three critical residues (S47, F6 5, and E94) on the surface of ferredoxin have large parallel effects o n 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 orie ntations of the proteins within the transient electron-transfer comple x.