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6 pages, figures, and tables statistics., Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

8 pages, figures, and tables statistics., The kinetics of reduction and intracomplex electron transfer in electrostatically stabilized and covalently crosslinked complexes between ferredoxin-NADP+ reductase (FNR) and flavodoxin (Fld) from the cyanobacterium Anabuena PCC 7119 were compared using laser flash photolysis. The second-order rate constant for reduction by 5-deazariboflavin semiquinone (dRfII) of FNR within the electrostatically stabilized complex at 10 mM ionic strength (4.0 X 10s M-’ s-‘) was identical to that for free FNR. This suggests that the FAD cofactor of FNR is not sterically hindered upon complex formation. A lower limit of approximately 7000 s-l was estimated for the first-order rate constant for intracomplex electron transfer from FNRmd to Fl&, under these conditions. In contrast, for the covalently crosslinked complex, a smaller secondorder rate constant (2.1 X lo* M-’ s-l) was obtained for the reduction of FNR by dRfII within the complex, suggesting that some steric hindrance of the FAD cofactor of FNR occurs due to crosslinking. A limiting rate constant of 1000 s-l for the intracomplex electron transfer reaction was obtained for the covalent complex, which was unaffected by changes in ionic strength. The substantially diminished limiting rate constant, relative to that of the electrostatic complex, may reflect either a suboptimal orientation of the redox cofactors within the covalent complex or a required structural reorganization preceding electron transfer which is not allowed once the proteins have been covalently linked. Thus, although the covalent complex is biochemically competent, it is not a quantitatively precise model for the catalytically relevant intermediate along the reaction pathway., This work was supported in part by Grant DK15057 from the National Institutes of Health (to G.T.) and Grant 0792/84 from the Comision Asesora de Investigation Cientifica y Tecnica, Spain (to C.G.-M.).