Characterization of the cross-linked complex formed between ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119

A covalent complex between ferredoxin-NADP+ reductase and flavodoxin, two flavoproteins isolated from the nitrogen-fixing cyanobacterium Anabaena, has been formed by a cross-linking reaction with a water-soluble carbodiimide. The complex has a 1:1 stoichiometry and an absorption spectrum similar to that of the mixture of the free proteins. Both proteins can be detected immunochemically when they are in the complex, since they react with antisera raised against the isolated proteins. The complex is shown to be active in the NADPH-cytochrome c reductase reaction, although with a lower turnover number than FNR when ferredoxin is used as the electron carrier. Stopped-flow experiments have shown a lower rate of electron transfer (approx. 20-fold) from the semiquinone form of flavodoxin, when bound into the complex, to cytochrome c, as compared to the free protein. Anaerobic titration of the reduced complex with NADP+ also indicates that there is transfer of electrons between the reductase and flavodoxin in the complex. Nevertheless, the covalent complex is found to be unable by itself to mediate the transfer of electrons from photosynthetic particles to NADP+. This indicates that the pathway for the entrance of electrons into the complex is partially or completely blocked depending on the degree of cross-linkage. However, some activity is shown when ferredoxin is added to the system. The covalent complex shows a limited ability to act as an electron transfer protein between photosynthetic membranes and either exogenous FNR or cytochrome c. The concentration of complex required in this case is also much higher than when free ferredoxin or flavodoxin are present. It is concluded that the covalent complex formed by ferredoxin-NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 can be used as a simplified model for the study of electron transfer reactions between two flavoproteins. It cannot be used for the study of reactions involving reduction of NADP+ by photosynthetic particles.