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. @I 1990 Academic Press, Inc. 76 Flavoproteins participate in a large number of oxidation/reduction reactions that function either in energy transduction or in the biosynthesis and degradation of metabolic intermediates (cf. (1)). Due to their unique ability to transfer either one or two electrons, they are utilized for electron transfer between pyridine nucleotides and the metal-containing heme or iron-sulfur proteins. They also participate in the transfer of electrons to (or from) other flavin-containing proteins, which raises interesting questions relating to the thermodynamic and structural requirements for efficient flavinto-flavin electron transfer. Among these requirements are: (i) the individual proteins must be able to form a productive complex; (ii) the redox potentials of both flavin cofactors must have the appropriate values for the electron transfer reaction to proceed in the required direction; (iii) the flavin cofactors should lie sufficiently close within the complex, and with the appropriate geometry, to facilitate the rapid transfer of electrons.Ferredoxin-NADP+ reductase (FNR)3 is an FAD containing flavoprotein that participates in the reductive i 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 ...
