Comparison of the kinetics of reduction and intramolecular electron transfer in electrostatic and covalent complexes of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119

Abstract

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 ...

Effects of chemical modification of Anabaena flavodoxin and ferredoxin‐NADP+ reductase on the kinetics of interprotein electron transfer reactions

“…The apparent complex dissociation constant and the limiting first-order rate constant for intracomplex electron transfer can be evaluated from these data by a nonlinear least-squares computer-fitting procedure as described previously [25, 261. This yielded results for the native proteins which were comparable to those reported in the previous studies [14,151.…”

“…Procedures were as described in Walker et al [14,15]. Photoexcitation of dRf was accomplished with a Photochemical Research Associates model LNIOO nitrogen laser which pumped a model LN102 dye laser using a BBQ dyc solution (PRA 2A386; emission wavelength, 395 nm; pulse duration approximately 1 ns).…”

“…The reduction of PG-modified Anahaena FNR by lasergenerated dRfJ3' was monitored by the absorbance decrease at 461 nm, which is in the region of maximum absorption for the oxidized FAD moiety of FNR [14]. This wavelength monitors both the formation and decay of dRfH' and the reduction of FAD, and thus is convenient for following the redox chemistry initiated by the laser flash.…”

“…Laser flash photolysis has previously been used to monitor the kinetics of reduction of protein -protein complexes and intermolecular electron transfer between FNR and Fld [14] and between Fd and FNR [15] from Anabaena PCC 7119. This technique is appropriate for this purpose since it involves the photochemical generation in situ of a strong reductant (5-deazariboflavin semiquinone), which allows the measurement of electron transfer reactions that are too fast to be determined by rapid-mixing methods [16-181. In the present paper we have used this approach to investigate the kinetics of reduction and intermolecular electron transfer between FNR modified by phenylglyoxal (PC) and Fld or Fd, and also between FNR and Fld modified by glycine ethyl ester (EtGly).…”

Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin‐NADP+ reductase

“…Complex formation is sensitive to the ionic strength of the medium and, therefore, it is proposed that electrostatic interactions are responsible for the stabilization of the complex. A number of studies have shown that such interactions are required for rapid electron transfer (Fillat et al, 1990;Walker et al, 1990). This transient kinetic complex can be stabilized by covalent cross-linkage of the purified proteins (Pueyo and Gomez-Moreno, 1991).…”