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

Abstract

The influcnce of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin -NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between PNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC 71 19. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNR,, (oxidized FNR) were dramatically decreased (z 100-fold) when phenylglyoxal-modified FNR was used, In contrast to the reaction involving the native proteins, no ionic strength effects on kohs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNR,, (FNR semiquinone) was used to reduce Fld,, (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fld,, (Fld semiquinone) formation via intracomplex electron transfer from FNR,, was approximately 12-fold smaller than that obtained for the native FNR (600 s-vs 7000 s-'). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNR,, to Fld,, which was approximately 7-fold smaller (1000 s-') than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.Ferredoxin -NADP' reductase (FNR) is a flavoenzyme that plays a key role in the metabolism of photosynthetic organisms by catalyzing the photoreduction of NADP+ using ferredoxin (Fd) or flavodoxin (Fld) as electron donors. It has also been implicated in the reverse electron transfer from NADPH to nitrogenase via Fd or Fld that occurs in nitrogenCorrespondence to G. Tollin, Department of Biochcmistry, UniFax: $1 602 621 9288. Abbreviations. FNR, FNR,, and FNR,,, ferredoxin -NADP' reductase and its oxidized and one-electron-reduced seiniq...

Structural and mechanistic aspects of flavoproteins: photosynthetic electron transfer from photosystem I to NADP+

“…Different interface propensities for the same FNR residues, with either Fd or Fld, are consistent with experimental observations indicating that, although FNR uses the same site for interaction with Fd and Fld, each individual residue does not participate to the same extent in interactions with each of the partners [100]. This is in agreement with the fact that, although multiple chemical modifications produced Flds less suitable for electron transfer [111], site-directed mutagenesis has not revealed any residues critical for the interaction with FNR [53,[88][89][90]. Replacement of the few Fld positions, T12, W57, N58 and Y94, with a high interface propensity produced opposing effects: some Fld:FNR complexes can be either weaker or stronger and less optimal for electron transfer than those with wild-type Fld, but others can appear more optimal for a particular electron transfer process.…”

Structural alignment of ferredoxin and flavodoxin based on electrostatic potentials: Implications for their interactions with photosystem I and ferredoxin-NADP reductase

“…Fld and FNR from Anabaena PCC 7119 can be cross‐linked in 1:1 stoichiometry 59. Chemical modification studies on Fld from Anabaena PCC 7119 suggest that the residues Asp123, Asp126, Asp129, Asp144, Asp145, and Asp146 of Fld interact with FNR 60. Also, arginine residues of Fld are involved in the interaction with FNR 61.…”

“…59 Chemical modification studies on Fld from Anabaena PCC 7119 suggest that the residues Asp123, Asp126, Asp129, Asp144, Asp145, and Asp146 of Fld interact with FNR. 60 Also, arginine residues of Fld are involved in the interaction with FNR. 61 Mutation studies reveal the involvement of residues Asp126 and Glu67 in the association reaction.…”

Involvement of Glutamic Acid 301 in the Catalytic Mechanism of Ferredoxin-NADP+ Reductase from Anabaena PCC 7119

“…When Anabaena is grown in a low-iron medium, flavodoxin replaces ferredoxin as electron donor to FNR (6). The electron-transfer reactions between Anabaena FNR and its physiological partners have been extensively studied (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Recently, the threedimensional structure of Anabaena PCC 7119 FNR and a model for the complex with NADP + were determined (18).…”