A functional heterologous electron-transfer protein complex: Desulfovibrio vulgaris flavodoxin covalently linked to spinach ferredoxin-NADP+ reductase.
The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide was found to readily promote formation of cross-links between spinach ferredoxin-NADP+ reductase and bacterial flavodoxins. The covalent complex between ferredoxin-NADP+ reductase and the Desulfovibrio vulgaris flavodoxin had a stoichiometry of 1 mol of flavodoxin per mole of the reductase, as assessed by denaturing electrophoresis, gel filtration and spectral analysis. The reductase moiety of the cross-linked complex gained the capacity to catalyze at a high rate the electron transfer from NADPH to cytochrome c without addition of free flavodoxin in the assay. The pH optimum for this activity was shifted to the alkaline region with respect to that for the noncovalent complex. FMN, the prosthetic group of flavodoxin, is required for electron transfer from the reductase FAD to cytochrome c. Structural studies carried out on the cross-linked complex allowed the identification of the peptide regions of the proteins involved in the interaction. The CNBr peptide 61-155 of the reductase was found cross-linked to the uncleaved flavodoxin, while the cross-linked region in flavodoxin appeared to be within the tryptic peptide 37-86. Treatment of flavodoxin with the carbodiimide in the presence of glycine ethyl ester brought about the modification of a few carboxyl groups and prevented its interaction with the reductase. It can be concluded that the bacterial flavodoxin binds to the reductase in a way similar to that of the physiological substrate ferredoxin (G. Zanetti, D. Morelli, S. Ronchi, A. Negri, A. Aliverti, and B. Curti, 1988, Biochemistry 27, 3753-3759). The cross-linked complex here described represents an useful model for studying electron transfer between the two flavoproteins.
Pirola
M C
MC
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
Monti
F
F
Aliverti
A
A
Zanetti
G
G
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D003574:Cytochrome c Group / Q000378:metabolism; D016969:Desulfovibrio vulgaris / Q000378:metabolism*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000302:isolation & purification / Q000378:metabolism*; D005418:Flavodoxin / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D013053:Spectrophotometry; D014675:Vegetables / Q000201:enzymology*
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D005418:Flavodoxin; D010446:Peptide Fragments; D005287:Ferredoxin-NADP Reductase; D003488:Cyanogen Bromide