Conformational changes in Chondrus crispus flavodoxin on dissociation of FMN and reconstitution with flavin analogues

• Published in: Biochemical journal Vol. 272; no. 3; pp. 775 - 779
• Main Authors: ROGERS, L. J, SYKES, G. A
• Format: Journal Article
• Language: English
• Published: Colchester Portland Press 15-12-1990
• Subjects: Analytical, structural and metabolic biochemistry; Apoproteins - isolation & purification; Apoproteins - metabolism; Binding and carrier proteins; Biological and medical sciences; Chromatography, High Pressure Liquid; Eukaryota - metabolism; Flavin Mononucleotide - metabolism; Flavins - metabolism; Flavodoxin - isolation & purification; Flavodoxin - metabolism; Fundamental and applied biological sciences. Psychology; Kinetics; Protein Binding; Proteins; Chondrus crispus; Chemical precipitation; Association dissociation; Gel electrophoresis; Algae; FMN; Association constant; Biological activity; Rhodophycophyta; Molecular parameter; Flavin; Apoproteins; Analog; Reconstituted system; Flavodoxin; Comparative study; Physicochemical properties; Thallophyta; Conformational transition
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2720775

The apoflavodoxin produced by precipitation of Chondrus crispus flavodoxin with trichloroacetic acid migrates as a single molecular species on non-denaturing PAGE, but at a much lower Rm than the flavoprotein. Values of s and D were significantly lower than for the flavodoxin, but their substitution in the Svedberg equation indicated the molecular mass was closely similar to that of the flavodoxin. This was confirmed by meniscus-depletion sedimentation-equilibrium studies. The Stokes radius of the apoflavodoxin was 3.65 nm, compared with 2.33 nm for the flavodoxin, and estimates of frictional coefficient ratio suggested the apoprotein was in extended conformation compared with the roughly globular shape of the flavodoxin. The Ka for FMN binding was 2.8 x 10(8)M, and the electrophoretic and physicochemical properties of the reconstituted flavoprotein were closely similar to those of the native flavodoxin. FAD, iso-FMN and thio-FMN were also bound effectively, but methyl-FMN and riboflavin were bound only weakly, if at all. The reconstituted flavoproteins were active to various extents in mediating electron transfer from NADPH to cytochrome c catalysed by flavodoxin-NADP+ oxidoreductase, the highest activity being with the thio-FMN flavodoxin.