title: 8-Mercaptoflavins as active site probes of flavoenzymes.
abstract: Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.
mesh_terms: D001665:Binding Sites; D003605:D-Amino-Acid Oxidase; D005287:Ferredoxin-NADP Reductase; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives; D005415:Flavins; D005418:Flavodoxin; D005420:Flavoproteins; D005949:Glucose Oxidase; D007536:Isomerism; D007770:L-Lactate Dehydrogenase; D009245:NADH Dehydrogenase; D009252:NADPH Dehydrogenase; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds
chemical_list: C021217:8-mercaptoflavins; D005415:Flavins; D005418:Flavodoxin; D005420:Flavoproteins; D013438:Sulfhydryl Compounds; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D007770:L-Lactate Dehydrogenase; D005949:Glucose Oxidase; D005287:Ferredoxin-NADP Reductase; D003605:D-Amino-Acid Oxidase; D009252:NADPH Dehydrogenase; D009245:NADH Dehydrogenase