Thc natural flavin of lactate oxidase, FMN, was removed and replaced by the synthetic flavin 2-thioriboflavin 5'-phosphate (Zthio FMN). Despite the differences in properties of the flavins, including an oxidation-reduction potential some 80 mV more positive than that of normal flavin the 2-thio-FMN enzyme behaves in practically all respects like the native enzyme. It catalyzes the oxidative decarboxylation of L-lactate with almost the same efficiency as the native enzyme and with similar kinetic constants for individual steps in the catalytic pathway. It forms covalent derivatives at the flavin N(5) and C(4a) positions in facile photochemical reactions analogous to those of the native enzyme. It also forms a flavin anion radical on photoreduction with 5-deazaflavin as catalyst and, as with the native enzyme, this radical is stabilized remarkably on formation of a complex with pyruvate. The spectral properties of the neutral flavin radical form of 2-thioflavin are also reported, as determined by photochemical reduction of 2-thio-FMN flavodoxin. Like native lactate oxidase, the 2-thio-FMN enzyme also forms a flavin N(5)-sulfite adduct in an equilibrium reaction with sulfite. These results demonstrate clearly with this enzyme that the native flavin may be removed and replaced by an artificial flavin, without altering the structural integrity of the protein.In recent years considerable use has been made of chemically modified flavins as probes of reaction mechanisms of flavin enzymes or of the protein environment around the bound flavin. This has been made possible by the broad specificity of the flavokinase/FAD synthetase system of Brevibacterium ummoniugenes, which is able to convert most artificial flavins from the more easily synthesized riboflavin level to the corresponding FMN and FAD level [l]. Fortunately most flavoproteins seem to be very permissive and bind a variety of ring-modified flavins, the main requirement for binding being the nature of the side chain at position N(10): ribityl phosphate if the original flavin were FMN and ribityl-diphospho-adenosine if the original flavin were FAD. Such replacement studies also rely on the ability to remove the native flavin without denaturing the protein, so that on adding flavin to the apoprotein the original holoenzyme is reconstituted. When this can be achieved with the native flavin, it is generally assumed that when an artificial flavin is found to bind to the apoprotein, it does so in a similar way to the native flavin. This is a crucial point in the concept of using such flavins as probes of the reaction mechanism, or as probes of the protein environment surrounding the bound flavin. Hence it is very important to show that the properties of such artificial flavoproteins mimic those of the native enzymes in importan1 details and to show that this can be achieved with