2-Thioriboflavin 5'-Phosphate (2-Thio-FMN) Lactate Osidase

Abstract

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

Carbon isotope effects on the decarboxylation of carboxylic acids. Comparison of the lactate oxidase reaction and the degradation of pyruvate by H2O2

“…As this is a very high value for the ratios of forward to backward reaction steps, we postulate an additional irreversible step prior to decarboxylation, which has for consequence that the carbon isotope fractionation on the carbon-carbon bond scission does not take effect. Actually, Lockridge et al (1972) determined the rate constant for the reaction with 02 (step k5) to be one of the largest in the overall reaction, and Choong & Massey (1983) proposed a reaction sequence in which addition of 02 and formation of the ternary enzyme-pyruvate-H202 complex (step k5) is the first irreversible step. Our result, that essentially no primary isotope effect is observed, is in line with this mechanism, and also indicates that the reaction with 02 and the decarboxylation are two subsequent reactions [a concerted step had been postulated by Lockridge et al (1972)].…”

4-thioflavins as active site probes of flavoproteins: reaction with sulphite and formation of 4-hydroxy-4-sulphonylflavins

“…With the flavins mentioned above the accessibility and environment of the benzenoid part of the flavin could be investigated. A similar approach using 2-thioflavins was employed to probe the N(l)-C(2)=0-N(3)H positions in various flavoproteins (Claiborne et al, 1982;Choong and Massey, 1983;Biemann et al, 1983). Vargo and coworkers (Vargo et al, 1981) have recently employed 5-deazaflavins and their reactions with peroxides as probes of the flavin 5-position.…”

The one- and two-electron reduction of 2-thioriboflavin by radical anions of CO2 and dithiothreitol

“…Chemically modified flavins (Fl)' have been used successfully in recent years as probes of reaction mechanisms and of the protein environment around the bound flavin in a number of flavoproteins (1)(2)(3)(4). In most cases the modified flavins have quite different absorption spectra, pKs, and redox potentials from their normal counterparts F A D or FMN.…”

“…This investigation is concerned with the formation and spectra of the semiquinone forms of 2-thioriboflavin, which has found use in several recent studies (1,4). The experiments were performed by the pulse radiolysis technique (5), which was also utilized in earlier examinations of flavin semiquinones (6)(7)(8)(9).…”

“…The reducing radicals .COT and DST. (the radical of dithiothreitol, see reaction [4] below and footnote l ) , were produced by the well-established sequence of reactions (5,10) For personal use only.…”