Fluorescence and optical characteristics of reduced flavines and flavoproteins

Abstract

ABSTRACT:The fluorescence and absorption properties of a series of reduced flavoproteins have been measured and compared with the properties of suitable model compounds. Contrary to common belief, a number of reduced flavoproteins have been found to exhibit appreciable fluorescence emission with maxima in the range 500-530 nm. In keeping with common observation, the reduced model flavines are devoid of fluorescence in solution at room temperature, but show marked fluorescence emission in the range 476-512 nm at 77 OK in rigid glasses. The fluorescence quantum yield of reduced lactate oxidase (emission x , , , 507 nm) is increased 4.7 times upon formation of covalent N5 adducts and the emission maximum is shifted to 476 nm. In the case of the nonfluores-T he electronic properties of the isoalloxazine chromophore system, which constitutes the redox active moiety of the flavine coenzymes, have been the object of several thorough theoretical and experimental investigations (Sun et al., 1972 ;Song, 1971, and literature cited therein). These studies have dealt mainly with the oxidized form of the free and proteinbound flavine as well as with model compounds. A distinctive characteristic of oxidized free flavines is their relatively strong fluorescence (quantum yield -0.3; Sun et al., 1972), with an emission maximum typically around 520 nm. The energy and intensity of this emission are dependent on solvent polarity and temperature (Koziol, 1969; Sun el al., 1972;Kotaki et al., 1967), on formation of complexes with a variety of molecules (Weber, 1950; Slikin, 1971), and on the position and properties of substituents Walker et al., 1972; Sun et al., 1972). Similar effects have been observed with riboflavine, FMN, or FAD1 bound to various apoproteins.Unfortunately the spectroscopy of reduced flavines has received far less attention than that of the oxidized flavoquinones. A reason for this could be the absence of wellresolved structure in the near-ultraviolet absorption spectrum and of fluorescence at ambient temperature in any solvent.On the other hand, the nonplanarity of the flavine ring in its reduced forms introduces difficulties in theoretical calculations

Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus

“…Although absorbance changes <400 nm cannot be accurately measured in NADPH-and dithionite reduced preparations, A 435 also increases in incubations with these reductants and 2 mM IPP, DMAPP, or (R)-[2-2 H]-IPP, suggesting that a similar flavin intermediate forms regardless of the reduction method or reaction direction (the dithionite reduced reaction spectra are shown in Figure 1B). The λ max value at 435 nm is unusual for flavoproteins but is most consistent with a neutral reduced FMN (as suggested previously for IDI-2 (22, 31)), which has been observed to have a λ max value anywhere from 390 to 450 nm, depending on the hydrophobicity and rigidity of the active site environment (32)(33)(34)(35). Consistent with previous photoreduction studies (22), a neutral semiquinone (E•FMN sem ) could be generated under anaerobic conditions if 2 mM IPP was pre-incubated with E•FMN ox prior to a 2 min photoreduction.…”

“…Previously, Rothman et al have suggested that a zwitterionic 5,5-dihydro reduced flavin tautomer could be involved in IDI-2 catalysis based on the appearance of an absorption band at ~320 nm that forms upon IPP binding, which is similar to the absorption λ max value reported for an N5-dimethylated reduced flavin (22). However, 1,5-dihydroflavin tautomers can also absorb in this region (32). It is also possible that the neutral reduced flavin that accumulates upon IPP binding is protonated at O4′ rather that at N1, giving the tautomer 10 (Scheme 3D).…”

Phage antibodies against an unstable hapten: Oxygen sensitive reduced flavin

“…Reduced flavin was titrated with o~Flred5 at pH 6 where the N(1) of Flied is protonated (as in the selection) and at pH 7.5 where N(1) is negatively charged. In both cases, the Flred fluorescence increased upon binding to 0~Flred5, presumably because the constraint induced by binding to the antibody restricts the number of accessible conformational states of Fl~d [16].…”

“…2A, curves b,c). This was unexpected as the rotational correlation time of a scFv molecule can be calculated according to Visser et al [17] as [10][11][12][13][14][15][16][17][18][19][20] ns. It appears that this discrepancy may be due to association of scFv molecules by hydrophobic interactions in the presence of salt, as in experiments performed in the absence of NaC1 (%crv shifts to about 20 ns (data not shown).…”

Kinetic and Spectroscopic Characterization of Type II Isopentenyl Diphosphate Isomerase from Thermus thermophilus: Evidence for Formation of Substrate-Induced Flavin Species

“…The reaction proceeds to give a series of spectra with an isosbestic point at 330 nm, indicating that the reaction occurs without the accumulation of an intermediate. The final species has an absorbance maximum at 352 nm (ε ~ 5500 M −1 cm −1 ) characteristic of anionic reduced FMN (Figure 3b), which has a peak at 342 nm when free in solution (26). FMNH − is the typical protonation state for the reduced cofactor in most flavoenzymes (34).…”