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Photolyases and cryptochromes (CRY) are structurally homologous flavoproteins with divergent functions. While photolyases repair UV-damaged DNA by photoinduced electron transfer from their FAD cofactor, CRY are involved in varied cellular processes, including light-dependent plant growth, regulation of mammalian circadian rhythm, and possibly magnetoreception. Despite their importance in Nature and human health, little is known about how they tune their FAD redox properties to achieve remarkable functional diversity. In this study, we reveal a kinetic mechanism, exploited by cyclobutane pyrimidine dimer photolyase (PL), for regulating the stability of its FAD semiquinone (sq). We find that the sq in CRY-DASH (Synechocystis) is substantially more reactive toward oxidation than in PL (Anacystis nidulans) and, using deuterium isotope and pH effects, show that rate-limiting proton transfer contributes to the exceptional kinetic stability of the PL sq. Through mutagenesis, we identify two PL-specific residues in the flavin binding pocket, Trp392 and Gly389 (Try398 and Asn395 in CRY-DASH, respectively), that ensure this kinetic stability, possibly through interactions with the adenine moiety of FAD and/or adjusting the polarity of the binding site. Significantly, these relatively distal residues have a much more profound impact than two amino acids closer to the FAD. By quantifying sq stability in a series of PL-CRY exchange mutants, our findings pave the way for investigations aimed at correlating sq stability with function in these proteins. As is being recognized with other flavoproteins, we expect that kinetic tuning of the rates of electron transfer will play a function-defining role in photolyases and cryptochromes. [ABSTRACT FROM AUTHOR]

The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin. A site-directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to ≤ 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and ≤ 5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone. [ABSTRACT FROM AUTHOR]

Presents evidence linking decrease in pH to a change in the electronic structure of the reduced flavin in Desulfovibrio vulgaris, Megasphaera elsdenii and Azotobacter vinelandii. Change in the ionization state of the flavin; Polarization of the flavin environment; Hydrogen-bonding network.