RESUMO
Drosophila melanogaster cryptochrome is one of the model proteins for animal blue-light photoreceptors. Using time-resolved and steady-state optical spectroscopy, we studied the mechanism of light-induced radical-pair formation and decay, and the photoreduction of the FAD cofactor. Exact kinetics on a microsecond to minutes timescale could be extracted for the wild-type protein using global analysis. The wild-type exhibits a fast photoreduction reaction from the oxidized FAD to the FAD(â¢-) state with a very positive midpoint potential of ~ +125 mV, although no further reduction could be observed. We could also demonstrate that the terminal tryptophan of the conserved triad, W342, is directly involved in electron transfer; however, photoreduction could not be completely inhibited in a W342F mutant. The investigation of another mutation close to the FAD cofactor, C416N, rather unexpectedly reveals accumulation of a protonated flavin radical on a timescale of several seconds. The obtained data are critically discussed with the ones obtained from another protein, Escherichia coli photolyase, and we conclude that the amino acid opposite N(5) of the isoalloxazine moiety of FAD is able to (de)stabilize the protonated FAD radical but not to significantly modulate the kinetics of any light-inducted reactions.
Assuntos
Criptocromos/química , Proteínas de Drosophila/química , Proteínas do Olho/química , Substituição de Aminoácidos , Animais , Criptocromos/genética , Criptocromos/efeitos da radiação , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/efeitos da radiação , Drosophila melanogaster/química , Drosophila melanogaster/genética , Transporte de Elétrons , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/efeitos da radiação , Flavina-Adenina Dinucleotídeo/química , Radicais Livres/química , Radicais Livres/efeitos da radiação , Luz , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Processos Fotoquímicos , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/efeitos da radiação , Espectrofotometria , Triptofano/químicaRESUMO
The NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with a translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in proton translocation. The recently published X-ray structures of the complex revealed the presence of a unique 110 Å "horizontal" helix aligning the membrane arm. On the basis of this finding, it was proposed that the energy released by the redox reaction is transmitted to the membrane arm via a conformational change in the horizontal helix. The helix corresponds to the C-terminal part of the most distal subunit NuoL. To investigate its role in proton translocation, we characterized the electron transfer and proton translocation activity of complex I variants lacking either NuoL or parts of the C-terminal domain. Our data suggest that the H+/2e- stoichiometry of the ΔNuoL variant is 2, indicating a different stoichiometry for proton translocation as proposed from structural data. In addition, the same H+/e- stoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.
