RESUMO
The photocycle of the light-activated channel, channelrhodopsin-2 C128T, has been studied by resonance Raman (RR) spectroscopy focussing on the intermediates P380 and P353 that constitute a side pathway in the recovery of the parent state. The P353 species displays a UV-vis absorption spectrum with a fine-structure reminiscent of the reduced-retro form of bacteriorhodopsin, whereas the respective RR spectra differ substantially. Instead, the RR spectra of the P380/P353 intermediate couple are closely related to that of a free retinal in the all-trans configuration. These findings imply that the parent state recovery via P380/P353 involves the transient hydrolysis and re-formation of the retinal-protein linkage.
Assuntos
Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Channelrhodopsins , Cor , Humanos , Concentração de Íons de Hidrogênio , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/genética , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
The electron shuttle heme protein Cyt-c(6) from the photosynthetic cyanobacterium Nostoc sp. PCC 7119 was immobilized on nanostructured Ag electrodes coated with SAMs that mimic different possible interactions with its natural reaction partner PSI. The structure, redox potential, and electron-transfer dynamics of the SAM-Cyt-c(6) complexes were investigated by TR-SERR spectroelectrochemistry. It is shown that the heterogeneous electron-transfer process is gated both in electrostatic and hydrophobic-hydrophilic complexes. At long tunneling distances, the reaction rate is controlled by the tunneling probability, while at shorter distances or higher driving forces, protein dynamics becomes the rate-limiting event.
Assuntos
Biomimética , Citocromos c6/química , Elétrons , Nostoc/enzimologia , Sítios de Ligação , Citocromos c6/genética , Cinética , Modelos Biológicos , Mutação , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
This article describes a method, based on surface-enhanced resonance Raman (SERR) spectroscopy, for studying the reaction dynamics of photoreceptors immobilized on metal electrodes. Time resolution and fresh sample conditions are achieved by synchronizing the rotational and translational motion of a novel kinematic electrode with the duration and time delay between the pump and probe events. The power and sensitivity of the method is illustrated by studying the photocycle of the sensory photoreceptor NpSRII and its sensitivity to the applied electric field. The results are compared with time-resolved resonance Raman measurements in solution.
