Vibrational spectroscopy of an algal Phot-LOV1 domain probes the molecular changes associated with blue-light reception

Abstract

The LOV1 domain of the blue light Phot1-receptor (phototropin homolog) from Chlamydomonas reinhardtii has been studied by vibrational spectroscopy. The FMN modes of the dark state of LOV1 were identified by preresonance Raman spectroscopy and assigned to molecular vibrations. By comparing the blue-light-induced FTIR difference spectrum with the preresonance Raman spectrum, most of the differences are due to FMN modes. Thus, we exclude large backbone changes of the protein that might occur during the phototransformation of the dark state LOV1-447 into the putative signaling state LOV1-390. Still, the presence of smaller amide difference bands cannot be excluded but may be masked by overlapping FMN modes. The band at 2567 cm(-1) is assigned to the S-H stretching vibration of C57, the residue that forms the transient thio-adduct with the chromophore FMN. The occurrence of this band is evidence that C57 is protonated in the dark state of LOV1. This result challenges conclusions from the homologous LOV2 domain from oat that the thiolate of the corresponding cysteine is the reactive species.

FIGURE 1

The chromophore FMN and the light-induced thiol adduct where the sulfur of C57 is covalently linked to the carbon at position 4a of the isoalloxazine ring.

FIGURE 2

Infrared difference spectra of LOV1 in the S-H stretching region obtained after continuous illumination with blue light. The data shown are a zoom-out from the original spectra that have been recorded across the whole midinfrared range (7000–800 cm⁻¹). The negative band shifts from 2567 cm⁻¹ in H₂O (continuous line) to 1867 cm⁻¹ in D₂O (dashed line) accompanied by a decrease in extinction. Note the different absorbance scale in the two panels.

FIGURE 3

Light-induced FTIR difference spectra of LOV1. The spectrum of a highly concentrated protein solution in H₂O (continuous line) is overlaid by the spectrum of the protein dissolved in D₂O (dashed line). The indicated frequencies correspond to the measurement in H₂O and are summarized in Table 1.

FIGURE 4

Resonance-Raman spectrum of LOV1 in the unilluminated dark state (top trace). The spectrum has been obtained under preresonant conditions with excitation at 752 nm. For comparison, the Raman spectrum of FMN dissolved in H₂O (middle trace) and of FMN in the solid state (bottom trace) are depicted. Dashed vertical lines correspond to the frequency of those LOV1 bands that are discussed in the text.

FIGURE 5

Comparison of the preresonance-Raman spectrum of the dark state of LOV1 (bottom trace) with the blue-light-induced FTIR difference spectrum. Spectra have been replotted from Figs. 3 and 4. Vertical dashed lines indicate bands of the chromophore of the dark (unilluminated) state of LOV1 that are detected by both vibrational techniques.