Abstract
The function of the biological photoswitch phytochrome is initiated by photoisomerization of the methine-bridged tetrapyrrole chromophore, followed by thermal relaxation steps. As a result of this reaction cascade, the protein interconverts between two parental state. These states, denoted as Pr (red absorbing) and Pfr (far-red absorbing), represent the physiologically inactive and active form of the protein, respectively. In this work we studied the primary photoprocesses of two bacterial phytochromes Agp1 and Agp2, in which either Pr or Pfr is the stable dark state, respectively. We employed cryogenic IR difference and resonance Raman spectroscopy between 4 K and 130 K to trap and characterize the species formed on the reaction pathways from Pfr to Lumi-F in Agp2 and Pr to Lumi-R in Agp1. The spectra analysis primarily focuses on the C[double bond, length as m-dash]O stretching modes, which are assigned based on isotopic labelling experiments. In both proteins, three sub-states were identified, which reveal similar patterns of sequential structural changes. In the first sub-state L1 of both photoreceptors, generated at 4 K, structural changes are restricted to the isomerization site including rings D and C. In L2, formed at 30 K in Agp2 but at the same temperature range with L1 in Agp1, the structural changes propagate to ring B, and in L3 also include ring A. Comparison with previously published studies demonstrates that the present approach of cryogenic vibrational spectroscopy provides important structural insights that complement results from crystallography and ultrafast time-resolved spectroscopy.