posted on 2021-09-27, 20:12authored byAnastasia Kraskov, Johannes von Sass, Anh Duc Nguyen, Tu Oanh Hoang, David Buhrke, Sagie Katz, Norbert Michael, Jacek Kozuch, Ingo Zebger, Friedrich Siebert, Patrick Scheerer, Maria Andrea Mroginski, Nediljko Budisa, Peter Hildebrandt
Phytochromes
switch between a physiologically inactive and active
state via a light-induced reaction cascade, which is initiated by
isomerization of the tetrapyrrole chromophore and leads to the functionally
relevant secondary structure transition of a protein segment (tongue).
Although details of the underlying cause–effect relationships
are not known, electrostatic fields are likely to play a crucial role
in coupling chromophores and protein structural changes. Here, we
studied local electric field changes during the photoconversion of
the dark state Pfr to the photoactivated state Pr of the bathy phytochrome
Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective
nitrile stretching modes in the various states of photoconversion
(vibrational Stark effect). Resonance Raman and IR spectroscopic analyses
revealed that both pCNF-substituted variants undergo the same photoinduced
structural changes as wild-type Agp2. Based on a structural model
for the Pfr state of F192pCNF, a molecular mechanical–quantum
mechanical approach was employed to calculate the electric field at
the nitrile group and the respective stretching frequency, in excellent
agreement with the experiment. These calculations serve as a reference
for determining the electric field changes in the photoinduced states
of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly
hydrogen bonded such that the theoretical approach is not applicable.
However, in both variants, the largest changes of the nitrile stretching
modes occur in the last step of the photoconversion, supporting the
view that the proton-coupled restructuring of the tongue is accompanied
by a change of the electric field.