posted on 2021-11-29, 19:08authored byAnastasia Kraskov, David Buhrke, Patrick Scheerer, Ida Shaef, Juan C. Sanchez, Melissa Carrillo, Moraima Noda, Denisse Feliz, Emina A. Stojković, Peter Hildebrandt
Phytochromes
are sensory photoreceptors that use light to drive
protein structural changes, which in turn trigger physiological reaction
cascades. The process starts with a double-bond photoisomerization
of the linear methine-bridged tetrapyrrole chromophore in the photosensory
core module. The molecular mechanism of the photoconversion depends
on the structural and electrostatic properties of the chromophore
environment, which are highly conserved in related phytochromes. However,
the specific role of individual amino acids is yet not clear. A histidine
in the vicinity of the isomerization site is highly conserved and
almost invariant among all phytochromes. The present study aimed at
analyzing its role by taking advantage of a myxobacterial phytochrome
SaBphP1 from Stigmatella aurantiaca, where this histidine is naturally substituted with a threonine
(Thr289), and comparing it to its normal, His-containing counterpart
from the same organism SaBphP2 (His275). We have carried out a detailed
resonance Raman and IR spectroscopic investigation of the wild-type
proteins and their respective His- or Thr-substituted variants (SaBphP1-T289H
and SaBphP2-H275T) using the well-characterized prototypical phytochrome
Agp1 from Agrobacterium fabrum as a
reference. The overall mechanism of the photoconversion is insensitive
toward the His substitution. However, the chromophore geometry at
the isomerization site appears to be affected, with a slightly stronger
twist of ring D in the presence of Thr, which is sufficient to cause
different light absorption properties in SaBphP1 and SaBphP2. Furthermore,
the presence of His allows for multiple hydrogen-bonding interactions
with the ring D carbonyl which may be the origin for the geometric
differences of the C–D methine bridge compared to the Thr-containing
variants. Other structural and mechanistic differences are independent
of the presence of His. The most striking finding is the protonation
of the ring C propionate in the Pfr states of SaBphP2, which is common
among bathy phytochromes but so far has not been reported in prototypical
phytochromes.