YSM Issue 95.1

Molecular Biology
FOCUS
VISUALIZING
THE HEART OF
PHOTOSYNTHESIS
Using photosystem II, a photosynthetic enzyme, to help
solve the mysteries of solar fuel production
experiments – in which targeted changes
are made to DNA – conducted in the last
fifty years. In these specific experiments,
scientists introduced mutations in the
PSII gene to assess the role of individual
amino acids, which comprise proteins, in
the enzyme’s function. These studies have
almost entirely been performed using Syn.
6803 cyanobacteria, which can survive
with altered PSII if supplemented with
glucose. This makes it an ideal model organism
for mutagenesis because in many
other species, mutations in PSII often led
to cell death, leaving researchers unable to
investigate function further.
However, the molecular structure of
PSII in Syn. 6803 had remained unsolved
because the organism is sensitive to the
harsh conditions required for techniques
like X-ray crystallography, which is used to
elucidate molecular structures. To this day,
the only reported structures for PSII have
come from thermophilic cyanobacteria, organisms
that thrive in high temperatures.
However, they are poor model organisms
for mutagenesis experiments due to their
intolerance of growing with altered PSII.
“All this work has been going on in parallel–mutagenesis
in organisms with no
known structures, and structural determination
in thermophiles that could not
be mutated,” Brudvig said. “People just assumed
that they were all the same and that
they could use the thermophile as a basis
for structure.” Scientists have therefore been
forced to proceed with this assumption to
interpret their functional data.
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But this approach may not be truly justified.
Firstly, there are obvious differences in
the DNA sequences of the PSII genes from
mesophilic and thermophilic organisms,
which implies diverging structure and function.
Moreover, membrane proteins from
mesophilic and thermophilic organisms are
generally known to have different molecular
characteristics. Thus, the study of PSII
function is greatly limited by the lack of a
high-resolution structure for the model organism
from which most biophysical data
comes: Syn. 6803.
A Structural Blueprint
Large, often unstable, protein structures
like PSII from Syn. 6803 are difficult, if not
downright impossible, to crystallize for use
in X-ray crystallography experiments. But
there is now an alternative technique to
visualize this three-dimensional structure:
cryo-EM. Single-particle cryo-EM bombards
a thin sheet of a protein solution with
electrons, using a camera to detect how
electron waves interact with the sample. A
computer then reconstructs a 3D model of
the protein from hundreds of thousands of
2D images in different orientations. “I like
to think of myself as a very, very high-resolution
photographer,” Gisriel said.
The Brudvig lab reported the structure
of PSII from Syn. 6803 with single-particle
cryo-EM at a resolution of 1.93 Angstroms
(Å). For reference, the average resolution
for published cryo-EM membrane protein
structures is ~5Å. At this unprecedented
resolution level, the Brudvig group could
even see the presence of some individual
protons within the complex.
PSII is biologically found in a dimeric
state, with two identical monomers, each containing
twenty one subunits. The core consists
of four subunits, with thirteen peripheral subunits
embedded in the membrane and four
“extrinsic” subunits found on the inner surface
of the membrane. With their novel structure
in hand, the Brudvig group could now
identify any major differences between the
thermophilic and Syn. 6803 PSII enzymes.
Cofactors are non-proteinous molecules
within an enzyme that promote its
catalytic activity. Most cofactors are indeed
conserved between the two species, except
for a pigment called BCR101, which helps
absorb light energy. Previous studies had
suggested that BCR101 was important to
allow PSII to dimerize, where two identical
PSII proteins chemically associate. However,
even without BCR101, Syn. 6803 still
retains a dimeric configuration, implying
that BCR101 is not as crucial for this role.
Interestingly, some peripheral and extrinsic
subunits, namely PsbO, PsbU, and PsbV,
are quite dissimilar between PSII from
the different species. This was unexpected
because these subunits surround the intricately
controlled “active site” of PSII, where
the enzyme’s catalytic activity occurs and
performs key functions in water oxidation.
The last remaining extrinsic subunit,
PsbQ, is found in both thermophilic and Syn.
6803 PSII. Notably, however, PsbQ had never
before been observed bound in complex with
March 2022 Yale Scientific Magazine 23