Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using ...time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a₃ iron atom is in a ferryl (Fe4+ = O2−) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.
Photosystem I coordinates more than 90 chlorophylls in its core antenna while achieving near perfect quantum efficiency. Low energy chlorophylls (also known as red chlorophylls) residing in the ...antenna are important for energy transfer dynamics and yield, however, their precise location remained elusive. Here, we construct a chimeric Photosystem I complex in Synechocystis PCC 6803 that shows enhanced absorption in the red spectral region. We combine Cryo-EM and spectroscopy to determine the structure
function relationship in this red-shifted Photosystem I complex. Determining the structure of this complex reveals the precise architecture of the low energy site as well as large scale structural heterogeneity which is probably universal to all trimeric Photosystem I complexes. Identifying the structural elements that constitute red sites can expand the absorption spectrum of oxygenic photosynthetic and potentially modulate light harvesting efficiency.
Photosynthetic organisms have adapted to survive a myriad of extreme environments from the earth's deserts to its poles, yet the proteins that carry out the light reactions of photosynthesis are ...highly conserved from the cyanobacteria to modern day crops. To investigate adaptations of the photosynthetic machinery in cyanobacteria to excessive light stress, we isolated a new strain of cyanobacteria,
0216, from the extreme light environment of the Sonoran Desert. Here we report the biochemical characterization and the 2.7 Å resolution structure of trimeric photosystem I from this high-light-tolerant cyanobacterium. The structure shows a new conformation of the PsaL C-terminus that supports trimer formation of cyanobacterial photosystem I. The spectroscopic analysis of this photosystem I revealed a decrease in far-red absorption, which is attributed to a decrease in the number of long- wavelength chlorophylls. Using these findings, we constructed two chimeric PSIs in
sp. PCC 6803 demonstrating how unique structural features in photosynthetic complexes can change spectroscopic properties, allowing organisms to thrive under different environmental stresses.
Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons ...can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth ('pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A
adenosine receptor (A
AR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A
AR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.
First evolving in cyanobacteria, the light reactions of oxygenic photosynthesis are carried out by the membrane proteins, photosystem II and photosystem I, located in the thylakoid membrane. Both ...utilize light captured by their core antenna systems to catalyze a charge separation event at their respective reaction centers and energizes electrons to be transferred energetically uphill, eventually to be stored as a high energy chemical bond. These protein complexes are highly conserved throughout different photosynthetic lineages and understanding the variations across species is vital for a complete understanding of how photosynthetic organisms can adapt to vastly different environmental conditions. Most knowledge about photosynthesis comes from only a handful of model organisms grown under laboratory conditions. Studying model organisms has facilitated major breakthroughs in understanding photosynthesis, however, due to the vast global diversity of environments where photosynthetic organisms are found, certain aspects of this process may be overlooked or missed by focusing on a select group of organisms optimized for studying in laboratory conditions. This dissertation describes the isolation of a new extremophile cyanobacteria, Cyanobacterium aponinum 0216, from the Arizona Sonoran Desert and its innate ability to grow in light intensities that exceed other model organisms. A structure guided approach was taken to investigate how the structure of photosystem I can influence the spectroscopic properties of chlorophylls, with a particular focus on long wavelength chlorophylls, in an attempt to uncover if photosystem I is responsible for high light tolerance in Cyanobacterium aponinum 0216. To accomplish this, the structure of photosystem I was solved by cryogenic electron microscopy to 2.7-anstrom resolution. By comparing the structure and protein sequences of Cyanobacterium aponinum to other model organisms, specific variations were identified and explored by constructing chimeric PSIs in the model organism Synechocystis sp. PCC 6803 to determine the effects that each specific variation causes. The results of this dissertation describe how the protein structure and composition affect the spectroscopic properties of chlorophyll molecules and the oligomeric structure of photosystem I, possibly providing an evolutionary advantage in the high light conditions observed in the Arizona Sonoran Desert.
Cytochrome
c
oxidase (C
c
O) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using ...time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine C
c
O. It is assigned to the P
R
-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme
a
3
iron atom is in a ferryl (Fe
4+
= O
2−
) configuration, and heme
a
and Cu
B
are oxidized while Cu
A
is reduced. A Helix-X segment is poised in an open conformational state; the heme
a
farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.
Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons ...can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ~100 times by a moderate increase in the bandwidth (`pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A2Aadenosine receptor (A2AAR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A2AAR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.
Shannon entropy is offered here to establish an urban diversity indicator using the city of Sheffield, UK as an illustrative case. Diversity is calculated for several time periods throughout the ...city's evolution and provides a means to evaluate the 'mix' of green and urban space throughout its expansion and development. Secondly, three statistics models are developed to open discussion around alternative forms of urban development extrapolating from early settlement origins to the present day; these include an entropic, deterministic, and a hybrid model. Through this approach, the paper aims to aid the hypothetical exploration of urban growth dynamics.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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