Photosynthesis

(1) Structure and Energy-Conserving
Function in Natural Photosynthesis

The goal of this program is to identify the structures and
structural dynamics that contribute to the high yield of
photochemical energy conversion in natural photosynthesis.
Natural photosynthesis relies upon structural organization on
multiple length scales, ranging from the specific atomic
environment surrounding electron donors and acceptors
within individual photosynthetic proteins, to the organization
of multiple photosynthetic proteins into cooperative units
within photosynthetic membranes. Precise control of structural
organization on multiple length scales is essential for the high
yield of solar energy conversion in natural photosynthesis, and
has not been fully duplicated in artificial systems. This project
investigates the contribution of structural hierarchy to
photochemical energy conversion in natural photosynthesis in
order to devise strategies for developing more efficient artificial
photosynthetic systems. Specifically, this project seeks to (i)
determine the structure of photosynthetic proteins in crystalline
and noncrystalline environments, (ii) correlate the structure of
photosynthetic assemblies with photochemical energy conversion
processes, and (iii) characterize rate-limiting structural dynamics
accompanying electron transfer. A highlight of this project is
the development of novel, high-precision, wide-angle X-ray
scattering techniques to resolve the structure of photosynthetic
assemblies under functional conditions in crystalline and
noncrystalline environments. These scattering techniques are
proving to be highly sensitive measures of global structure and
structural change, and nicely complement the precise, localized
structural information obtained from X-ray and pulsed electron
paramagnetic resonance spectroscopies supported by other
projects in this summary (see Sections 2 and 4). Hence, the
combination of all of these structural tools provides a
comprehensive means to characterize the structure and structural
dynamics of photosynthetic assemblies under functional conditions.

Measurement of Structure and Structural Dynamics in
Noncrystalline Environments. We are developing new,
multidimensional X-ray and neutron scattering techniques for
quantitatively measuring molecular structure and reaction-linked
structural change for photochemical systems in “real-world”
liquid and noncrystalline media using the DOE-BESSRC
time-resolved/anomalous X-ray scattering beam line at the
Advanced Photon Source (APS) and the Intense Pulsed
Neutron Source (IPNS). The extremely high flux and exceptional
stability of the pulsed X-ray beam at APS has opened up new
opportunities for collecting time-resolved, wide-angle X-ray
scattering data for probing the fine structure of macromolecules
in solution on the distance scale of 3 to 10 Å. We have found
that wide-angle solution scattering experiments provide a
meaningful measure of molecular structure under conditions in
which traditional crystallography or solution NMR methodologies
fail. This work is important because it provides a new, quantitative
technique for measuring macromolecular structure, reaction-linked
structural change. During FY2000 this project made significant
progress in developing experimental and computational techniques
for the collection and analysis of time-resolved, wide-angle
X-ray scattering data at APS. Highlights of this work include the
following.

• Development of instrumentation and techniques at the Basic
  Energy Sciences Synchrotron Radiation Center at the
  Advanced Photon Source, in collaboration with Seifert
  (CHM), Winans (CHM), and Thiyagarajan (PNS), optimized
  for high-precision measurement of small- and wide-angle
  scattering for macromolecules in solution. We developed
  techniques to collect high-resolution scattering data protein
  samples using flowed, temperature-controlled samples in
  volumes as small as 20 µl, with protein concentrations in
  the range 0.5 mg/ml to 5 mg/ml.
• Demonstrated the sensitivity of wide-angle scattering data
  to the details of protein structure by a preliminary survey of
  the wide-angle scattering patterns for a number of
  water-soluble and membrane-associated proteins. Two
  general conclusions were derived from this survey. First,
  scattering patterns for individual proteins are found to be
  distinguishable, even among the highly homologous series
  of c-cytochromes. This finding suggests that wide-angle
  scattering patterns accurately reflect details of protein
  folding. Second, large variations are found in the similarity
  between scattering patterns calculated from crystal
  coordinate data and experimental solution scattering
  data. This result indicates that individual proteins vary
  significantly in the fidelity of transfer of protein structure
  from the crystalline to solution state, and demonstrates
  the importance of collecting structural information for
  proteins in solution in order to understand their physiological
  function.
• Demonstrated the sensitivity of our techniques for resolving
  protein structural change by detecting the oxidation state
  dependent changes in cytochrome c, which show that our
  scattering techniques are capable of resolving a subtle,
  delocalized structural change as small as 0.2 Å rms difference
  in protein backbone atom positions, or a larger 5 Å change in
  atomic position occurring among a small, 1% subset of atoms.
  These results establish the basis for using high-precision,
  wide-angle scattering measurements to detect subtle,
  reaction-linked structural change.
• Developed computer algorithms for quantitative calculation
  of small- and wide-angle X-ray scattering profiles for proteins
  based upon crystal coordinates or other molecular models.
  This work is the first step in the development of a new
  technique for determining protein structure in solution by
  refining crystal coordinate data to fit high-precision,
  wide-angle scattering patterns.
• Characterized the structure of the reaction center-cytochrome
  c electron transfer complex in solution by small angle neutron
  scattering, in collaboration with Littrell (PNS) and
  Thiyagarajan (PNS), and correlated variations in the structure
  of this complex to variations in electron transfer rate. This
  work established a foundation for extending this technique
  to the characterization of the structure of other photosynthetic
  bimolecular electron transfer complexes, such as the
  PSII-oxygen evolving protein complex.

Future research in this area will focus upon correlating structure
and structural dynamics with photochemical energy conversion
in natural photosynthesis. This work will exploit new
opportunities for resolving the structure of photosynthetic
assemblies using new, high-precision X-ray and neutron
scattering techniques. Photochemical assemblies will be
characterized in photosynthetically relevant media, including
liquid solutions, organized biomimetic membranes, and
liquid-crystalline media. Specific projects include the following:

Reaction-linked Structural Reorganization Associated
with Quinone Photochemistry. This project will use
high-precision, wide-angle X-ray scattering techniques to
analyze the structural changes that are associated with quinone
photochemistry in reaction centers. A considerable amount of
biochemical and spectroscopic data indicates that the electron
transfer between quinones in the reaction center is a
conformationally gated reaction. Crystal structures for reaction
centers in the quinone oxidized and reduced states show structural
differences. These structural changes are controversial, and the
question as to whether these changes occur under normal
photosynthetic function remains to be determined. Our calculations
show that the structural reorganization seen in crystals would be
resolved in solution scattering patterns. We will use time-resolved
scattering techniques to detect reaction-linked conformational
changes associated with quinone photochemistry, and correlate
the kinetics and temperature-dependencies of these conformational
changes to rate-limiting steps in electron transfer.

Structures of Reaction Center-Electron Donor Complexes.
Techniques will be developed for resolving the structures of
reaction center-electron donor complexes using “multidimensional,
high-resolution scattering patterns” as fitting criteria for iterative
configurational searches. X-ray scattering patterns of the bacterial
reaction center - cytochrome c₂ complex will be obtained to
complement neutron scattering data collected earlier, permitting
the development of a more refined structural model. An analogous
project will be initiated in collaboration with Berry (U. of
Minnesota) to investigate the structure of the PSII-oxygen
evolving complex. This aspect of the project will develop a
model using the available crystallographic data on the structure
of the PSII core complex, and will provide the first structural
characterization of the oxygen-evolving complex.

Structural Basis for Metal Ion Activity in Photosynthesis.
This project will use scattering techniques to identify the structural
basis for divalent metal ion regulation of reaction center
photochemistry, and metal ion function as photosynthetic electron
donors/acceptors. Specifically, this project will investigate the
changes in reaction center structure and dynamics induced by
binding divalent metal ions to a surface site on bacterial reaction
centers. As described more fully in Section 2, this metal ion binding
is significant because kinetic assays suggest that it modifies the
conformational gating controlling the electron transfer between
quinones buried in the core of the reaction center. In addition, this
project will investigate the structure of hybrid photochemical
assemblies between PSI reaction centers and hydrogen evolving
colloidal metal catalysts.

Low-temperature, High-precision, Wide-angle X-ray
Scattering of Photosynthetic Proteins. Techniques will be
developed for measuring high-precision, wide-angle X-ray
scattering patterns for photosynthetic assemblies as a function
of temperature from room temperature to 77 K. Our current
measurements have shown that a significant broadening of
wide-angle scattering patterns occur in the temperature range
280 K to 350 K, due to temperature-dependent variation in
protein dynamics. We expect to see enhanced resolution of
wide-angle scattering features upon cryogenic cooling, which
will offer new opportunities to resolve the structure of
photosynthetic proteins. To the best of our knowledge, the
recording of scattering patterns for proteins at cryogenic
temperatures will be the first-of-a-kind experiment. This new
technique will be used to investigate temperature-dependent
changes in reaction center structure, which will be used to
identify the cause of temperature-dependent changes in
spectroscopic and electron transfer properties of reaction centers.

Contact: D. M. Tiede

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