Photosynthesis

(2) Electron Transfer in Hierarchical
Photochemical Systems

The primary goal of this work is to determine how structural,
dynamic, and energetic features of the natural photosynthetic
reaction center proteins of green plants and photosynthetic
bacteria influence the sequential electron transfer reactions that
lead to efficient photoinduced charge separation. Various methods
(e.g., isotopic substitution, mutagenesis, metal exchange) are
employed to modify the natural photosynthetic proteins in ways
that affect these parameters. Specialized electron paramagnetic
resonance (EPR) techniques are used to monitor the paramagnetic
intermediates involved in photosynthetic charge separation.
Theoretical modeling of electron transfer mechanisms and
electron spin polarized (ESP) EPR line shapes provide structural
and kinetic information about requirements for efficient charge
separation. The principles are applied to design systems that can
undergo efficient photoinduced charge separation; thereby
addressing the DOE goals of understanding photochemical
energy conversion and developing chemical systems and
processes to efficiently convert light into chemical energy.

The structurally characterized photosynthetic bacterial reaction
center (RC) protein provides an important and useful native system
to explore the nature of protein motion coupled to biological
electron transfer. While it is widely accepted that protein
conformational changes play an important part in biological
electron transfer, there is little experimental information on the
role of anisotropic local protein environments in modulating
electron transfer. Recently, we detected a local protein
environment, a Zn²⁺ site, that apparently controls protein
dynamics important for electron transfer in the RC. Isolated RCs
from Rhodobacter sphaeroides R26 were found to bind Zn²⁺
stoichiometrically and in a site distinct from the well-characterized
non-heme high spin Fe²⁺ site which is buried in the protein interior
between the quinones Q_A and Q_B. Zn²⁺ binding to the new site
dramatically slows electron transfer from Q_A to Q_B with the
room temperature kinetics becoming distributed across the
microsecond to millisecond time domain. We proposed that Zn²⁺
binding alters the dynamics of conformational changes in the RC,
thereby influencing electron transfer. Since our initial report of
this second metal site, others have extended this work to show
that Zn²⁺ and Cd²⁺ binding influences the proton uptake of
Q_B. From these studies, proposed mechanisms for inhibition
of proton transfer include that the metal ion is binding to ligands
that act as proton donors, or that the metal ion electrostatically
hinders proton uptake. In order to delineate the mechanism of
metal ion-induced modulation of electron and proton transfer
we have investigated the influence of Cu²⁺-binding to the RC
by utilizing a combination of bioinorganic, transient optical,
and magnetic resonance techniques.

Transient optical measurements show that Cu²⁺ slows Q_A^-Q_B ®
Q_AQ_B^- electron transfer in native Fe-containing RCs, consistent
with Cu²⁺ binding at or near the Zn²⁺ site that regulates
Q_A^-Q_B ® Q_AQ_B^- electron transfer and Q_B proton uptake.
Thus, methods were developed to specifically bind Cu²⁺ to two
distinct metal sites on the RC; the surface metal ion binding site
on native Fe-containing RCs and, for comparison, to the native
non-heme Fe-site in biochemically Fe-removed RCs. Cu²⁺ EPR
spectra were obtained to provide information about the metal
ion coordination environment and surrounding protein for each
sample. The continuous wave (cw) EPR results clearly indicate
two spectroscopically different Cu²⁺ environments on the RC.
A cw EPR spectrum typical of a type 2 or normal copper center
was observed for RCs with Cu²⁺ bound to the surface site. The
spectrum is indicative of a tetragonal Cu²⁺ environment. The Cu²⁺
spectrum of RCs with Cu²⁺ substituted into the Fe-site is nearly
identical to that previously reported for chaotropically treated
Fe-removed RCs, and was interpreted as arising from coordination
to three nitrogen ligands in a distorted octahedral environment.
This Cu²⁺ bound to the Fe-site is magnetically coupled by
exchange and dipole-dipole interactions with the unpaired spin of
Q_A^-, as shown in the light-induced EPR spectrum. In contrast,
the Cu²⁺ EPR signal does not change in the light when Cu²⁺ is
bound to the surface site. Thus, the Cu²⁺ does not magnetically
interact with the light-induced radicals and, therefore, the surface
site must be at least 23 Å removed from the primary donor, P⁺,
and reduced quinone acceptor, Q_A^-.

Pulsed EPR spectroscopic experiments of Cu-RCs were obtained
with our recently rebuilt homodyne pulsed X-band EPR
spectrometer. The pulsed spectrometer is designed to perform
several types of experiments, including echo detected field
swept EPR experiments, echo decay and free induction decay
experiments, delay after laser flash experiments and two
dimensional experiments. We have used electron spin echo
envelope modulation (ESEEM) spectroscopy to characterize
the magnetic interactions between Cu²⁺ and weakly coupled
magnetic nuclei in the RC. These ESEEM spectra represent the
first reported ESEEM spectra of Cu-RCs, and provide the first
direct solution structural information about the ligands at the
surface metal site. From these pulsed EPR experiments,
modulations were observed that are consistent with multiple
weakly hyperfine coupled ¹⁴N nuclei in close proximity to
Cu²⁺, indicating that two or more histidines ligate the Cu²⁺ at
the surface site. These ESEEM results agree with our proposal
that histidines H68, H126, H128, or L211 may be metal ligands
at the surface site. These histidines are positioned beneath the
Q_B binding pocket and surround a water channel that is
proposed to be a proton pathway to Q_B. Thus, metal, cw, and
pulsed EPR analyses confirm that we have developed reliable
methods for stoichiometrically and specifically binding Cu²⁺ to
a surface site that is distinct from the well characterized Fe-site
and support the view that Cu²⁺ is bound at or near the Zn²⁺
site that modulates electron transfer between the Q_A and Q_B
and proton uptake by Q_B.

The structure of the charge separated state P⁺A₁^- of photosystem
I, where P⁺ is the oxidized chlorophyll donor and A₁^- the reduced
quinone acceptor, has been studied with high time resolution EPR
at W-band (with G. Kothe). The electron spin polarized (ESP)
EPR signal observed for the light induced state P⁺A₁^- allows us to
directly examine the geometry of the electron donor and acceptor.
Transient EPR spectra were obtained for lyophilized, whole cells
of deuterated, ¹⁵N-labeled cyanobacteria. The observed spin-
polarized W-band EPR spectra of P⁺A₁^- indicate a magnetic-field
-induced orientation of the photosynthetic RCs in the field of the
EPR spectrometer. This is an important observation, and critical
for interpreting high-field EPR results. Photosystem I apparently
aligns when frozen in the magnetic field, resulting in a slightly
different polarized spectrum than when frozen without the field.
When taking the orientation effect into account, the correlated
radical pair model satisfactorily describes the observed EPR
spectra, providing new structural information about the charge
separated state P⁺A₁^- in frozen solution.

We have installed a D-band (140 GHz) spectrometer, capable
of operating in the pulsed and cw modes. This new capability will
greatly aid our analysis of the transient and triplet radical species
we study in the photosynthetic and model systems.

Future Research

Planned research of the electron transfer processes in photosynthesis
will focus on studying the surface metal ion binding site of bacterial
reaction center samples. Key questions remain concerning the
coordination environment, location, and role of this metal site in
regulating electron and proton transfer. Transient optical
experiments show that both Zn²⁺ and Cu²⁺ slow Q_A^-Q_B ®
Q_AQ_B^- electron transfer, providing important evidence that
Cu²⁺ and Zn²⁺ bind in the same vicinity of the RC. We cannot
say, however, that the Cu⁺² and Zn²⁺ sites are identical. In fact,
we will explore the possibility that the metal site in Rb. sphaeroides
R26 RCs is binuclear. Several experiments indicate the possibility
of a binuclear site: Zn²⁺ and Cu²⁺ competitive metal-binding
experiments were unsuccessful and under specific conditions it
is possible to bind a total of 2 mole equivalent (Zn²⁺ + Cu²⁺)
of metal ion to RCs, in addition to the native Fe²⁺. We plan to
correlate structural information obtained from EPR spectroscopic
experiments (cw and pulsed) to the solution kinetics for a series
of RC samples with defined metal ion ratios. In addition, we will
initiate metal-binding studies of RCs from different bacterial
strains. We will use purified RCs from both Rp. viridis and
Rb. capsulatus. The amino acid sequences of each differ in
the histidine-rich protein region proposed for the surface site.
Thus, these experiments could provide important insight into the
location of the surface metal site as well as the specific mechanism
for metal-induced regulation of electron and proton transport for
RCs from several species of purple photosynthetic bacteria.

The usefulness of the Cu²⁺ ion as a spin-probe will be exploited
in several other ways. We are in the process of obtaining software
for modeling ESEEM spectra to determine the number of histidine
ligands bound to the Cu⁺² in the surface and Fe sites. Furthermore,
relaxation measurements obtained with pulsed EPR methodologies
will allow us to determine the distance of the bound Cu⁺² to the
unpaired spins of photoinduced chromophores of the protein.
High-field EPR at D-band will provide detailed information about
the symmetry of the metal site and nature of the metal ligands. We
will also use our expertise in isotopic labeling to look for evidence
of a water channel in the vicinity of the surface metal site. Subtle
changes in the Cu²⁺ coordination sphere in the presence of H₂O
or D₂O for protonated and deuterated RCs could potentially be
monitored with pulsed (X- and D-band) EPR techniques. These
experiments could provide important information regarding the
mechanism of proton movement to the Q_B proton acceptor in the
RC. In addition, conditions for Cu- and Zn-binding to the RC in
the presence of precipitating lipids will be determined. These
experiments will allow us to ascertain the feasibility of preparing
quality Cu- and Zn-RC samples for X-ray absorption spectroscopic
experiments (XAFS) at APS. These XAFS experiments would
provide structural information that is both similar and
complementary to that obtained from EPR.

Determining the role of the metal ion in regulating electron and
proton transfer will require correlation of the metal site structure
with the electron and proton transfer events. One could envision
with a surface site, protein flexibility such that different metal ions
would coordinate in inherently favorable geometries. Such
structural differences could potentially provide mechanistic insight
into the role of the metal ion and/or protein in electron transfer.
Thus, methodologies for binding different transition metal ions, i.e.
Co²⁺, Ni²⁺, and Cd²⁺, to RCs will be developed. Spectroscopic
techniques to concomitantly investigate metal coordination
environment and solution kinetics (transient optical measurements)
for a wide variety of metal-RC complexes will be developed.

In a related study, we plan to investigate the role of the non-heme
Fe²⁺. A definitive role for this Fe²⁺ has yet to be determined. In
1986, it was reported that the substitution of different divalent
metal ions into the Fe-site does not significantly alter the Q_A^-Q_B ®
Q_AQ_B^- electron transfer rate. We propose to revisit these
studies for several reasons. In a recently reported X-ray crystal
structure, significant movement of Q_B and surrounding protein
matrix was observed for the “light” structure vs. the ground state
“dark” structure. It was proposed that this movement is necessary
to facilitate the electron transfer from Q_A to Q_B. Thus, the
protein structure in the vicinity of the quinones could be intimately
connected to reorganization energies important for electron
transport. In fact, preliminary X-ray absorption fine structure
(XAFS) studies indicate preparation dependent structural changes
in the Fe-site of RCs that can be related to changes in the
surrounding protein matrix and possibly to the rate of the electron
transfer from Q_A to Q_B. Substitution of different divalent metal
ions into the Fe-site will provide a means to further interrogate
the region of the protein between the quinones. We have
developed reproducible and reliable preparations of iron-removed,
zinc-substituted, and most recently, copper-substituted bacterial
reaction center protein. Thus, we have an extensive knowledge
base to aid us in developing new procedures for incorporating
metal ions, e.g. Ni²⁺, Mn²⁺, Cd²⁺, and Co²⁺, into the Fe-site.
Initially, we plan to correlate transient optical experimental results
with the type of metal in the Fe-site. The structures of each metal
center will be characterized with EPR and XAFS spectroscopic
techniques. These proposed studies will be an extension of our
previous time-resolved EPR studies of kinetically-characterized
Fe-removed RCs that exhibited either the native 200 ps^-1 ("fast")
or 3-6 ns^-1 ("slow") k_Q electron transfer rates from the
bacteriopheophytin to the quinone (Q_A). Detailed analysis of the
electron spin polarized EPR spectra of these Fe-removed RCs
implicated a role for protein reorganization energy in regulating
the k_Q electron transfer rate. Examination of specifically metal-
substituted bacterial reaction center (RC) proteins will help
elucidate critical factors (structure, reorganization energy)
that consistently regulate the electron transfer in this system.

The location of the iron-sulfur clusters F_A and F_B in photosystem I
will be investigated with our Very High Frequency (VHF) 140 GHz
CW/Pulsed EPR spectrometer (with G. Brudvig). Photosystem I
mediates electron-transfer from plastocyanin to ferredoxin. In
addition to three [4Fe-4S] clusters (F_x/A/B), the multisubunit
protein photosystem I contains other redox active cofactors
including a P₇₀₀ chlorophyll dimer, a monomeric chlorophyll
electron acceptor (A₀) and a phylloquinone (A₁). The sequence
of events between the iron-sulfur cluster, F_x, and ferredoxin
remains unclear. We plan to resolve the EPR signals from the
charge-separated spin pairs (P₇₀₀-F^red_x/A/B) at high field.
These experiments will help resolve if F_A and/or F_B is the
terminal electron acceptor to ferredoxin, providing information
about the chain of terminal electron transfer steps in photosystem I.

The extent to which the protein and solvent environment optimize
yield of primary photosynthetic electron transfer in oxygen-evolving
organisms will be investigated with various isotopically-labeled
photosystem I samples. Nuclear coherences observed from the
P⁺A^- state suggests that the structure of this state differs at low
temperature vs. room temperature (with G. Kothe). This is consistent
with our finding that A₁ is labile. To examine the possibility of the
existence of two or more conformations of P⁺A^-, we have prepared
samples under controlled sample freezing conditions. Zero quantum
coherences will be measured to investigate any temperature-induced
structural changes in the P⁺A^- state. Furthermore, preliminary
transient EPR results suggest that there is a water channel in the
vicinity of the quinone acceptor. We have prepared samples with
protonated protein in a D₂O surrounding and deuterated protein
in a H₂O surrounding to examine the roles of H-bonding and a
water channel in the A₁ acceptor site. A deuteron matrix ENDOR
line that we have observed in previous experiments will be
investigated to see if it can be used to characterize water channels
inside the reaction center protein.

The photoprotection mechanism of photosystem II will be
investigated with high-field EPR (with G. Brudvig). It is believed
that a secondary electron-transfer pathway exists to prevent
photoinhibition of photosystem II. Recent resonance Raman
spectroscopic results implicate a role for carotenoid cation
radical in the secondary electron-transfer pathway that consists
of alternate donors to P⁺₆₈₀, including cytochrome b₅₅₉ and
an accessory chlorophyll (Chl_z). Unfortunately, the electron
paramagnetic resonance spectra of carotenoid and chlorophyll
radicals are virtually identical. We will use our VHF 140 GHz
CW/Pulsed EPR spectrometer to spectroscopically resolve the
carotenoid cation radical from the oxidized chlorophyll radical.
Deuterated photosystem II core complexes will be used to
increase spectral resolution. This EPR characterization will
provide important information about the structure of the carotenoid
cation radical and possibly the nature of the secondary electron
transfer pathway in photosystem II. In addition, we hope to
obtain data relevant to understanding how carotenoids play
different roles in natural systems (donor or acceptor of excited
state energy, molecular wire).

1. Molecular Structure Determination for Photogenerated
   Intermediates in Photoinduced Electron Transfer Reactions
   using Steady-State and Transient XAFS, L. X. Chen,
   M. R. Wasielewski, T. Rajh, P. L. Lee, M. C. Thurnauer
   and P. Montano, J. Phys. IV France, 7, C2-569-C2-572
   (1997)
2. Influence of Iron-Removal Procedures on Sequential
   Electron Transfer in Photosynthetic Bacterial Reaction
   Centers Studied by Transient EPR Spectrscopy, L. M. Utschig,
   S. R. Greenfield, J. Tang, P. D. Laible and M. C. Thurnauer,
   Biochem., 36, 8548-8558 (1997)
3. Spectroscopic Characterization of Quinone-Site
   Mutants of the Bacterial Photosynthetic Reaction Center,
   P. D. Laible, Y. Zhang, A. L. Morris, S. W. Snyder,
   C. Ainsworth, S. R. Greenfield, M. R. Wasielewski,
   P. Parot, B. Schoepp, M. Schiffer, D. K. Hanson and
   M. C. Thurnauer, Photosyn. Res., 52, 93-103 (1997)
4. Anomalous Pulse-Angle and Phase Dependence of Hahn's
   Electron Spin Echo and Multiple-Quantum Echoes of the
   Spin Correlated Radical Pair P₇₀₀⁺A₁^- in Photosystem I,
   H. Hara, J. Tang, A. Kawamori, S. Itoh and M. Iwaki,
   Appl. Magn. Reson., 14, 367-379 (1998)
5. Structural Implications of Transient X-, K- and W-Band
   EPR Spectra of Deuterated and Protonated Reaction
   Centers of Rhodobacter sphaeroides R-26, J. Tang,
   Chem. Phys. Lett., 290, 49-57 (1998)
6. Protein Modifications Affecting Triplet Energy Transfer
   in Bacterial Photosynthetic Reaction Centers, P. D. Laible,
   V. Chynwat, M. C. Thurnauer, M. Schiffer, D. K. Hanson
   and H. A. Frank, Biophys. J., 74, 2623-2637 (1998)
7. Transient W-Band EPR Study of Sequential Electron
   Transfer in Photosynthetic Bacterial Reaction Centers,
   J. Tang, L. M. Utschig, O. Poluektov and M. C. Thurnauer,
   J. Phys. Chem. B, 103, 5145-5150 (1999)
8. Magnetic Field Induced Orientation of Photosynthetic
   Reaction Centers as Revealed by Time-Resolved W-Band
   EPR of Spin-Correlated Radical Pairs, T. Berthold,
   M. Bechtold, U. Heinen, G. Link, O. Poluektov, L. Utschig,
   J. Tang, M. C. Thurnauer and G. Kothe, J. Phys. Chem. B,
   103, 10733-10736 (1999)
9. EPR Investigation of Cu²⁺-Substituted Photosynthetic
   Bacterial Reaction Centers: Evidence for Histidine Ligation
   at the Surface Metal Site, L. M. Utschig, O. Poluektov,
   D. M. Tiede and M. C. Thurnauer, Biochem., 39,
   2961-2969 (2000)
10. High-Field EPR Study of Carotenoid and Chlorophyll
    Cation Radicals in Photosystem II, K. V. Lakshmi,
    M. J. Reifler, G. W. Brudvig, O. G. Poluektov,
    A. M. Wagner, and M. C. Thurnauer, J. Phys. Chem.,
    104 (45), 10445-10448 (2000)
11. Cu²⁺ Site in Photosynthetic Bacterial Reaction Centers
    from Rhodobacter sphaeroides, Rhodobacter capsulatus,
    and Rhodopseudomonas viridis, L. M. Utschig,
    O. Poluektov, S. L. Schlesselman, M. C. Thurnauer,
    and D. M. Tiede., Biochem., 40, 6132-6141 (2001)
12. Structure of the P⁺₇₀₀A^-₁ Radical Pair Intermediate
    in Photosystem I by High Time Resolution Multifrequency
    Electron Paramagnetic Resonance: Analysis of Quantum
    Beat Oscillations, G. Link, T. Berthold, M. Bechtold,
    J.-U. Weidner, E. Ohmes, J. Tang, O. Poluektov,
    L. Utschig, S. L. Schlesselman, M. C. Thurnauer, and
    G. Kothe, J. Am. Chem. Soc., 123, 4211-4222 (2001)

Contacts: M. C. Thurnauer, L. Utschig, O. Poluektov

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