In 1971, Norris and co-workers noticed that the line width of the P700+ EPR spectrum is
narrower than that of Chl-a monomers and is consistent with an unpaired spin delocalization over an entity containing two chlorophyll molecules. This led to the conclusion that P700 is a Chl-
a dimer [38]. This proposal was also supported by optical spectroscopy studies. P700 absorbs at
700 nm which is 30 nm further to the red than that of Chl-a in solution and such a shift suggests a multimeric nature to P700. Also, the light-minus-dark difference circular dichroism (CD)
spectrum of PS I particles shows two approximately equal bands at 696.5(+) and 688(-) nm [33, 39] which was again an indication of a dimeric structure. FTIR difference spectra obtained from PS I particles show a broad positive difference band centered near 3200 cm-1, which is
characteristic of the electronic transition of a dimeric species. Also two difference bands are 1.2.1 A Brief Historical Review
The primary electron donor in PS I was named P700 by Kok, B. in 1956 (P for ‘pigment’
and ‘700’ because he observed a light induced absorption change in spinach chloroplast around 700 nm) [32].
Early research on the structure and properties of the primary electron donor were mainly based on optical and EPR studies in comparison with Chl-a [33]. The first EPR signal from the P700+ was obtained by Commoner in 1956 [34]. Later EPR signals associated with P700+ and their
kinetics, as well as the kinetics of optical signals were also obtained and correlated quantitatively [33, 35-37]. These early studies established P700 as one or more Chl-a molecule(s).
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observed for P700 FTIR DS in the region of the ester absorption for Chl-a in solution, which also
indicate that two different pigments contribute to the spectra. 1.2.2 X-Ray Crystallography of P700
The first X-ray crystal structural model of PS I at 6Å resolution was isolated from Synechococcus sp. (now named Thermo-synechococcus elongatus) in 1987 [40]. Detailed structure at 4Å followed soon and a dimeric nature for P700 was proposed based on these X-ray
crystallographic studies. A more complete structure of PS I at 2.5Å came out in 2001 [6, 7, 41- 45], in which the two chlorophylls of P700 were clearly resolved and it became evident that P700
was a hetrodimer of a Chl-a and Chl-a’ molecule (which is an epimer at the C13 position of the
chlorin ring system), located on PsaB (PB) and PsaA (PA) respectively. The resolution of the
crystal structure was also sufficient to analyze the protein-cofactor interactions and shows that P700 is an asymmetric dimer with the two chlorophyll molecules having different extent of
interactions with the protein environment. 1.2.3 Structure of Chlorophyll-a
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The molecular structure and IUPAC numbering scheme for Chl-a is shown in Figure 1.4. Chl-a consists of a penta-pyrrolic porphyrin body with a long phytyl tail attached at the 17 position. Chl-a is a magnesium containing porphyrin, with the magnesium coordinated by four nitrogen atoms and characteristically contains a vinyl group at position 3. The four pyrrol rings are joined by methelene bridges and the system of double bonds forms a closed, conjugated macrocyclic loop [46, 47].
1.2.4 The Structure of P700
Figure 1.5: (a) Structure of P700 showing the Mg-Mg distance (b) The angle between the lines formed by the Mg-N4 bonds
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The primary electron donor P700, is a heterodimer consisting of a Chl-a (PB) and a Chl-
a’(PA) molecule. Chl-a’ is an epimer of Chl-a at the C13 position of the chlorin ring system
(Figure 1.4). The macrocycles of two chlorophylls of P700 are parallel and is separated by 3.6 Å.
Pyrole rings I and II of PA and PB overlap, and the Mg2+ ions are separated by 6.3 Å and the N4A-
MgA-MgB-N4B dihedral angle is ~57° (Figure 1.5).
1.2.5 The Protein Environment of P700
Figure 1.6: Structure of P700 obtained from the X-ray crystallographic structure analysis at 2.5Å resolutions.
The Chl-a and Chl-a’ molecules of P700 are bound to PsaA and PsaB and the amino acid
environment around these molecules is decidedly asymmetric (Figure 1.6). Both the chlorophylls of P700 are axially ligated to histidine residues symmetrically (HisA680 and HisB660 in Thermo-
synechococcus elongatus sequence numbering scheme) but their hydrogen bonding interaction with the protein back bone is asymmetric.
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Figure 1.7: (a) View of ring V of PA showing possible H-bond interactions to the 131 keto and 133 ester C=O groups. (b)
View of ring V of PB showing the nearby amino acids. These residues does not provide any H-bond to PB.
Figure 1.7(a) shows that the hydroxyl oxygen of ThrA743 (The amino acid numbering is according to the sequence of the cyanobacterium Thermo-synechococcus elongates, for Synechocystis sp. PCC 6803, and C. reinhardtii sequence numbering see Table 1.1) is 2.98 Å from the 131 keto carbonyl (C=O) oxygen of P
A and is suitably positioned to form a hydrogen
bond. In addition, the ThrA743 hydroxyl oxygen is 2.7 Å from the oxygen atom of a water molecule (H2O-19). The oxygen atom of H2O-19 water molecule is 3.28 Å away from the
methoxy oxygen of the 133 ester C=O of P
A, and is also within hydrogen bonding distance to
TyrA603 and SerA607.The C=O groups of PB on the other hand is free of any hydrogen bonding
interactions (Figure 1.7(b)).
Table 1.1: Sequence numbering of some PsaA and PsaB aminoacids in close proximity of P700 in Thermo-synechococcus elongates, Synechocystis sp. PCC 6803, and Chlamydomonas reinhardtii.
S. elongatus Synechocystis C. reinhardtii Axial ligands to P700 HisA680
HisB660 HisA676 HisB651 HisA676 HisB656 Vicinity of 131 keto
C=O of P700
ThrA743
TyrB727 ThrA739 TyrB718 ThrA739 TyrB722 Vicinity of 133 ester
C=O of P700
SerA607, TyrA603
GlyB594, LeuB590 SerA603, TyrA599 GlyB585,LeuB581 SerA604,TyrA600 GlyB589,LeuB585 Vicinity of 173 ester
C=O of P700
TyrA735
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