An aggregate of chlorophyll a′ (Chi a′, C132‐epimer of ChI a) formed in a methanovwater (40160, vol/vol) mixed solvent was examined by visible absorption, circular dichro‐ism (CD), fluorescence and resonance Raman spectrosco‐pies in relation to its possible involvement in the core of photosystem I reaction center. The Chl a′ aggregate exhibited a sharp, double‐peaked absorption spectrum (690 and 715 nm) accompanied by an intense, conservative CD signal. The fluorescence excitation polarization spectrum showed that the doublet results from the exciton splitting in a single aggregate species. Time‐dependent changes in the spectroscopic properties clearly point to a simple transformation process from one molecular species to another, though a minor component appears to coexist. This conclusion was supported also by the principal multicom‐ponent spectral estimation analysis of the transients of absorption spectra. The species formed at the initial stage is most probably a T‐shaped dimer or oligomer, which is then gradually converted into the final major product, presumably a stacked dimer. In both of these states, the Chl molecules are linked together via direct coordination of the C13’keto carbonyl oxygen onto the Mg atom of neighboring molecules, as suggested by almost identical resonance Raman spectra in ordinary and deuterated methanovwater mixed solvents. The stacked dimers probably further associate to form a colloidal state in this solvent system. Based on these results, a model for the Chl a′ aggregation is proposed.

The “phase behavior” of chlorophyll a‘ (Chl a‘, C132-epimer of Chl a) dissolved in aqueous methanol was examined in terms of the composition of the solvent. This study aimed at elucidating the property of Chl a‘, the exotic pigment found in a photosynthetic reaction center complex, as well as at clarifying the nature of the Chl aggregation in aqueous media. Visible absorption, circular dichroism (CD), fluorescence and resonance Raman spectroscopies, dynamic light-scattering measurements, and electron microscopy were utilized. Chl a‘ formed either of two types of colloids depending on the solvent composition. The one formed over a wide methanol volume percentage (ca. 73−30%) commonly possessed a single microscopic structural unit that yielded the double-peaked absorption (ca. 690 and 715 nm) accompanied by a symmetric dispersed-type CD spectrum. Increasing methanol concentration within this solvent composition range enhanced the size of the colloid and finally caused critical opalescence, which was reminiscent of the critical behavior of the aqueous solution of nonionic surfactants. These findings indicate that the microscopic structure of the Chl a‘ aggregate was independent of the size and shape of the colloid. This is in sharp contrast to the solvent composition dependence of the Chl a aggregation in the same medium:  an increase in methanol concentration in going from 40% to 70% (vol/vol) shifted a broad red-most absorption band from ca. 700 to 750 nm, which was correlated to the enhancement of the aggregation number and the colloidal size. The difference between the aggregation behaviors of Chl a and a‘ suggests a narrower choice of possible molecular arrangements in the Chl a‘ aggregate as an inherent property of the pigment. The nature of the Chl aggregation in aqueous media is discussed in relation to the micellization of nonionic surfactant.

Reported crystallographic data and calculated molecular models indicated that chlorophyll (Chl) a and bacteriochlorophyll (BChl) a tend to bind the fifth ligand on the side of the macrocycle where the C13(2)-(R)-methoxycarbonyl moiety protrudes (denoting the 'back' side). The crystal structures of 34 photosynthetic proteins possessing (B)Chl cofactors revealed that most of Chl a and BChl a (and b) are coordinated by any peptidyl residue (e.g., histydyl-imidazolyl group), peptidyl backbone or water from the 'back' side. Almost all the cofactors that bind a water molecule as the fifth ligand in these proteins have a 'back' configuration. Theoretical model calculations for methyl chlorophyllide a (MeChlid a) and methyl bacteriochlorophyllide a (MeBChlid a) bound to an imidazole molecule indicated that the 'back' side is energetically favored for the ligand binding. These results are consistent with the fact that ethyl chlorophyllide a (EtChlid a) dihydrate crystal consists of the 'back' complex. The modeling also showed that both removal and stereochemical inverse of the C13(2)-methoxycarbonyl group affect the relative stability between the 'back' and 'face' complexes. The effect of the C13(2)-moiety on the choice of the macrocycle side for the ligand binding is discussed in relation to the function of P700.

The supramolecular structure of the aggregate of chlorophyll a′ (Chl a′, the C13 2 -(S)-epimer of Chl a) was examined in comparison with that of the Chl a aggregate. The Chl a/a′ aggregate colloids were formed in aqueous alcohols, and IR, resonance Raman, and small-angle X-ray scattering (SAXS) measurements were performed on lyophilized precipitates of the aggregate colloids. The analyses on the Chl a colloidal precipitate obtained from a 26/74 2-propanol/H 2 O solution demonstrated that the supramolecular structure was similar to those of the aggregates of hydrated Chl a. On the other hand, the vibrational spectra of the Chl a′ colloidal precipitate formed in 40/60 MeOH/H 2 O were quite similar to those of anhydrous Chl a aggregates. A trace hydroxyl stretching IR absorption (at around 3350 cm -1 ) of the Chl a′ aggregate precipitate was as small as that which the anhydrous Chl a aggregates could show. A SAXS profile of the Chl a′ colloidal precipitate demonstrated a lamellar structure with a 52-Å bilayer spacing, 8 Å greater than that of the Chl a aggregate formed in the 2-propanol/water. It was supposed that the Chl a′ aggregate was essentially anhydrous even in aqueous alcohols, while the Chl a aggregate was hydrated or swollen. A possible model of the supramolecular structure of the Chl a′ aggregate precipitate is presented. It is concluded that the difference between the supramolecular structures of the Chl a and a′ aggregates does not depend simply on the steric hindrance between the bulky substituents at the C13 2 and C17 positions but also on the possibility to form rigid intermolecular hydrogen-bonding networks. This is the origin of the diastereoselective separation of Chl a and a′, and it is also closely correlated to a structural degradation of the Chl a′ aggregate that occurs during the preparation of the colloidal precipitate.

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KeywordsChlorophyll a, chlorophyll a', fast atom chromatography bombardment mass spectrometry, high-performance liquidWe have been utilizing silica normal-phase HPLC for the preparation and analysis of chlorophylls (Chls)1, and have detected two molecules of Chl a' (C132 epimer of Chl a) in close association with P700, the primary donor of photosystem (PS) I of higher plants and cyanobacteria.2-4 However, this claim has occasionally met serious skepticism based on previous warnings against the use of siliceous adsorbents for the chromatography of chlorophylls.5,6The questions raised so far concern the identity of each peak, denaturation and recovery in the column, and the integrity of the pigments during extraction.Though we have repeatedly addressed these questions"2, we now feel that it is appropriate to assure the assignments and to compare the performance of our normal-phase HPLC with that of reversed-phase HPLC, which is generally considered to be sufficiently inert with respect to the elution behaviors of chlorophyllous pigments. Experimental InstrumentationFour HPLC systemsl''-9, of which the operational conditions are summarized in Table 1, were employed in the present work.1H-NMR spectra were recorded on a 270 MHz NMR spectrometer (JEOL GSX-270) for acetone-d6 solutions with tetramethylsilane as an internal standard. The pulse angle was 45° (4.3 µs), the acquisition time 5.5 s, and 64 transients were accumulated. The measurements were carried out at 25° C. Resonance peaks were assigned by comparisons with those reported by Hynninen and Lotjonen.10 Positive-ion FAB-MS measurements were performed with a JEOL JMS-700 MStation. Pigments (25 nmol) were dissolved in chloroform (10 µl), and 1 sl of the solution was submitted to analysis using 3-nitrobenzyl alcohol as the matrix.ll-13 Ions were produced by

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We conducted a meta-analysis of (bacterio)chlorophyll [(B)Chl] molecules in photosynthetic pigment-protein complexes from the viewpoint of coordination chemistry. We surveyed the ligand species and site in the axial coordination of 146 Chl and 21 BChl molecules in 42 reported crystal structures of 12-type proteins. The imidazolyl moiety of histidine (His) is the most abundant ligand, and the second is water, a much weaker ligand. We focused on the positions, the circumstances, and the macrocycle sides for the coordination of the 31 hydrated (B)Chl molecules found in these proteins. A ligand water molecule of a hydrated (B)Chl is not necessarily hydrogen-bonded to the surrounding protein residues. A hydrated (B)Chl seems to occupy the redundant space where more strongly coupled His-Chl complexes cannot be formed. It is noted that 28 of 31 hydrated (B)Chl molecules (90) were coordinated from the α-side of the (bacterio)chlorin macrocycle, the opposite side from which the C 17-propionic ester protrudes. Among them, all five hydrated Chl molecules at the edges of the proteins were coordinated from the α-side, suggesting that (B)Chl molecules prefer this side for the coordination bondings to the β-side. The analysis also revealed that each (B)Chl binding site was composed of both the protein residues and the neighboring pigment molecules contributing roughly equally. It can be safely said that the cofactor pigments aggregated even in the proteins. Penta-coordination is advantageous to flexible adjustment of intermolecular orientations of (B)Chl molecules in the aggregates.