An additional component in the purified core lightharvesting complex (LH1) from wild-type purple photosynthetic bacterium Rhodospirillum rubrum has been identified as an oxidized species of a-polypeptide by MALDI-TOF mass spectrometry. This component appears as a slightly earlier-eluting peak in the RP-HPLC chromatogram compared with the authentic a-polypeptide. The oxidation site has been determined to be the N-terminal methionine residue by high-resolution NMR spectroscopy, where the methionine is oxidized to methionine sulfoxide in a diastereoisomeric form. Interconversion between the oxidized and authentic a-polypeptides has been confirmed by selective oxidation and reduction. The oxidative modification of methionine is shown to have discernible effects on the ability to form B820 subunit with b-polypeptide and bacteriochlorophyll a, and on the stability of the reconstituted B820 subunit. Both the ability and the stability for the samples using the oxidized a-polypeptide are moderately reduced, indicating that the oxidation-induced conformational change in the N-terminal domain of a-polypeptide may affect the pigment-binding environment through a long-range interaction. The MALDI-TOF mass results also reveal that the N-terminus of a-polypeptide is formylated and no phosphorylation has occurred in this polypeptide.Keywords: antenna complex; membrane protein; methionine sulfoxide; self-assembly; TOF-MS.Light-harvesting (LH) complex serves as highly efficient molecular machinery for the collection and transfer of solar energy to photochemical reaction centers in photosynthetic organisms. In purple photosynthetic bacteria, the LH complexes are classified into either of two major types depending on their in vivo locations to the reaction center, namely, the core (LH1) and peripheral (LH2) complexes, both comprising two small polypeptides a and b (plus g in some species) along with bacteriochlorophyll (BChl) and carotenoid molecules [1]. In contrast to most wild-type species that have both LH1 and LH2, purple nonsulfur photosynthetic bacterium Rhodospirillum rubrum has only LH1 with a Q y absorbance at about 880 nm and a stoichiometry of 1 : 1 : 2 : 1 for the a and , b polypeptides, BChl a and spirilloxanthin molecules for the wild-type [2,3]. The LH1 complex from R. rubrum is one of the most commonly studied integral pigment-membrane protein complexes and possesses a unique property, i.e. the ability to form a structural subunit characterized by a Q y absorption band at 820 nm by dissociation of the LH1 complex with detergents [4]. In addition, both the subunit (referred to as B820) and LH1 complexes can be reversibly dissociated and reconstituted from the individual components [5]. Biochemical and spectroscopic properties of the subunit and LH1 complexes from various species have been extensively investigated by using a wide variety of modified and synthesized polypeptides and pigment molecules in order to correlate the functions with the structural features [6±17]. Despite an increase in our knowledge on the st...

High-resolution solution NMR spectra have been obtained for bacteriochlorophyll (BChl) a molecules in a biologically functional subunit of a bacterial core light-harvesting complex based on a modified reconstitution method. The reconstituted subunit of pigment-integral membrane polypeptides is stable and homogeneous at high concentrations at room temperature and exhibits a Q(y) absorption peak at 818 nm. (1)H and (13)C chemical shifts have been specifically assigned for BChl a using the fully and selectively (13)C-labeled pigments incorporated with natural abundance polypeptides in deuterated detergent solution. Remarkable signal broadening has been observed upon reconstitution, where the bacteriochlorin macrocycle is shown in a highly restricted molecular motion while the phytol side chain remains relatively mobile. Two sets of resonances are revealed for 3(2), 8(1), 10, 12(1), and 13(4) protons, and 8(2) methyl protons exhibit four resonances with large upfield complexation shifts. The result indicates a nonequivalent state for the two BChl a molecules in the subunit and can be best interpreted in terms of a parallel face-to-face configuration with partial overlap over the pyrrolic rings II, III, and V. In comparison with BChl a in acetone, 8(2), 13(2), and 13(4) protons are largely perturbed, and the propionic and phytol side chain may adopt a different conformation in the reconstituted subunit. The (13)C chemical shift of 3(1) carbonyl carbon shows a large change downfield, indicating strong hydrogen bonding for all the acetyl carbonyls. Carbonyl carbons at 13(1) give rise to two (13)C resonances with equal intensities, suggesting that the keto carbonyl in one BChl a molecule within a subunit forms a stronger hydrogen bond than that in another BChl a molecule.

The B820 subunit is an integral pigment-membrane protein complex and can be obtained by both dissociation of the core light-harvesting complex (LH1) in photosynthetic bacteria and reconstitution from its component parts in the presence of n-octyl -D-glucopyranoside (OG). Intrinsic size of the B820 subunit from Rhodospirillum rubrum LH1 complex was measured by small-angle neutron scattering in perdeuterated OG solution and evaluated by Guinier analysis. Both the B820 subunits prepared by dissociation of LH1 and reconstitution from apopolypeptides and pigments were shown to have a molecular weight of 11 400 ( 500 and radius of gyration of 11.0 ( 1.0 Å, corresponding to a heterodimer consisting of one pair of R -polypeptides and two bacteriochlorophyll a molecules. Molecular weights of micelles formed by OG alone in solutions were determined in a range from 30 000 to 50 000 over concentrations of 1-5% (w/v), and thus are much larger than that of the B820 subunit. Similar measurement on the pigment-depleted apopolypeptides revealed highly heterogeneous behavior in the OG solutions, indicating that aggregates with various sizes were formed. The result provides evidence that bacteriochlorophyll a molecules play a crucial role in stabilizing and maintaining the B820 subunits in the dimeric state in solution. Further measurements on individual R-and -polypeptides exhibited a marked difference in aggregation property between the two polypeptides. The R-polypeptides appear to be uniformly dissolved in OG solution in a monomeric form, whereas the -polypeptides favor a self-associated form and tend to form large aggregates even in the presence of detergent. The difference in aggregation tendency was discussed in relation to the different behavior between R-and -polypeptides in reconstitution with bacteriochlorophyll a molecules.Despite intense interest and continuing investigation, controversy over the nature of an intermediate form of bacterial core light-harvesting complexes (LH1 1 ) has not been settled. The LH1 complex, serving as highly efficient molecular machinery for collection and transfer of solar energy to photochemical reaction centers in photosynthetic bacteria, is a large multimer of a minimal unit composed of two small integral membrane polypeptides (R and , ca. 6 kDa) associated with two bacteriochlorophyll (BChl) and one carotenoid molecule. Upon addition of detergents, the LH1 multimer can be dissociated into a structural subunit characterized by a Q y absorption band at 820 nm (1). Both the subunit (referred to as B820) and LH1 complex can be reversibly dissociated and reconstituted from the their components (2). Early chromatographic studies by gel filtration estimated a range of molecular weight from 32 to 65 kDa for the B820 subunit, roughly corresponding to a tetramer composed of two minimal structural units (1, 3). In contrast, spectroscopic evidence suggested that the B820 is a heterodimer consisting of two interacting BChl a molecules linked to one R -polypeptide pair (4,5). Recently, ide...

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