Methionine oxidation and its effect on the stability of a reconstituted subunit of the light‐harvesting complex from Rhodospirillum rubrum

Abstract

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...

Gene sequencing and characterization of the light-harvesting complex 2 from thermophilic purple sulfur bacterium Thermochromatium tepidum

“…Part of the a1-polypeptide appeared to be oxidized, probably at the Met residue in the N-terminal domain as this is the sole Met in the a1-polypeptide. The Met-oxidation has also been reported on LH1 a-polypeptides of purple photosynthetic bacteria (Wang et al 2001a). Based on the TOF/MS result, methylation seems to have occurred on LH2 b1-and b2-polypeptides, but not on b3-polypeptide.…”

“…Separation of the polypeptides in LH2 was performed using a similar method described elsewhere (Wang et al 2001a). The purified LH2 complexes were treated with benzene and methanol to remove carotenoids and BChl a.…”

N‐terminal methylation of the core light‐harvesting complex in purple photosynthetic bacteria

“…rubrum and a carotenoid‐less mutant Rs. rubrum G9 essentially followed the method B of Picorel et al [7], and some details were given elsewhere [8]. LH1 complex from Rb.…”

“…The LH1–RC fraction was then eluted by a linear gradient of NaCl from 100 to 200 mM. The β‐polypeptides from LH1 and LH1–RC complexes were obtained using a reverse‐phase high performance liquid chromatography (HPLC) column with a mixed organic solvent system as described previously [8]. Edman degradation analyses for determination of the N‐terminal amino acids of the β‐polypeptides from Tch.…”

Generating oxidation-resistant variants of Bacillus kaustophilus leucine aminopeptidase by substitution of the critical methionine residues with leucine

“…Methionine residues are remarkable for their high susceptibility to chemical oxidation with methionine sulfoxide as the general product ͑Vogt 1995͒. Methionine sulfoxidation has been reported to affect the function of a number of biologically active proteins ͑Stauffer and Etson 1969; Hayes et al 1998;Hatakeyama et al 2000;Wang et al 2001͒. Recently, a thermophilic Bacillus kaustophilus leucine aminopeptidase ͑bkLAP͒ gene encoding a protein of 497 amino acids with a calculated molecular mass of 53.7 kDa was cloned and expressed in Escherichia coli ͑Lin et al 2004͒.…”