A functional proteomic analysis of the intracytoplasmic membrane (ICM) development process was performed in Rhodobacter sphaeroides during adaptation from high-intensity illumination to indirect diffuse light. This initiated an accelerated synthesis of the peripheral light-harvesting 2 (LH2) complex relative to that of LH1-reaction center (RC) core particles. After 11 days, ICM vesicles (chromatophores) and membrane invagination sites were isolated by rate-zone sedimentation and subjected to clear native gel electrophoresis. Proteomic analysis of gel bands containing the RC-LH1 and -LH2 complexes from digitonin-solubilized chromatophores revealed high levels of comigrating electron transfer enzymes, transport proteins, and membrane assembly factors relative to their equivalent gel bands from cells undergoing adaptation to direct low-level illumination. The GroEL chaperonin accounted for >65% of the spectral counts in the RC-LH1 band from membrane invagination sites, which together with the appearance of a universal stress protein suggested that the viability of these cells was challenged by light limitation. Functional aspects of the photosynthetic unit assembly process were monitored by near-IR fast repetition rate analysis of variable fluorescence arising from LH-bacteriochlorophyll a components. The quantum yield of the primary charge separation during the early stages of adaptation showed a gradual increase (variable/maximal fluorescence = 0.78-0.83 between 0 and 4 h), while the initial value of ∼70 for the functional absorption cross section (σ) gradually increased to 130 over 4 days. These dramatic σ increases showed a direct relation to gradual slowing of the RC electron transport turnover rate (τQA) from ∼1.6 to 6.4 ms and an ∼3-fold slowing of the rate of reoxidation of the ubiquinone pool. These slowed rates are not due to changes in UQ pool size, suggesting that the relation between increasing σ and τQA reflects the imposition of constraints upon free diffusion of ubiquinone redox species between the RC and cytochrome bc 1 complex as the membrane bilayer becomes densely packed with LH2 rings.
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