Millisecond delayed light as a probe of membrane energization and redox poise in intact cells of photosynthetic bacteria.

Millisecond delayed light (DL) of bacteriochlorophylls was measured following rectangular light excitation of variable duration from intact purple photosynthetic bacteria with different electron donor configurations: the cytochrome-less mutant cycA of Rhodobacter sphaeroides (single turnover), wild-type Rubrivivax gelatinosus and Blastochloris viridis (multiple turnovers with bound tetraheme cytochrome), and the pufC mutant (loosely bound alternative donors). All strains exhibited similar dominant DL decay components (1-10 ms) but with markedly different amplitudes. In cycA, DL decayed much faster than the P+QA- → PQA charge-pair recombination (≈ 60 ms) and the saturation of its amplitude was delayed relative to prompt fluorescence (PF) by more than an order of magnitude relative to the rise time. Millisecond DL in cycA resulted from direct deactivation-type radiative recombination (via nuclear tunneling through P*) of a small subpopulation (α ≈ 2 × 10-6) of P+QA- states, as determined from the DL-to-PF intensity ratio. Strains capable of multiple turnovers exhibited ∼100-fold enhanced DL (α ≈ 10-4 in Rvx. gelatinosus), attributed to redox equilibrium between P/P+ and cytochrome c2+/c3+ that continuously repopulated P+QA-. This enhancement was modulated by proton-motive force (pmf), as demonstrated by sensitivity to protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Electron donor ferrocene quenched DL in all strains, consistent with perturbation of the redox equilibrium. Millisecond DL as an optical reporter of redox poise and membrane energization in intact photosynthetic bacteria offers valuable contribution to understanding energy dissipation pathways in bacterial photosynthesis and energization state of photosynthetic membranes.