Intensity Dependence of the Excited State Lifetimes and Triplet Conversion Yield in the Fenna–Matthews–Olson Antenna Protein

The Fenna–Matthews–Olson (FMO) protein is a soluble light-harvesting, bacteriochlorophyll <i>a</i> (BChl <i>a</i>) containing antenna complex found in green sulfur bacteria. We have measured time-resolved fluorescence and transient absorption at variable laser intensities at 298 and 77 K using FMO protein from Chlorobaculum tepidum prepared in both oxidizing and reducing environments. Fitting of the spectroscopic data shows that high laser intensities (i.e., above 10¹³ photons × cm^–2 delivered per laser pulse) distort the intrinsic decay processes in this complex. At high laser intensities, both oxidized and reduced FMO samples behave similarly, exhibiting high levels of singlet–singlet annihilation. At lower laser intensities, the reduced protein mainly displays a singlet excited state lifetime of 2 ns, although upon oxidation, a 60 ps lifetime dominates. We also demonstrate that the apparent quantum yield of singlet–triplet intersystem crossing in the reduced FMO complex is ∼11% in the most favorable low laser intensities, with this yield decreasing and the probability of singlet–singlet annihilation yield increasing as laser intensity increases. After correcting for stimulated emission effects in the experiments, the actual maximum triplet yield is calculated to be ∼27%. Experiments at 77 K demonstrate that BChl <i>a</i> triplet states in FMO are localized on pigments no. 4 or 3, the lowest energy pigments in the complex. This study allows for a discussion of how BChl triplets form and evolve on the picosecond-to-nanosecond time scale, as well as whether triplet conversion is a physiologically relevant process

Bacteriochlorophyll f: properties of chlorosomes containing the “forbidden chlorophyll”

The chlorosomes of green sulfur bacteria (GSB) are mainly assembled from one of three types of bacteriochlorophylls (BChls), BChls c, d, and e. By analogy to the relationship between BChl c and BChl d (20-desmethyl-BChl c), a fourth type of BChl, BChl f (20-desmethyl-BChl e), should exist but has not yet been observed in nature. The bchU gene (bacteriochlorophyllide C-20 methyltransferase) of the brown-colored green sulfur bacterium Chlorobaculum limnaeum was inactivated by conjugative transfer from Eshcerichia coli and homologous recombination of a suicide plasmid carrying a portion of the bchU. The resulting bchU mutant was greenish brown in color and synthesized BChl fF. The chlorosomes of the bchU mutant had similar size and polypeptide composition as those of the wild type (WT), but the Qy absorption band of the BChl f aggregates was blue-shifted 16 nm (705 nm vs. 721 nm for the WT). Fluorescence spectroscopy showed that energy transfer to the baseplate was much less efficient in chlorosomes containing BChl f than in WT chlorosomes containing BChl e. When cells were grown at high irradiance with tungsten or fluorescent light, the WT and bchU mutant had identical growth rates. However, the WT grew about 40% faster than the bchU mutant at low irradiance (10 μmol photons m−2 s-1). Less efficient energy transfer from BChl f aggregates to BChl a in the baseplate, the much slower growth of the strain producing BChl f relative to the WT, and competition from other phototrophs, may explain why BChl f is not observed naturally

Dynamics of Energy and Electron Transfer in the FMO-Reaction Center Core Complex from the Phototrophic Green Sulfur Bacterium <i>Chlorobaculum tepidum</i>

The reaction center core (RCC) complex and the RCC with associated Fenna–Matthews–Olson protein (FMO-RCC) complex from the green sulfur bacterium <i>Chlorobaculum tepidum</i> were studied comparatively by steady-state and time-resolved fluorescence (TRF) and femtosecond time-resolved transient absorption (TA) spectroscopies. The energy transfer efficiency from the FMO to the RCC complex was calculated to be ∼40% based on the steady-state fluorescence. TRF showed that most of the FMO complexes (66%), regardless of the fact that they were physically attached to the RCC, were not able to transfer excitation energy to the reaction center. The TA spectra of the RCC complex showed a 30–38 ps lifetime component regardless of the excitation wavelengths, which is attributed to charge separation. Excitonic equilibration was shown in TA spectra of the RCC complex when excited into the BChl <i>a</i> Q_<i>x</i> band at 590 nm and the Chl <i>a</i> Q_<i>y</i> band at 670 nm, while excitation at 840 nm directly populated the low-energy excited state and equilibration within the excitonic BChl <i>a</i> manifold was not observed. The TA spectra for the FMO-RCC complex excited into the BChl <i>a</i> Q_<i>x</i> band could be interpreted by a combination of the excited FMO protein and RCC complex. The FMO-RCC complex showed an additional fast kinetic component compared with the FMO protein and the RCC complex, which may be due to FMO-to-RCC energy transfer