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

Primary and Higher Order Structure of the Reaction Center from the Purple Phototrophic Bacterium Blastochloris viridis: A Test for Native Mass Spectrometry

The reaction center (RC) from the phototrophic bacterium Blastochloris viridis was the first integral membrane protein complex to have its structure determined by X-ray crystallography and has been studied extensively since then. It is composed of four protein subunits, H, M, L, and C, as well as cofactors, including bacteriopheophytin (BPh), bacteriochlorophyll (BCh), menaquinone, ubiquinone, heme, carotenoid, and Fe. In this study, we utilized mass spectrometry-based proteomics to study this protein complex via bottom-up sequencing, intact protein mass analysis, and native MS ligand-binding analysis. Its primary structure shows a series of mutations, including an unusual alteration and extension on the C-terminus of the M-subunit. In terms of quaternary structure, proteins such as this containing many cofactors serve to test the ability to introduce native-state protein assemblies into the gas phase because the cofactors will not be retained if the quaternary structure is seriously perturbed. Furthermore, this specific RC, under native MS, exhibits a strong ability not only to bind the special pair but also to preserve the two peripheral BCh’s

Structural Analysis of the Homodimeric Reaction Center Complex from the Photosynthetic Green Sulfur Bacterium <i>Chlorobaculum tepidum</i>

The reaction center (RC) complex of the green sulfur bacterium <i>Chlorobaculum tepidum</i> is composed of the Fenna–Matthews–Olson antenna protein (FMO) and the reaction center core (RCC) complex. The RCC complex has four subunits: PscA, PscB, PscC, and PscD. We studied the FMO/RCC complex by chemically cross-linking the purified sample followed by biochemical and spectroscopic analysis. Blue-native gels showed that there were two types of FMO/RCC complexes, which are consistent with complexes with one copy of FMO per RCC and two copies of FMO per RCC. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the samples after cross-linking showed that all five subunits of the RC can be linked by three different cross-linkers: bissulfosuccinimidyl suberate, disuccinimidyl suberate, and 3,3-dithiobis-sulfosuccinimidyl propionate. The interaction sites of the cross-linked complex were also studied using liquid chromatography coupled to tandem mass spectrometry. The results indicated that FMO, PscB, PscD, and part of PscA are exposed on the cytoplasmic side of the membrane. PscD helps stabilize FMO to the reaction center and may facilitate transfer of the electron from the RC to ferredoxin. The soluble domain of the heme-containing cytochrome subunit PscC and part of the core subunit PscA are located on the periplasmic side of the membrane. There is a close relationship between the periplasmic portions of PscA and PscC, which is needed for the efficient transfer of the electron between PscC and P840

Excitonic Energy Landscape of the Y16F Mutant of the <i>Chlorobium tepidum</i> Fenna–Matthews–Olson (FMO) Complex: High Resolution Spectroscopic and Modeling Studies

We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a non-Markovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna–Matthews–Olson (FMO) trimer from the green sulfur bacterium <i>Chlorobium tepidum</i>. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub₈₂₁ and Sub₈₁₇) provide new insight into extensive changes induced by the single-point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher exciton state(s) in the Y16F mutant depends on frequency and occurs on a picosecond time scale

The Fate of the Triplet Excitations in the Fenna–Matthews–Olson Complex

The fate of triplet excited states in the Fenna–Matthew–Olson (FMO) pigment–protein complex is studied by means of time-resolved nanosecond spectroscopy and exciton model simulations. Experiments reveal microsecond triplet excited-state energy transfer between the bacteriochlorophyll (BChl) pigments, but show no evidence of triplet energy transfer to molecular oxygen, which is known to produce highly reactive singlet oxygen and is the leading cause of photo damage in photosynthetic proteins. The FMO complex is exceptionally photo stable despite the fact it contains no carotenoids, which could effectively quench triplet excited states of (bacterio)­chlorophylls and are usually found within pigment–protein complexes. It is inferred that the triplet excitation is transferred to the lowest energy pigment, BChl 3, within the FMO complex, whose triplet state energy is shifted by pigment–protein interactions below that of the singlet oxygen excitation. Thus, the energy transfer to molecular oxygen is blocked and the FMO does not need carotenoids for photo protection

Modeling of Various Optical Spectra in the Presence of Slow Excitation Energy Transfer in Dimers and Trimers with Weak Interpigment Coupling: FMO as an Example

We present an improved simulation methodology to describe nonphotochemical hole-burned (NPHB) spectra. The model, which includes both frequency-dependent excitation energy transfer (EET) rate distributions and burning following EET, provides reasonable fits of various optical spectra including resonant and nonresonant holes in the case of FMO complex. A qualitative description of the NPHB process in light of a very complex protein energy landscape is briefly discussed. As an example, we show that both resonant and nonresonant HB spectra obtained for the 825 nm band of the trimeric FMO of <i>C. tepidum</i> are consistent with the presence of a relatively slow EET between the lowest energy states of the monomers of the trimer (mostly localized on BChl <i>a</i> 3), with a weak (∼1 cm^–1) coupling between these states revealed via calculated emission spectra. We argue that the nature of the so-called 825 nm absorption band of the FMO trimer, contrary to the presently accepted consensus, cannot be explained by a single transition

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

Structural Analysis of Diheme Cytochrome <i>c</i> by Hydrogen–Deuterium Exchange Mass Spectrometry and Homology Modeling

A lack of X-ray or nuclear magnetic resonance structures of proteins inhibits their further study and characterization, motivating the development of new ways of analyzing structural information without crystal structures. The combination of hydrogen–deuterium exchange mass spectrometry (HDX-MS) data in conjunction with homology modeling can provide improved structure and mechanistic predictions. Here a unique diheme cytochrome <i>c</i> (DHCC) protein from <i>Heliobacterium modesticaldum</i> is studied with both HDX and homology modeling to bring some definition of the structure of the protein and its role. Specifically, HDX data were used to guide the homology modeling to yield a more functionally relevant structural model of DHCC

Origin of the S* Excited State Feature of Carotenoids in Light-Harvesting Complex 1 from Purple Photosynthetic Bacteria

This spectroscopic study investigates the origin of the transient feature of the S* excited state of carotenoids bound in LH1 complexes from purple bacteria. The studies were performed on two RC-LH1 complexes from <i>Rba. sphaeroides</i> strains that bound carotenoids with different carbon–carbon double bond conjugation <i>N</i>, neurosporene (<i>N</i> = 9) and spirilloxanthin (<i>N</i> = 13). The S* transient spectral feature, originally associated with an elusive and optically silent excited state of spirilloxanthin in the LH1 complex, may be successfully explained and mimicked without involving any unknown electronic state. The spectral and temporal characteristics of the S* feature suggest that it is associated with triplet–triplet annihilation of carotenoid triplets formed after direct excitation of the molecule via a singlet fission mechanism. Depending on pigment homogeneity and carotenoid assembly in the LH1 complex, the spectro-temporal component associated with triplet–triplet annihilation may simply resolve a pure T-S spectrum of a carotenoid. In some cases (like spirilloxanthin), the T-S feature will also be accompanied by a carotenoid Stark spectrum and/or residual transient absorption of minor carotenoid species bound into LH1 antenna complex