Magnetic field dependence of photo-CIDNP MAS NMR on photosynthetic reaction centers of Rhodobacter sphaeroides WT

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is observed in frozen and quinone depleted photosynthetic reaction centers of the purple bacteria Rhodobacter sphaeroides wild type (WT) by (13)C solid-state NMR at three different magnetic fields. All light-induced signals appear to be emissive at all three fields. At 4.7 T (200 MHz proton frequency), the strongest enhancement of NMR signals is observed, which is more than 10 000 above the Boltzmann polarization. At higher fields, the enhancement factor decreases. At 17.6 T, the enhancement factor is about 60. The field dependence of the enhancement appears to be the same for all nuclei. The observed field dependence is in line with simulations that assume two competing mechanisms of polarization transfer from electrons to nuclei, three-spin mixing (TSM) and differential decay (DD). These simulations indicate a ratio of the electron spin density on the special pair cofactors is 3:2 in favor of the L-BChl during the radical cation state. The good agreement of simulations with the experiments raises expectations that artificial solid reaction centers can be tuned to show photo-CIDNP in the near future.     

Electron spin density distribution in the special pair triplet of Rhodobacter sphaeroides R26 revealed by magnetic field dependence of the solid-state photo-CIDNP effect

Photo-CIDNP (photochemically induced dynamic nuclear polarization) can be observed in frozen and quinone-blocked photosynthetic reaction centers (RCs) as modification of magic-angle spinning (MAS) NMR signal intensity under illumination. Studying the carotenoidless mutant strain R26 of Rhodobacter sphaeroides, we demonstrate by experiment and theory that contributions to the nuclear spin polarization from the three-spin mixing and differential decay mechanism can be separated from polarization generated by the radical pair mechanism, which is partially maintained due to differential relaxation (DR) in the singlet and triplet branch. At a magnetic field of 1.4 T, the latter contribution leads to dramatic signal enhancement of about 80,000 and dominates over the two other mechanisms. The DR mechanism encodes information on the spin density distribution in the donor triplet state. Relative peak intensities in the photo-CIDNP spectra provide a critical test for triplet spin densities computed for different model chemistries and conformations. The unpaired electrons are distributed almost evenly over the two moieties of the special pair of bacteriochlorophylls, with only slight excess in the L branch.     

Signals in solid-state photochemically induced dynamic nuclear polarization recover faster than signals obtained with the longitudinal relaxation time

During the photocycle of quinone-blocked photosynthetic reaction centers (RCs), photochemically induced dynamic nuclear polarization (photo-CIDNP) is produced by polarization transfer from the initially totally electron polarized electron pair and can be observed by 13C magic-angle spinning (MAS) NMR as a strong modification of signal intensities. The same processes creating net nuclear polarization open up light-dependent channels for polarization loss. This leads to coherent and incoherent enhanced signal recovery, in addition to the recovery due to light-independent longitudinal relaxation. Coherent mixing between electron and nuclear spin states due to pseudosecular hyperfine coupling within the radical pair state provides such a coherent loss channel for nuclear polarization. Another polarization transfer mechanism called differential relaxation, which is based on the long lifetime of the triplet state of the donor, provides an efficient incoherent relaxation path. In RCs of the purple bacterium Rhodobacter sphaeroides R26, the photochemical active channels allow for accelerated signal scanning by a factor of 5. Hence, photo-CIDNP MAS NMR provides the possibility to drive the NMR technique beyond the T1 limit.     

Photochemically induced dynamic nuclear polarization in photosystem I of plants observed by 13C magic-angle spinning NMR

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in photosystem I of spinach by (13)C magic angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a single Chl a molecule has been obtained which is assigned to the P2-cofactor of the primary electron donor P700. Since all light-induced (13)C NMR signals appear to be emissive, a predominance of the three-spin mixing mechanism over the differential decay mechanism is proposed. The origin of the strong contribution of the three-spin mixing mechanism and the differences with photosystem II are discussed.     

Orientation-resolving pulsed electron dipolar high-field EPR spectroscopy on disordered solids: I. Structure of spin-correlated radical pairs in bacterial photosynthetic reaction centers

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.     

Lipid binding to the carotenoid binding site in photosynthetic reaction centers

Lipid binding to the carotenoid binding site near the inactive bacteriochlorophyll monomer was probed in the reaction centers of carotenoid-less mutant, R-26 from Rhodobacter sphaeroides. Recently, a marked light-induced change of the local dielectric constant in the vicinity of the inactive bacteriochlorophyll monomer was reported in wild type that was attributed to structural changes that ultimately lengthened the lifetime of the charge-separated state by 3 orders of magnitude (Deshmukh, S. S.; Williams, J. C.; Allen, J. P.; Kalman, L. Biochemistry 2011, 50, 340). Here in the R-26 reaction centers, the combination of light-induced structural changes and lipid binding resulted in a 5 orders of magnitude increase in the lifetime of the charge-separated state involving the oxidized dimer and the reduced primary quinone in proteoliposomes. Only saturated phospholipids with fatty acid chains of 12 and 14 carbon atoms long were bound successfully at 8 °C by cooling the reaction center protein slowly from room temperature. In addition to reporting a dramatic increase of the lifetime of the charge-separated state at physiologically relevant temperatures, this study reveals a novel lipid binding site in photosynthetic reaction center. These results shed light on a new potential application of the reaction center in energy storage as a light-driven biocapacitor since the charges separated by ～30 ? in a low-dielectric medium can be prevented from recombination for hours.     

Stability of integral membrane proteins under high hydrostatic pressure: the LH2 and LH3 antenna pigment-protein complexes from photosynthetic bacteria

The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure, quite some of them remain apparently intact. The pigmented photosynthetic antenna complexes thus constitute a suitable model system for studying in detail the stability of integral membrane proteins.     

Analysis of the Electronic Structure of the Special Pair of a Bacterial Photosynthetic Reaction Center by 13C Photochemically Induced Dynamic Nuclear Polarization Magic‐Angle Spinning NMR Using a Double‐Quantum Axis

The origin of the functional symmetry break in bacterial photosynthesis challenges since several decades. Although structurally very similar, the two branches of cofactors in the reaction center (RC ) protein complex act very differently. Upon photochemical excitation, an electron is transported along one branch, while the other remains inactive. Photochemically induced dynamic nuclear polarization (photo‐CIDNP ) magic‐angle spinning (MAS ) ¹³C NMR revealed that the two bacteriochlorophyll cofactors forming the “Special Pair” donor dimer are already well distinguished in the electronic ground state. These previous studies are relying solely on ¹³C‐¹³C correlation experiments as radio‐frequency‐driven recoupling (RFDR ) and dipolar‐assisted rotational resonance (DARR ). Obviously, the chemical‐shift assignment is difficult in a dimer of tetrapyrrole macrocycles, having eight pyrrole rings of similar chemical shifts. To overcome this problem, an INADEQUATE type of experiment using a POST C7 symmetry‐based approach is applied to selectively isotope‐labeled bacterial RC of Rhodobacter (R.) sphaeroides wild type (WT ). We, therefore, were able to distinguish unresolved sites of the macromolecular dimer. The obtained chemical‐shift pattern is in‐line with a concentric assembly of negative charge within the common center of the Special Pair supermolecule in the electronic ground state.     

细菌光合反应中心突变体原初电子转移机理的理 论研究

利用量子化学DFT从头计算方法,计算经过突变的细菌光合反应中心HM202L原始电子给体和其他色素分子的电子结构,然后对其原初电子转移机理进行探讨。结果表明：1)超分子D-2A的HOMO主要是由定域在其组成单元BChl~L分子上的原子轨道组成,而它的LUMO主要是由定域在其组成单元MBPheo~M分子上的原子轨道组成。这表明它在基态的激发态时分别存在超分子内的电荷分离态[BChl~L^--MBPheo~M^+]和[BChl~L^+-MBPheo~M^-]。同时也说明了D-2A阳离子态的正电荷完全分布在组成单元细菌叶绿素分子BChl~L上,与实验事实相符。2)HM202L细菌光合反应中心原初电子转移反应存在由ABCha~L^h^*驱动的电子转移反应。     

Theoretical investigation of the electronic asymmetry of the special pair cation radical in the photosynthetic type-II reaction center

The electronic asymmetry of the special pair cation radical in the photosynthetic reaction center was studied, using density functional calculations with a polarizable continuum model and a point charge model as the protein environment. The calculated spin density distribution between the halves of the special pair from Rhodobacter sphaeroides agreed well with the experimental value due to the protein polarity effect. The differences in the specific orientations of the ester carbonyl groups of the phytyl groups, as well as the methyl ester groups, are one of the origins of the electronic asymmetry. The orientations of these groups are specific, as revealed by the 14 recent X-ray structures of a variety of type-II reaction centers, with a few exceptions. In addition to the spin density distributions of the special pair cation radical from other species, the structural features of the protein surrounding these groups of a variety of type-II reaction centers were investigated to elucidate the generality and mechanisms of the specific orientations of the groups by a structural alignment and a multiple sequence alignment. The determining factors of the electronic asymmetry among type-II reaction centers are discussed.     

Electron transfer kinetics in photosynthetic reaction centers embedded in polyvinyl alcohol films

The coupling between electron transfer and protein dynamics has been studied at room temperature in isolated reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in polyvinyl alcohol (PVA) films of different water/RC ratios. The kinetic analysis of charge recombination shows that dehydration of RC-containing PVA films causes reversible, inhomogeneous inhibition of electron transfer from the reduced primary quinone acceptor (Q(A)(-)) to the secondary quinone Q(B). A more extensive dehydration of solid PVA matrices accelerates electron transfer from Q(A)(-) to the primary photooxidized electron donor P(+). These effects indicate that incorporation of RCs into dehydrated PVA films hinders the conformational dynamics gating Q(A)(-) to Q(B) electron transfer at room temperature and slows down protein relaxation which stabilizes the primary charge-separated state P(+)Q(A)(-). A comparison with analogous effects observed in trehalose-coated RCs suggests that protein motions are less severely reduced in PVA films than in trehalose matrices at comparable water/RC ratios.     

Porous silicon/photosynthetic reaction center hybrid nanostructure

The purified photosynthetic reaction center protein (RC) from Rhodobacter sphaeroides R-26 purple bacteria was bound to porous silicon microcavities (PSiMc) either through silane-glutaraldehyde (GTA) chemistry or via a noncovalent peptide cross-linker. The characteristic resonance mode in the microcavity reflectivity spectrum red shifted by several nanometers upon RC binding, indicating the protein infiltration into the porous silicon (PSi) photonic structure. Flash photolysis experiments confirmed the photochemical activity of RC after its binding to the solid substrate. The kinetic components of the intraprotein charge recombination were considerably faster (τ(fast) = 14 (±9) ms, τ(slow) = 230 (±28) ms with the RC bound through the GTA cross-linker and only τ(fast) = 27 (±3) ms through peptide coating) than in solution (τ(fast) = 120 (±3) ms, τ(slow) = 1387 (±2) ms), indicating the effect of the PSi surface on the light-induced electron transfer in the protein. The PSi/RC complex was found to oxidize the externally added electron donor, mammalian cytochrome c, and the cytochrome oxidation was blocked by the competitive RC inhibitor, terbutryne. This fact indicates that the specific surface binding sites on the PSi-bound RC are still accessible to external cofactors and an electronic interaction with redox components in the aqueous environment is possible. This new type of biophotonic material is considered to be an excellent model for new generation applications at the interface of silicon-based electronics and biological redox systems designed by nature.     

Electrochemical reactions of redox cofactors in Rhodobacter sphaeroides reaction center proteins in lipid films.

Cyclic voltammetry of thin films made from the lipid dimyristoylphosphatidyl choline and reaction centers from the purple bacterium Rhodobacter sphaeroides on pyrolytic graphite electrodes in bromide-free pH 8 buffers at 4 degrees C revealed an oxidation peak at 0.98 V and a reduction peak at -0.17 V vs. NHE. No reverse CV peaks were found, suggesting chemical irreversibility. The reduction peak disappeared for reaction centers depleted of quinones, suggesting that the peak represents reduction of this cofactor. The oxidation peak showed a catalytic current increase in the presence of small amounts of ferrous cytochrome c, and decreased by 85% when illuminated by visible light, suggesting assignment to the primary donor (P) cofactor. While oxidized primary donor P(+) is destroyed upon electrochemical formation in the film, reaction of ferrous cyt c with P(+) suggests its persistence in the films on the microsecond time scale.     

ACOUSTIC DETECTION OF TRIPLET STATES FORMED BY RADICAL PAIR RECOMBINATION IN QUINONE-DEPLETED PHOTOSYNTHETIC REACTION CENTERS, BY MAGNETIC FIELD MODULATION (MAGNETOPHOTOACOUSTIC EFFECT)-A FEASIBILITY STUDY

Abstract: An acoustic method is outlined to detect triplet states formed by radical pair recombination in photosynthetic reaction centers. It is based on magnetic field effect on the probability of triplet state formation by recombination. Using a periodically modulated magnetic field in the presence of constant exciting light, a periodic modulation of the triplet state concentration is set in the sample, which is detected through the corresponding modulated heat emission, transduced to acoustic vibration of the gas phase around the sample. This effect is similar to the photoacoustic effect, except that here the light is not modulated. The feasibility of detecting such an effect was proven experimentally, by obtaining a signal from quinone-depleted reaction centers of Rhodobacter sphaeroides. The signal had twice the frequency of the magnetic field modulation; it was proportional to the light intensity and significantly stronger at the lower temperatures (in the investigated range 113–278 K). No signal was obtained from quinone-containing reaction centers, which do not produce triplets. A theoretical outline of the effect and the experimental set-up are described. The magnitude of the effect was calibrated against ordinary photoacoustic measurements, allowing numerical evaluation of certain parameters of the triplet state ( e.g. triplet energy or yield) with the aid of auxiliary information from the literature.     

Triplet State Energy Transfer Between the Primary Donor and the Carotenoid in Rhodobacter sphaeroides R-26.1 Reaction Centers Exchanged with Modified Bacteriochlorophyll Pigments and Reconstituted with Spheroidene

Abstract— The dynamics of triplet energy transfer between the primary donor and the carotenoid were measured on several photosynthetic bacterial reaction center preparations from Rhodobacter sphaeroides : (a) wild-type strain 2.4.1, (b) strain R-26.1, (c) strain R-26.1 exchanged with 13²-hy-droxy-[Zn]-bacteriochlorophyll at the accessory bacteriochlorophyll (BChl) sites and reconstituted with spheroidene and (d) strain R-26.1 exchanged with P-vinyl]-13²-hydroxy-bacteriochlorophyll at the accessory BChl sites and reconstituted with spheroidene. The rise and decay times of the primary donor and carotenoid triplet-triplet absorption signals were monitored in the visible wavelength region between 538 and 555 run as a function of temperature from 4 to 300 K. For the samples containing carotenoids, all of the decay times correspond well to the previously observed times for spheroidene (5 ± 2 us). The rise times of the carotenoid triplets were found in all cases to be biexponen-tial and comprised of a strongly temperature-dependent component and a temperature-independent component. From a comparison of the behavior of the carotenoid-con-taining samples with that from the reaction center of the carotenoidless mutant Rb. sphaeroides R-26.1, the temperature-independent component has been assigned to the buildup of the primary donor triplet state resulting from charge recombination in the reaction center. Arrhenius plots of the buildup of the carotenoid triplet states were used to determine the activation energies for triplet energy transfer from the primary donor to the carotenoid. A model for the process of triplet energy transfer that is consistent with the data suggests that the activation barrier is strongly dependent on the triplet state energy of the accessory BChl pigment, BChl_B.     

Structure of the charge separated state P865(+)Q(A)- in the photosynthetic reaction centers of Rhodobacter sphaeroides by quantum beat oscillations and high-field electron paramagnetic resonance: evidence for light-induced Q(A)- reorientation

The structure of the secondary radical pair, P865(+)Q(A)-, in fully deuterated and Zn-substituted reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides R-26 has been determined by high-time resolution and high-field electron paramagnetic resonance (EPR). A computer analysis of quantum beat oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the orientation of the various magnetic tensors of P865(+)Q(A)- with respect to a magnetic reference frame. The orientation of the g-tensor of P865(+) in an external reference system is adapted from a single-crystal W-band (95 GHz) EPR study [Klette, R.; T?rring, J. T.; Plato, M.; M?bius, K.; B?nigk, B.; Lubitz, W. J. Phys. Chem. 1993, 97, 2015-2020]. Thus, we obtain the three-dimensional structure of the charge separated state P865(+)Q(A)- on a nanosecond time scale after light-induced charge separation. Comparison with crystallographic data reveals that the position of the quinone is essentially the same as that in the X-ray structure. However, the head group of Q(A)- has undergone a 60 degrees rotation in the ring plane relative to its orientation in the crystal structure. Analysis suggests that the two different QA conformations are functionally relevant states which control the electron-transfer kinetics from Q(A)- to the secondary quinone acceptor QB. It appears that the rate-limiting step of this reaction is a reorientation of Q(A)- in its binding pocket upon light-induced reduction. The new kinetic model accounts for striking observations by Kleinfeld et al. who reported that electron transfer from Q(A)- to QB proceeds in RCs cooled to cryogenic temperature under illumination but does not proceed in RCs cooled in the dark [Kleinfeld, D.; Okamura, M. Y.; Feher, G. Biochemistry 1984, 23, 5780-5786].     

Light-generated paramagnetic intermediates in BLUF domains

Blue-light sensitive photoreceptory BLUF domains are flavoproteins, which regulate various, mostly stress-related processes in bacteria and eukaryotes. The photoreactivity of the flavin adenine dinucleotide (FAD) cofactor in three BLUF domains from Rhodobacter sphaeroides, Synechocystis sp. PCC 6803 and Escherichia coli have been studied at low temperature using time-resolved electron paramagnetic resonance. Photoinduced flavin triplet states and radical-pair species have been detected on a microsecond time scale. Differences in the electronic structures of the FAD cofactors as reflected by altered zero-field splitting parameters of the triplet states could be correlated with changes in the amino-acid composition of the various BLUF domains' cofactor binding pockets. For the generation of the light-induced, spin-correlated radical-pair species in the BLUF domain from Synechocystis sp. PCC 6803, a tyrosine residue near the flavin's isoalloxazine moiety plays a critical role.     

Attenuated total reflection Fourier transform infrared spectroscopy of redox transitions in photosynthetic reaction centers: comparison of perfusion- and light-induced difference spectra

Chemically induced Fourier transform infrared difference spectra associated with redox transitions of several primary electron donors and acceptors in photosynthetic reaction centers (RCs) have been compared with the light-induced FTIR difference spectra involving the same cofactors. The RCs are deposited on an attenuated total reflection (ATR) prism and form a film that is enclosed in a flow cell. Redox transitions in the film of RCs can be repetitively induced either by perfusion of buffers poised at different redox potentials or by illumination. The perfusion-induced ATR-FTIR difference spectra for the oxidation of the primary electron donor P in the RCs of the purple bacteria Rb. sphaeroides and Rp. viridis and P700 in the photosystem 1 of Synechocystis 6803, as well as the Q(A)/Q(A) transition of the quinone acceptor (Q(A)) in Rb. sphaeroides RCs are reported for the first time. They are compared with the light-induced ATR-FTIR difference spectra P+Q(A)/PQ(A) for the RCs of Rb. sphaeroides and P700+/P700 for photosystem 1. It is shown that the perfusion-induced and light-induced ATR-FTIR difference spectra recorded on the same RC film display identical signal to noise ratios when they are measured under comparable conditions. The ATR-FTIR difference spectra are very similar to the equivalent FTIR difference spectra previously recorded upon photochemical or electrochemical excitation of these RCs in the more conventional transmission mode. The ATR-FTIR technique requires a smaller amount of sample compared with transmission FTIR and allows precise control of the aqueous environment of the RC films.     

Stabilization effect of single-walled carbon nanotubes on the functioning of photosynthetic reaction centers

The interaction between single-walled carbon nanotubes and photosynthetic reaction centers purified from purple bacterium Rhodobacter sphaeroides R-26 has been investigated. Atomic force microscopy studies provide evidence that reaction center protein can be attached effectively to the nanotubes. The typical diameter of the nanotube is 1-4 nm and 15 +/- 2 nm without and with the reaction centers, respectively. Light-induced absorption change measurements indicate the stabilization of the P+(Q(A)Q(B))- charge pair, which is formed after single saturating light excitation after the attachment to nanotubes. The separation of light-induced charges is followed by slow reorganization of the protein structure. The stabilization effect of light-initiated charges by the carbon nanotubes opens a possible direction of several applications, the most promising being in energy conversion and storage devices.     

Triplet Energy Transfer between the Primary Donor and Carotenoids in Rhodobacter sphaeroides R-26.1 Reaction Centers Incorporated with Spheroidene Analogs Having Different Extents of π-Electron Conjugation

Abstract— Three carotenoids, spheroidene, 3,4-dihydrospheroidene and 3,4,5,6-tetrahydrospheroidene, having 8, 9 and 10 conjugated carbon-carbon double bonds, respectively, were incorporated into Rhodobacter (Rb.) sphaeroides R-26.1 reaction centers. The extents of binding were found to be 95±5% for spheroidene, 65±5% for 3,4-dihydrospheroidene and 60±10% for 3,4,5,6-tetrahydrospheroidene. The dynamics of the triplet states of the primary donor and carotenoid were measured at room temperature by flash absorption spectroscopy. The carotenoid, spheroidene, was observed to quench the primary donor triplet state. The triplet state of spheroidene that was formed subsequently decayed to the ground state with a lifetime of 7.0±0.5 μs. The primary donor triplet lifetime in the Rb. sphaeroides R-26.1 reaction centers lacking carotenoids was 60±5 μs. Quenching of the primary donor triplet state by the carotenoid was not observed in the Rb. sphaeroides R-26.1 reaction centers containing 3,4-dihydrospheroidene nor in the R-26.1 reaction centers containing 3,4,5,6-tetrahydrospheroidene. Triplet-state electron paramagnetic resonance was also carried out on the samples. The experiments revealed carotenoid triple-state signals in the Rb. sphaeroides R-26.1 reaction centers incorporated with spheroidene, indicating that the primary donor triplet is quenched by the carotenoid. No carotenoid signals were observed from Rb. sphaeroides R-26.1 reaction centers incorporating 3,4-dihydrospheroidene nor in reaction centers incorporating 3,4,5,6-tetrahydrospheroidene. Circular dichroism, steady-state absorbance band shifts accompanying the primary photochemistry in the reaction center and singlet energy transfer from the carotenoid to the primary donor confirm that the carotenoids are bound in the reaction centers and interacting with the primary donor. These studies provide a systematic approach to exploring the effects of carotenoid structure and excited state energy on triplet transfer between the primary donor and carotenoids in reaction centers from photosynthetic bacteria.