Sphaeroides R26 Research Articles

Carotenoids Do Not Protect Bacteriochlorophylls in Isolated Light-Harvesting LH2 Complexes of Photosynthetic Bacteria from Destructive Interactions with Singlet Oxygen.

The effect of singlet oxygen on light-harvesting (LH) complexes has been studied for a number of sulfur (S+) and nonsulfur (S−) photosynthetic bacteria. The visible/near-IR absorption spectra of the standard LH2 complexes (B800-850) of Allochromatium (Alc.) vinosum (S+), Rhodobacter (Rba.) sphaeroides (S−), Rhodoblastus (Rbl.) acidophilus (S−), and Rhodopseudomonas (Rps.) palustris (S−), two types LH2/LH3 (B800-850 and B800-830) of Thiorhodospira (T.) sibirica (S+), and an unusual LH2 complex (B800-827) of Marichromatium (Mch.) purpuratum (S+) or the LH1 complex from Rhodospirillum (Rsp.) rubrum (S−) were measured in aqueous buffer suspensions in the presence of singlet oxygen generated by the illumination of the dye Rose Bengal (RB). The content of carotenoids in the samples was determined using HPLC analysis. The LH2 complex of Alc. vinosum and T. sibirica with a reduced content of carotenoids was obtained from cells grown in the presence of diphenylamine (DPA), and LH complexes were obtained from the carotenoidless mutant of Rba. sphaeroides R26.1 and Rps. rubrum G9. We found that LH2 complexes containing a complete set of carotenoids were quite resistant to the destructive action of singlet oxygen in the case of Rba. sphaeroides and Mch. purpuratum. Complexes of other bacteria were much less stable, which can be judged by a strong irreversible decrease in the bacteriochlorophyll (BChl) absorption bands (at 850 or 830 nm, respectively) for sulfur bacteria and absorption bands (at 850 and 800 nm) for nonsulfur bacteria. Simultaneously, we observe the appearance of the oxidized product 3-acetyl-chlorophyll (AcChl) absorbing near 700 nm. Moreover, a decrease in the amount of carotenoids enhanced the spectral stability to the action of singlet oxygen of the LH2 and LH3 complexes from sulfur bacteria and kept it at the same level as in the control samples for carotenoidless mutants of nonsulfur bacteria. These results are discussed in terms of the current hypothesis on the protective functions of carotenoids in bacterial photosynthesis. We suggest that the ability of carotenoids to quench singlet oxygen (well-established in vitro) is not well realized in photosynthetic bacteria. We compared the oxidation of BChl850 in LH2 complexes of sulfur bacteria under the action of singlet oxygen (in the presence of 50 μM RB) or blue light absorbed by carotenoids. These processes are very similar: {[BChl + (RB or carotenoid) + light] + O2} → AcChl. We speculate that carotenoids are capable of generating singlet oxygen when illuminated. The mechanism of this process is not yet clear.

Effect of chemical substitution on the surface charge of the photosynthetic Reaction Center from Rhodobacter sphaeroides: an in-silico investigation.

The Reaction Centers (RCs) proteins are membrane proteins representing the key component so flight energy transduction in photosynthetic organisms. Upon photon absorption, these photoenzymes produce a long lasting intra protein hole electron couples whose charges are separated by 3 nanometers. The dipoles formed within the RCs can be effectively employed as transducing cores of several biological-organic hybrid devices whose design can accomplish photocurrents generation or act as phototransistor. To widen the application of the RCs to as many substrate as possible one valuable strategy is the bioconjugation of the protein with specific molecules ad-hoc selected to improve enzymatic performance and/or integration in proper scaffolding. In the present manuscript, we investigate the changes of the isoelectric point of the RC from the carotenoidless strain of the photosynthetic bacterium Rhodobacter sphaeroides R26 by inducing “in silico” mutations to predict on the role of the aminoacids involved in the bioconjugation.

15N–1H Transfer of Light-Induced Nuclear Hyperpolarization in Frozen Photosynthetic Reaction Centers

Recently, we have demonstrated the possibility to transfer the light-induced hyperpolarization occurring on select 13C nuclei of photosynthetic cofactors due to the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect into the proton pool via inverse 13C–1H cross-polarization Bielytskyi et al. (J Magn Reson 293: 82–91, 2018). Such approach allowed us to observe the selective response from the protons belonging to photochemically active cofactors in their native protein environment. In the present study, we demonstrate that also 15N nuclei can be used as a source of polarization in a similar type of experiments. We present the 2D photo-CIDNP 15N–1H heteronuclear correlation (HETCOR) experiment acquired on uniformly 15N-labeled quinone-blocked reaction center (RC) from Rhodobacter sphaeroides R26. Obtained 1H chemical shifts match with previously observed ones on selectively 13C-labeled RC from Rhodobacter sphaeroides WT. We expect that using 15N as a source of polarization in potential heteronuclear spin-torch experiments could be advantageous for systems where the procedure for selective 13C labeling is not yet established.

Unveiling the compositional variety of cardiolipins in Rhodobacter sphaeroides by liquid chromatography with electrospray ionization and multistage collision-induced dissociation mass spectrometry.

Cardiolipins (CL) contained in the lipid extracts of the photosynthetic bacterium Rhodobacter sphaeroides (strain R26) were systematically characterized by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry, performed in single (MS), tandem (MS/MS) and sequential (MS3) modes using a linear ion trap mass spectrometer. The total number of carbon atoms and C=C bonds of each CL and, subsequently, those related to each of the constituting phosphatidic acid (PA) units, along with the location of the latter on the central glycerol backbone, were inferred from MS and MS/MS data, respectively. Moreover, the composition and location of both acyl chains on the glycerol backbone of each PA unit was obtained by MS3 measurements, an approach used for the first time for the structural elucidation of CL in R. sphaeroides. As a result, an unprecedented profile of CL in this bacterium was obtained, with 27 main species characterized, many of which are represented by compositional or regiochemical isomers. Interestingly, such a variability is generated from a limited number of different acyl chains, either saturated (i.e. 12:0, 16:0, 17:0, 18:0, 19:0) or mono-unsaturated (16:1, 18:1). The absence of polyunsaturated chains, more susceptible to oxidation damage, appeared to be indirectly related to the lack of carotenoids potentially acting as antioxidant agents, a specific feature of R. sphaeroides R26. The occurrence of odd-numbered acyl chains was ascribed to the need to guarantee membrane fluidity, through a less compact packing of CL, thus compensating for the lack of CL bearing polyunsaturated side chains. Graphical abstract Representation of MS signals due to carboxylate anions that would be detected, as separate couples, in the fragmentation spectra of the anions resulting from the two phosphatidic acid units included in a cardiolipin molecule bearing four different acyl chains.

Electron-Transfer Secondary Reaction Matrices for MALDI MS Analysis of Bacteriochlorophyll a in Rhodobacter sphaeroides and Its Zinc and Copper Analogue Pigments.

Bacteriochlorophyll a (BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol-ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M]+•) and negative ([M]-•) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward. Graphical Abstract ᅟ.

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MSn)

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MSn, n = 2–4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M−H]− at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M−H]− ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides.

Time-Resolved Electron Paramagnetic Resonance Study on Cofactor Geometries and Electronic Couplings after Primary Charge Separations in the Photosynthetic Reaction Center

To elucidate how cofactor geometries after photoinduced primary charge-separations influence electronic couplings (VCR) for primary charge-recombination (CR) processes in the photosynthetic reaction center, we have analyzed time-resolved electron paramagnetic resonance (TREPR) spectra both of the primary charge-separated (CS) state (P+•HA–•) and its charge-recombined triplet state (3P*) of the special pair in Rhodobacter sphaeroides R26. To determine the CS-state geometry, quantum mechanical modeling has been performed on the spin polarization of the 3P* generated by the spin dynamics due to the anisotropies of the hyperfine and the spin–spin dipolar interactions in the primary CS state. From transverse magnetizations of the primary CS state, we have also determined the VCR value leading to the singlet excited state (1P*) of the special pair. The above analyses have revealed that while the primary charge separation does not largely modulate the cofactor conformations, it leads to significant enhancement i...

Photoactive film by covalent immobilization of a bacterial photosynthetic protein on reduced graphene oxide surface

ABSTRACTCovalent functionalization of reduced graphene oxide (rGO) with the photosynthetic reaction center (RC) from Rhodobacter sphaeroides R26 via click chemistry reaction has been performed. The hybrid system was characterized by flash photolysis and infrared spectroscopy and the RC was found to retain its photoactivity and structural integrity. The strategy is applicable for the fabrication of hybrid bio-electronic devices capable of absorbing and converting solar energy.

The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress

A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co(2+) and CrO4 (2-) is reported and changes in membrane lipid profiles were rationalised.

Characterization of ‘pinky’ strain grown in culture of Rhodobacter sphaeroides R26.1

In the process of cultivating Rhodobater sphaeroides R26.1, some of which turned from blue to pink due to the irradiation of a beam of leaking white light. The mutant strains were named ‘pinky’ strains, which were cultivated in the red light and in the dark for a comparative study. It turned out that the strains did not grow in the dark, so they might be photosynthetic bacteria. The electronic absorption spectrum of the ‘pinky’ strains was measured, which shows they contained two main photosynthetic pigments, carotenoids(Cars) and bacteriochlorophylls(BChls). And then they were extracted and analyzed. It proves that Bchls included Bchl a and Bchl a′. Nuclear magnetic resonance (NMR) spectra were exploited to determine the chemical structure of Cars. The results indicate that there were seven kinds of Cars, including lycopene, rhodopin, anhydrorhodovibrin, 3,4-dihydroanhydrorhodovibrin, 3,3,4-dihydrospirilloxanthin, 3,4,3′,4′-tetrahydrospirilloxanthin and spirilloxanthin. Based on the above results, it was found that most identified Cars formed via spirilloxanthin biosynthesis pathway. The analyzed results of 16S rRNA gene show that the homology of ‘pinky’ strains with Rhodopseudomonas palusteris was 99%. Rhodopseudomonas palusteris has been cultivated in our laboratory. Because of its strong vitality, it did not become extinct with so many years passing. When Rhodobater sphaeroides R26.1 was cultivated, it got rejuvenated under the appropriate conditions and caused Rhodobater sphaeroides R26.1 to be contaminated.

Triplet state of the primary donor in reaction centers of the phototrophic bacterium Rhodobacter sphaeroides R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T0 mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.

Rates of the initial two steps of electron transfer in reaction centers from Rhodobacter sphaeroides as determined by singular-value decomposition followed by global fitting

The pump–probe spectral data matrices of reaction centers from Rhodobacter sphaeroides R26.1 and 2.4.1 in the 650–1265 nm region, that have been obtained by excitation of P at 868 nm with ∼0.1 ps pulses, were analyzed by singular-value decomposition followed by global fitting by the use of a three-component sequential model. The species-associated difference spectra showed the P∗BH → P +B −H → P +BH − sequential electron transfer and the rates of electron transfer were determined to be (1.9 ps) −1 and (2.7 ps) −1 for the first and the second steps, respectively. The P∗BH ⇌ P +B −H reversible electron transfer was also identified.

Oligomeric Characterization of the Photosynthetic Apparatus of Rhodobacter sphaeroides R26.1 by Nondenaturing Electrophoresis Methods

Blue and colorless native gel electrophoresis in combination with LC-ESI-MS/MS are powerful tools in the analysis of protein networks in biological membranes. We used these techniques in the present study to generate a comprehensive overview on a proteome-wide scale of intracytoplasmic membrane (ICM) associated proteins in order to investigate the native supramolecular organization of Rhodobacter sphaeroides R26.1 photosynthetic apparatus. The results obtained were compared with past proteomic data, as well as with models for the topology of photosynthetic membranes as derived from previously published atomic force microscopy studies. We identified 52 proteins organized in 10 different multiprotein complexes. We were able to demonstrate the existence of different oligomeric states for the integral membrane pigment-protein complexes dedicated to bacterial photosynthesis. Specifically, we found dimers and trimers, as well as supercomplexes of light-harvesting (LH) 2 at very high molecular weights (around 10,000 kDa). We recovered the monomeric form of the photochemical reaction center (RC), as well as the monomer and dimer of the reaction center-light harvesting 1-PufX (RC-LH1-PufX) complex. Curiously, no type of LH1 complex was detected. Lastly, ATP synthase and cytochrome bc(1) complexes were only recovered in their monomeric states. Purified ICM vesicles were shown to be rich in newly discovered gene products, including three proteins with unknown functions (RSP_2125, RSP_3238, RSP_6207), a possible alkane hydroxylase and a spheroidene monooxygenase. Other multiprotein complexes were found to be localized in the ICM, including succinate dehydrogenase in trimeric form and sarcosine oxidase in two different aggregation states. These findings contribute to the growing body of evidence that the bacterial ICM is a specialized bioenergetic membrane hosting, not only photosynthesis, but many other critical activities.

Nanosecond-Flash 15N Photo-CIDNP MAS NMR on Reaction Centers of Rhodobacter sphaeroides R26

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is observed with time-resolved 15N magic-angle spinning (MAS) nuclear magnetic resonance (NMR) in uniformly 15N-labeled reaction centers (RCs) of the purple bacterium Rhodobacter (Rb.) sphaeroides R26 under illumination with nanosecond laser flashes. The 15N NMR signals enhanced by the solid-state photo-CIDNP effect are related to electron spin densities in the primary radical pair and allow for reconstruction of the electronic structure at atomic scale. New assignments for the photochemically active nitrogens are proposed based on simulations of the intensities. The ratio of electron spin densities between cofactors PL and PM is observed to be about 4:1. The origin of the high asymmetry is discussed.

Spin labeling EPR

Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational dynamics of biomolecules under native-like conditions. This article summarizes the basics as well as recent progress of site-directed spin labeling. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis and the photosynthetic reaction center from Rhodobacter sphaeroides R26.

Electron Paramagnetic Resonance Study of Radiation Damage in Photosynthetic Reaction Center Crystals

Electron paramagnetic resonance (EPR) was used to simultaneously study radiation-induced cofactor reduction and damaging radical formation in single crystals of the bacterial reaction center (RC). Crystals of Fe-removed/Zn-replaced RC protein from Rhodobacter ( R.) sphaeroides R26 were irradiated with varied radiation doses at cryogenic temperature and analyzed for radiation-induced free radical formation and alteration of light-induced photosynthetic electron transfer activity using high-field (HF) D-band (130 GHz) and X-band (9.5 GHz) EPR spectroscopies. These analyses show that the formation of radiation-induced free radicals saturated at doses 1 order of magnitude smaller than the amount of radiation at which protein crystals lose their diffraction quality, while light-induced RC activity was found to be lost at radiation doses at least 1 order of magnitude lower than the dose at which radiation-induced radicals exhibited saturation. HF D-band EPR spectra provide direct evidence for radiation-induced reduction of the quinones and possibly other cofactors. These results demonstrate that substantial radiation damage is likely to have occurred during X-ray diffraction data collection used for photosynthetic RC structure determination. Thus, both radiation-induced loss of photochemical activity in RC crystals and reduction of the quinones are important factors that must be considered when correlating spectroscopic and crystallographic measurements of quinone site structures.

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.

Two Stereoisomers of Spheroidene in the Rhodobacter sphaeroides R26 Reaction Center: A DFT Analysis of Resonance Raman Spectra

From a theoretical analysis of the resonance Raman spectra of 19 isotopomers of spheroidene reconstituted into the reaction center (RC) of Rhodobacter sphaeroides R26, we conclude that the carotenoid in the RC occurs in two configurations. The normal mode underlying the resonance Raman transition at 1239 cm −1, characteristic for spheroidene in the RC, has been identified and found to uniquely refer to the cis nature of the 15,15′ carbon-carbon double bond. Detailed analysis of the isotope-induced shifts of transitions in the 1500–1550 cm −1 region proves that, besides the 15,15′- cis configuration, spheroidene in the RC adopts another cis-configuration, most likely the 13,14- cis configuration.

Formation and geminate quenching of singlet oxygen in purple bacterial reaction center

The phosphorescence of singlet oxygen ( 1X ∗) photosensitized by the carotenoidless reaction center (RC) of Rhodobacter sphaeroides R26.1 has been investigated, using H 2O and D 2O as the suspending media. To enhance (under neutral conditions) the triplet quantum yield of the special pair P 870 (P) by the radical pair mechanism, the quinone acceptor Q A was removed by means of a chemical treatment. The phosphorescence signal fits the functional form P 0[exp (− t/ τ) − exp(− t/ ζ)], regardless of whether 1X ∗ is sensitized by P † or M † (where the dagger denotes triplet excitation and M is a water-soluble molecule). The time constant ζ was identified with the decay time of 1X ∗; when P † is the sensitizer, one finds ζ P ( 1 ) = 3.3 ± 0.3 μ s , and ζ P ( 2 ) = 34 ± 3 μ s , where the superscripts 1 and 2 refer to H 2O and D 2O, respectively; the corresponding values for sensitization by M † (in the absence of RC) are ζ M ( 1 ) = 3.7 ± 0.4 μ s , and ζ M ( 2 ) = 75 ± 5 μ s . The addition of RC’s to the solution of M in D 2O reveals that the RC is a quencher of 1X ∗; however, for equal concentrations of the RC, ζ P ( 2 ) < ζ M ( 2 ) , showing that 1X ∗ is deactivated, after its entry into the suspending medium, mainly by the solvent or the same RC which acts as the sensitizer. The values of τ P are similar in both solvents, ca. 2 μs, but this time constant does not figure in the disappearance of P †, which follows a bi-exponential course, α 1exp(− t/ τ 1) + α 2exp(− t/ τ 2). The time constants τ 1 and τ 2 (72 ± 5 μs and 12 ± 1 μs, respectively) as well as the factor α 2 are insensitive to the oxygen content, and quenching of P † is manifested only through a threefold reduction in the magnitude of α 1; these data imply the absence of dynamic quenching and heterogeneity of the RC. The mean lifetime of 1X ∗ inside the protein matrix is identified with τ P, and the absence of a prompt component in the phosphorescence signal rationalized by proposing that the radiative decay of 1X ∗ within the RC is much slower than that in an aqueous environment.

13C−1H heteronuclear dipolar correlation studies of the hydrogen bonding of the quinones inRhodobacter sphaeroides R26 reaction centers

The photosynthetic reaction center (RC) of the photosynthetic bacteriumRhodobacter sphaeroides R26 contains two quinones, QA and QB. Solid-state heteronuclear (1H−13C) dipolar correlation spectroscopy has been used to study the binding of the quinones in the ground state for RCs reconstituted with l-13C ubiquinone-10. Lee-Goldburg cross-polarization buildup curves are recorded to determine distancesrCH between the l-13C carbon labels and the protons involved in the polarization transfer. The l-13C of both QA and QB have intermolecular correlations with protons that resonate downfield, in the region of hydrogen-bonding protons. The distances between the carbon labels and the correlated protons are short, 0.21±0.01 nm. Hence the nuclear magnetic resonance provides evidence for strong hydrogen-bonding interactions at the l-C=O of both QA and QB for RCs in the ground state. The environment of the l-13C of the QB is structurally heterogeneous compared to that of the QA. The data can be reconciled with a strong H-bonding interaction of the l-C=O of QA with Ala M260 NH, and with complex hydrogen bonding involving NH of Ile-L224 and of Gly-L225, and possibly the Ser-L223 hydroxyl group of the l-C=O of the QB, in the proximal site.