Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile²²⁹ Met, Ser²²³ Pro and Tyr²²² Gly. The mutations of Ser²²³ is analogous to the mutation of Ser²⁶⁴ in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q _b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q _b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ₀ and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.Abbreviations ATP adenosine 5-triphosphate - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - bp basepair - cyt c²⁺ reduced form of cytochrome c - DEAE diethylami-noethyl - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - Pipes piperazine-N,N-bis-2-ethane-sulfonic acid - PSII photosystem II - RC reaction center - SDS sodium dodecylsulfate - Tris tris(hydroxy-methyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Glucocorticoids in muscular dystrophy: beneficial effects of dexamethasone on avian myopathy

A corticosteroid with mixed glucocorticoid-mineralocorticoid actions was previously shown to improve neuromuscular function in muscular dystrophic chickens. The significance of that finding was recently underscored by reports that a mixed-action corticosteroid improved muscle function in Duchenne dystrophy patients, albeit at high doses. In the present study a pure glucocorticoid improved function and retarded muscle histopathology in the chicken, but a pure mineralocorticoid did not. These observations suggest that elucidation of mechanisms by which glucocorticoids beneficially affect dystrophic muscle could lead to development of more effective therapies.     

ENDOR spectroscopy reveals light induced movement of the H-bond from Ser-L223 upon forming the semiquinone (Q(B)(-)(*)) in reaction centers from Rhodobacter sphaeroides

Proton ENDOR spectroscopy was used to monitor local conformational changes in bacterial reaction centers (RC) associated with the electron-transfer reaction DQB --> D+*QB-* using mutant RCs capable of photoreducing QB at cryogenic temperatures. The charge separated state D+*QB-* was studied in mutant RCs formed by either (i) illuminating at low temperature (77 K) a sample frozen in the dark (ground state protein conformation) or (ii) illuminating at room temperature prior to and during freezing (charge separated state protein conformation). The charge recombination rates from the two states differed greatly (>10(6) fold) as shown previously, indicating a structural change (Paddock et al. (2006) Biochemistry 45, 14032-14042). ENDOR spectra of QB-* from both samples (35 GHz, 77 K) showed several H-bond hyperfine couplings that were similar to those for QB-* in native RCs indicating that in all RCs, QB-* was located at the proximal position near the metal site. In contrast, one set of hyperfine couplings were not observed in the dark frozen samples but were observed only in samples frozen under illumination in which the protein can relax prior to freezing. This flexible H-bond was assigned to an interaction between the Ser-L223 hydroxyl and QB-* on the basis of its absence in Ser L223 --> Ala mutant RCs. Thus, part of the protein relaxation, in response to light induced charge separation, involves the formation of an H-bond between the OH group of Ser-L223 and the anionic semiquinone QB-*. These results show the flexibility of the Ser-L223 H-bond, which is essential for its function in proton transfer to reduced QB.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: I. Identification of the ENDOR lines associated with the hydrogen bonds to the primary quinone QA*-

Hydrogen bonds are important in determining the structure and function of biomolecules. Of particular interest are hydrogen bonds to quinones, which play an important role in the bioenergetics of respiration and photosynthesis. In this work we investigated the hydrogen bonds to the two carbonyl oxygens of the semiquinone QA*- in the well-characterized reaction center from the photosynthetic bacterium Rhodobacter sphaeroides R-26. We used electron paramagnetic resonance and electron nuclear double resonance techniques at 35 GHz at a temperature of 80 K. The goal of this study was to identify and assign sets of 1H-ENDOR lines to protons hydrogen bonded to each of the two oxygens. This was accomplished by preferentially exchanging the hydrogen bond on one of the oxygens with deuterium while concomitantly monitoring the changes in the amplitudes of the 1H-ENDOR lines. The preferential deuteration of one of the oxygens was made possible by the different 1H --> 2H exchange times of the protons bonded to the two oxygens. The assignment of the 1H-ENDOR lines sets the stage for the determination of the geometries of the H-bonds by a detailed field selection ENDOR study to be presented in a future article.     

Trapped conformational states of semiquinone (D+*QB-*) formed by B-branch electron transfer at low temperature in Rhodobacter sphaeroides reaction centers

The reaction center (RC) from Rhodobacter sphaeroides captures light energy by electron transfer between quinones QA and QB, involving a conformational gating step. In this work, conformational states of D+*QB-* were trapped (80 K) and studied using EPR spectroscopy in native and mutant RCs that lack QA in which QB was reduced by the bacteriopheophytin along the B-branch. In mutant RCs frozen in the dark, a light induced EPR signal due to D+*QB-* formed in 30% of the sample with low quantum yield (0.2%-20%) and decayed in 6 s. A small signal with similar characteristics was also observed in native RCs. In contrast, the EPR signal due to D+*QB-* in mutant RCs illuminated while freezing formed in approximately 95% of the sample did not decay (tau >107 s) at 80 K (also observed in the native RC). In all samples, the observed g-values were the same (g = 2.0026), indicating that all active QB-*'s were located in a proximal conformation coupled with the nonheme Fe2+. We propose that before electron transfer at 80 K, the majority (approximately 70%) of QB, structurally located in the distal site, was not stably reducible, whereas the minority (approximately 30%) of active configurations was in the proximal site. The large difference in the lifetimes of the unrelaxed and relaxed D+*QB-* states is attributed to the relaxation of protein residues and internal water molecules that stabilize D+*QB-*. These results demonstrate energetically significant conformational changes involved in stabilizing the D+*QB-* state. The unrelaxed and relaxed states can be considered to be the initial and final states along the reaction coordinate for conformationally gated electron transfer.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Respiratory abdominal muscle recruitment and chest wall motion in myotonic muscular dystrophy.

Abdominal muscles are selectively active in normal subjects during stress and may increase the potential energy for inspiration by reducing the end-expiratory lung volume (EELV). We hypothesized that a similar process would occur in subjects with myotonic muscular dystrophy (MMD), but would be less effective, because of to their weakness and altered chest wall mechanics. Fine-wire electromyography (EMG) of the transversus abdominis (TA), internal oblique (IO), external oblique, and rectus abdominis was recorded in 10 MMD and 10 control subjects. EMG activity, respiratory inductive plethysmography, and gastric pressure were recorded during static pressure measurement and at increasing levels of inspiratory resistance breathing. EELV was reduced and chest wall motion was synchronous only in controls. Although the TA and IO were selectively recruited in both groups, EMG activity of the MMD group was twice that of controls at the same inspiratory pressure. In MMD subjects with mildly reduced forced vital capacity, significant differences can be seen in abdominal muscle recruitment, wall motion, work of breathing, and ventilatory parameters.     

X-Ray structure determination of three mutants of the bacterial photosynthetic reaction centers from Rb. sphaeroides; altered proton transfer pathways

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the reduction of a bound quinone molecule Q(B) is coupled with proton uptake. When Asp-L213 is replaced by Asn, proton transfer is inhibited. Proton transfer was restored by two second-site revertant mutations, Arg-M233-->Cys and Arg-H177-->His. Kinetic effects of Cd(2+) on proton transfer showed that the entry point in revertant RCs to be the same as in the native RC. The structures of the parental and two revertant RCs were determined at resolutions of 2.10, 1.80, and 2.75 A. From the structures, we were able to delineate alternate proton transfer pathways in the revertants. The main changes occur near Glu-H173, which allow it to substitute for the missing Asp-L213. The electrostatic changes near Glu-H173 cause it to be a good proton donor and acceptor, and the structural changes create a cavity which accommodates water molecules that connect Glu-H173 to other proton transfer components.     

Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26: localization of the binding site by chemical cross-linking and immunochemical studies

The location of the cytochrome binding site on the reaction center of Rhodopseudomonas sphaeroides was studied by two different approaches. In one, cross-linking agents, principally dithiobis(propionimidate) and dimethyl suberimidate, were used to link cytochrome c and cytochrome c2 to reaction centers; in the other, the inhibition of electron transfer by antibodies against the subunits was investigated. Cytochrome c (horse) cross-linked to the L and M subunits, whereas cytochrome c2 (R. sphaeroides) cross-linked only to the L subunit. The cross-linked reaction center-cytochrome complexes were isolated by affinity chromatography. The rate of electron transfer in the cross-linked cytochrome c2 complex was the same as that in the un-cross-linked complex. However, when cytochrome c was used, the rate in the cross-linked complex was about 15 times slower than that in the un-cross-linked complex. Fab fragments of antibodies specific against the L and M subunits blocked electron transfer from both cytochrome c (horse) and cytochrome c2 (R. sphaeroides). Antibodies specific for the H subunit did not block either reaction. We conclude that the cytochrome binding site on the reaction center is close (approximately 10 A) to both the L and M subunits, possibly in a cleft between them.