No abstract

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX þ and PufX À strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX À cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1b. This arrangement, coupled with a flexible ring, would give the RC Q B site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc 1 complex.

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.

This investigation was motivated by a desire to get a deeper insight into the mechanism of carotenoiod-to-bacteriochlorophyll (Car-to-BChl) energy transfer proceeding via the carotenoid S1 state. (Here, we call the 2Ag - and 1Bu + states “the S1 and S2 states” according to the notation presently accepted.) To systematically examine the effect of the conjugation length of carotenoid on the rate and efficiency of the Car(S1)-to-BChl(Qy) energy transfer, we performed the following experiments. (1) Subpicosecond time-resolved absorption spectroscopy was employed to measure the S1-state lifetimes of lycopene (number of conjugated CC bonds, n = 11), spheroidene (n = 10), and neurosporene (n = 9), both free in n-hexane and bound to the LH2 complexes from Rhodospirillum molischianum (Rs. molischianum), Rhodobactor sphaeroides (Rb. sphaeroides) 2.4.1, and Rb. sphaeroides G1C, respectively. The lifetime of each free (bound) carotenoid was determined to be 4.7(3.4) ps for lycopene, 9.3(1.7) ps for spheroidene, and 21.2(1.3) ps for neurosporene. It was found that the rate and the efficiency of the Car(S1)-to-BChl(Qy) energy transfer increase systematically when the number of conjugated CC bonds decreases. (2) High-sensitivity steady-state fluorescence spectroscopy was used to measure the spectra of dual emission from the S2 and S1 states for the above carotenoids dissolved in n-hexane. The fluorescence data, combined with the above kinetic data, allowed us to evaluate the magnitudes of the transition-dipole moments associated with the Car(S1) emission. It was found that the S1 emissions of the above carotenoids carry noticeably large oscillator strengths (transition-dipole moments). In the case of the LH2 complex from Rs. molischianum, whose structural information is now available, the time constant of the Car(S1)-to-BChl(Qy) energy transfer (18.6 ps), which was predicted on the basis of a Car(S2)-to-BChl(Qy) full Coulombic coupling scaled by the ratio of the S1 vs S2 transition dipole moments, was in good agreement with the one spectroscopically determined (12.3 ps). The oscillator strength associated with the Car(S1) emission was discussed in terms of the state mixing between the carotenoid S2 and S1 states.

Reaction center-light harvesting 1 (RC-LH1) complexes are the fundamental units of bacterial photosynthesis, which use solar energy to power the reduction of quinone to quinol prior to the formation of the proton gradient that drives ATP synthesis. The dimeric RC-LH1-PufX complex of Rhodobacter sphaeroides is composed of 64 polypeptides and 128 cofactors, including 56 LH1 bacteriochlorophyll a (BChl a) molecules that surround and donate energy to the two RCs. The 3D structure was determined to 8 Å by X-ray crystallography, and a model was built with constraints provided by electron microscopy (EM), nuclear magnetic resonance (NMR), mass spectrometry (MS), and site-directed mutagenesis. Each half of the dimer complex consists of a RC surrounded by an array of 14 LH1 αβ subunits, with two BChls sandwiched between each αβ pair of transmembrane helices. The N- and C-terminal extrinsic domains of PufX promote dimerization by interacting with the corresponding domains of an LH1 β polypeptide from the other half of the RC-LH1-PufX complex. Close contacts between PufX, an LH1 αβ subunit, and the cytoplasmic domain of the RC-H subunit prevent the LH1 complex from encircling the RC and create a channel connecting the RC QB site to an opening in the LH1 ring, allowing Q/QH₂ exchange with the external quinone pool. We also identified a channel that connects the two halves of the dimer, potentially forming a long-range pathway for quinone migration along rows of RC-LH1-PufX complexes in the membrane. The structure of the RC-LH1-PufX complex explains the crucial role played by PufX in dimer formation, and it shows how quinone traffic traverses the LH1 complex as it shuttles between the RC and the cytochrome bc₁ complex.

Abstract-Endothelin A (ET A ) receptor blockade has prevented vascular remodeling in aldosterone and salt-induced hypertension. To evaluate effects of the ET A receptor antagonist, BMS 182874, compared with the aldosterone antagonist, spironolactone, on vascular remodeling in aldosterone-infused rats not exposed to a high salt diet, Sprague-Dawley rats were infused subcutaneously with aldosterone (0.75 g/h) and treated with BMS 182874 (40 mg · kg, spironolactone, or hydralazine (both 25 mg · kg Ϫ1 · d Ϫ1 ) while receiving a normal salt diet for 6 weeks. Aldosterone increased systolic BP (PϽ0.01), plasma endothelin (3.33Ϯ0.32 versus 1.85Ϯ0.40 pmol/L in control, PϽ0.05), systemic oxidative stress as shown by plasma thiobarbituric acid-reacting substances and vascular nicotinamide adenine dinucleotide phosphate (NADPH) activity. Aldosterone increased small artery media thickness (17.7Ϯ0.9 versus 13.6Ϯ0.8 m in control, PϽ0.05) and media/lumen ratio (7.6Ϯ0.4 versus 5.5Ϯ0.4% in control, PϽ0.05), with growth index of 21% indicating hypertrophic remodeling. Laser confocal microscopy showed increased collagen and fibronectin deposition and intercellular adhesion molecule-1 (ICAM-1) content in the vessel wall of aldosterone-infused rats. The 3 treatments lowered BP, although hydralazine was slightly less effective. BMS 182874 and spironolactone decreased oxidative stress, normalized the hypertrophic remodeling, decreased collagen and fibronectin deposition, and reduced ICAM-1 abundance in the vascular wall of aldosterone-infused rats, whereas hydralazine only reduced NADPH activity in aorta but did not affect the remaining parameters. Vascular remodeling of small arteries occurs in aldosterone-infused rats exposed to a normal salt diet and may be mediated in part by ET-1 via stimulation of ET A receptors. Endothelin blockade may exert beneficial effects on vascular remodeling, fibrosis, oxidative stress, and adhesion molecule expression in aldosterone-induced hypertension.

The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

Two-dimensional crystals of the reaction-centre-light-harvesting complex I (RC-LH1) of the purple non- sulfur bacterium Rhodospirillum rubrum have been formed from detergent-solubilized and purified protein complexes. Unstained samples of this intrinsic membrane protein complex have been analysed by electron cryomicroscopy (cryo EM). Projection maps were calculated to 8.5 A from two different crystal forms, and show a single reaction centre surrounded by 16 LH1 subunits in a ring of approximately 115 A diameter. Within each LH1 subunit, densities for the alpha- and beta-polypeptide chains are clearly resolved. In one crystal form the LH1 forms a circular ring, and in the other form the ring is significantly ellipsoidal. In each case, the reaction centre adopts preferred orientations, suggesting specific interactions between the reaction centre and LH1 subunits rather than a continuum of possible orientations with the antenna ring. This experimentally determined structure shows no evidence of any other protein components in the closed LH1 ring. The demonstration of circular or elliptical forms of LH1 indicates that this complex is likely to be flexible in the bacterial membrane.