The cytochrome bc1 complex from Rhodovulum sulfidophilum is a dimer with six quinones per monomer and an additional 6-kDa component. (1/78)

A highly active, large-scale preparation of cytochrome bc1 complex has been obtained from the photosynthetic purple bacterium Rhodovulum (Rhv.) sulfidophilum. It has been characterized using mass spectrometry, quinone and lipid analysis as well as inhibitor binding. About 35 mg of pure complex can be obtained from 1 g of membrane protein. EPR spectroscopy and optical titrations have been used to obtain the redox midpoint potentials of the cofactors. The Em-value of 310 mV for the Rieske protein is the most positive midpoint potential for this protein in a bc1 complex so far. The bc1 complex from Rhv. sulfidophilum is very stable and consists of three subunits and a 6-kDa polypeptide. The complex appears as a dimer in solution and contains six quinone molecules per monomer which are tightly bound. EPR spectroscopy shows that the Q(o) site is highly occupied. High detergent concentrations convert the complex into an inactive, monomeric form that has lost the Rieske protein as well as the quinones and the 6-kDa component.  (+info)

A new cytochrome subunit bound to the photosynthetic reaction center in the purple bacterium, Rhodovulum sulfidophilum. (2/78)

The nucleotide sequence of the puf operon, which contains the genes encoding the B870 light-harvesting protein and the reaction center complex of the purple photosynthetic bacterium, Rhodovulum sulfidophilum, was determined. The operon, which consisted of six genes, pufQ, pufB, pufA, pufL, pufM, and pufC, is a new variety in photosynthetic bacteria in the sense that pufQ and pufC coexist. The amino acid sequence of the cytochrome subunit of the reaction center deduced from the pufC sequence revealed that this cytochrome contains only three possible heme-binding motifs; the heme-1-binding motif of the corresponding tetraheme cytochrome subunits was not present. This is the first exception of the "tetraheme" cytochrome family in purple bacteria and green filamentous bacteria. The pufC sequence also revealed that the sixth axial ligands to heme-1 and heme-2 irons were not present in the cytochrome either. This cytochrome was actually detected in membrane preparation as a 43-kDa protein and shown to associate functionally with the photosynthetic reaction center as the immediate electron donor to the photo-oxidized special pair of bacteriochlorophyll. This new cytochrome should be useful for studies on the role of each heme in the cytochrome subunit of the bacterial reaction center and the evolution of proteins in photosynthetic electron transfer systems.  (+info)

Comparative sequence analysis of the DNA packaging, head, and tail morphogenesis modules in the temperate cos-site Streptococcus thermophilus bacteriophage Sfi21. (3/78)

The temperate Streptococcus thermophilus bacteriophage Sfi21 possesses 15-nucleotide-long cohesive ends with a 3' overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry. Over the DNA packaging, head and tail morphogenesis modules, the Sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. A nearly one to one gene correlation was found with the phage lambda genes Nu1 to H, except for gene B-to-E complex, where the Sfi21 map resembled that of coliphage HK97. The similarity between Sfi21 and HK97 was striking: both major head proteins showed an N-terminal coiled-coil structure, the mature major head proteins started at amino acid positions 105 and 104, respectively, and both major head genes were preceded by genes encoding a possible protease and portal protein. The purported Sfi21 protease is the first viral member of the ClpP protease family. The prediction of Sfi21 gene functions by reference to the gene map of intensively investigated coliphages was experimentally confirmed for the major head and tail gene. Phage Sfi21 shows nucleotide sequence similarity with Lactococcus phage BK5-T and a lactococcal prophage and amino acid sequence similarity with the Lactobacillus phage A2 and the Staphylococcus phage PVL. PVL is a missing link that connects the portal proteins from Sfi21 and HK97 with respect to sequence similarity. These observations and database searches, which demonstrate sequence similarity between proteins of phage from gram-positive bacteria, proteobacteria, and Archaea, constrain models of phage evolution.  (+info)

A spectroscopic method for observing the domain movement of the Rieske iron-sulfur protein. (4/78)

The g-tensor orientation of the chemically reduced Rieske cluster in cytochrome bc(1) complex from Rhodovulum sulfidophilum with respect to the membrane was determined in the presence and absence of inhibitors and in the presence of oxidized and reduced quinone in the quinol-oxidizing-site (Q(o)-site) by EPR on two-dimensionally ordered samples. Almost identical orientations were observed when oxidized or reduced quinone, stigmatellin, or 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole was present. Occupancy of the Q(o)-site by myxothiazole induced appearance of a minority population with a substantially differing conformation and presence of E-beta-methoxyacrylate-stilbene significantly reduced the contribution of the major conformation observed in the other cases. Furthermore, when the oxidized iron-sulfur cluster was reduced at cryogenic temperatures by the products of radiolysis, the orientation of its magnetic axes was found to differ significantly from that of the chemically reduced center. The "irradiation-induced" conformation converts to that of the chemically reduced center after thawing of the sample. These results confirm the effects of Q(o)-site inhibitors on the equilibrium conformation of the Rieske iron-sulfur protein and provide evidence for a reversible redox-influenced interconversion between conformational states. Moreover, the data obtained with the iron-sulfur protein demonstrate that the conformation of "EPR-inaccessible" reduction states of redox centers can be studied by inducing changes of redox state at cryogenic temperatures. This technique appears applicable to a wide range of comparable electron transfer systems performing redox-induced conformational changes.  (+info)

Transcriptional control of expression of genes for photosynthetic reaction center and light-harvesting proteins in the purple bacterium Rhodovulum sulfidophilum. (5/78)

The purple photosynthetic bacterium Rhodovulum sulfidophilum synthesizes photosynthetic apparatus even under highly aerated conditions in the dark. To understand the oxygen-independent expression of photosynthetic genes, the expression of the puf operon coding for the light-harvesting 1 and reaction center proteins was analyzed. Northern blot hybridization analysis showed that puf mRNA synthesis was not significantly repressed by oxygen in this bacterium. High-resolution 5' mapping of the puf mRNA transcriptional initiation sites and DNA sequence analysis of the puf upstream regulatory region indicated that there are three possible promoters for the puf operon expression, two of which have a high degree of sequence similarity with those of Rhodobacter capsulatus, which shows a high level of oxygen repression of photosystem synthesis. Deletion analysis showed that the third promoter is oxygen independent, but the activity of this promoter was not enough to explain the aerobic level of mRNA. The posttranscriptional puf mRNA degradation is not significantly influenced by oxygen in R. sulfidophilum. From these results, we conclude that puf operon expression in R. sulfidophilum is weakly repressed by oxygen, perhaps as a result of the following: (i) there are three promoters for puf operon transcription, at least one of which is oxygen independent; (ii) readthrough transcripts which may not be affected by oxygen may be significant in maintaining the puf mRNA levels; and (iii) the puf mRNA is fairly stable even under aerobic conditions.  (+info)

Intrinsic conformation of lipid A is responsible for agonistic and antagonistic activity. (6/78)

Lipopolysaccharides (LPS, endotoxin) represent a major virulence factor of Gram-negative bacteria, which can cause septic shock in mammals, including man. The lipid anchor of LPS to the bacterial outer membrane, lipid A, exhibits a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is also responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against endotoxically active LPS. By applying orientation measurements with attenuated total reflectance (ATR) infrared spectroscopy on hydrated lipid A samples, we show here that these different biological activities are directly correlated to the intrinsic conformation of lipid A. Bisphosphoryl-hexaacyl lipid A molecules with an asymmetric (4/2) distribution of the acyl chains linked to the diglucosamine backbone have a large tilt angle (> 45 degrees ) of the diglucosamine backbone with respect to the membrane surface, a conical molecular shape (larger cross-section of the hydrophobic than the hydrophilic moiety), and are endotoxically highly active. Monophosphoryl hexaacyl lipid A has a smaller tilt angle, and the conical shape is less expressed in favour of a more cylindrical shape. This correlates with decreasing endotoxic activity. Penta- and tetraacyl lipid A or hexaacyl lipid A with a symmetric acyl chain distribution (3/3) have a small tilt angle (< 25 degrees ) and a cylindrical shape and are endotoxically inactive, but may be antagonistic.  (+info)

Proton pumping by cytochrome oxidase: progress, problems and postulates. (7/78)

The current status of our knowledge about the mechanism of proton pumping by cytochrome oxidase is discussed. Significant progress has resulted from the study of site-directed mutants within the proton-conducting pathways of the bacterial oxidases. There appear to be two channels to facilitate proton translocation within the enzyme and they are important at different parts of the catalytic cycle. The use of hydrogen peroxide as an alternative substrate provides a very useful experimental tool to explore the enzymology of this system, and insights gained from this approach are described. Proton transfer is coupled to and appears to regulate the rate of electron transfer steps during turnover. It is proposed that the initial step in the reaction involves a proton transfer to the active site that is important to convert metal-ligated hydroxide to water, which can more rapidly dissociate from the metals and allow the reaction with dioxygen which, we propose, can bind the one-electron reduced heme-copper center. Coordinated movement of protons and electrons over both short and long distances within the enzyme appear to be important at different parts of the catalytic cycle. During the initial reduction of dioxygen, direct hydrogen transfer to form a tyrosyl radical at the active site seems likely. Subsequent steps can be effectively blocked by mutation of a residue at the surface of the protein, apparently preventing the entry of protons.  (+info)

Deletion of the 6-kDa subunit affects the activity and yield of the bc1 complex from Rhodovulum sulfidophilum. (8/78)

The cytochrome bc1 complex from Rhodovulum sulfidophilum purifies as a four-subunit complex: the cytochrome b, cytochrome c1 and Rieske iron-sulphur proteins, which are encoded together in the fbc operon, as well as a 6-kDa protein. The gene encoding the 6-kDa protein, named fbcS, has been identified. It is located within the sox operon, which encodes the subunits of sarcosine oxidase. The encoded 6-kDa protein is very hydrophobic and is predicted to form a single transmembrane helix. It shows no sequence homology to any known protein. The gene has been knocked-out of the genome and a three-subunit complex can be purified. This deletion leads to a large reduction in the yield of the isolated complex and in its activity compared to wild-type. The high quinone content found in the wild-type complex is, however, maintained after removal of the 6-kDa protein. Surprisingly, a fourth subunit of approximately 6 kDa is again found to copurify with the Rhv. sulfidophilum bc1 complex when only the fbc operon is expressed heterologously in a near-relative, Rhodobacter capsulatus, which lacks this small subunit in its own bc1 complex.  (+info)