Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase

The three-dimensional structure of Wolinella succinogenes quinol:fumarate reductase (QFR), a dihaem-containing member of the superfamily of succinate:quinone oxidoreductases (SQOR), has been determined at 2.2 A resolution by X-ray crystallography [Lancaster et al., Nature 402 (1999) 377-385]. The structure and mechanism of W. succinogenes QFR and their relevance to the SQOR superfamily have recently been reviewed [Lancaster, Adv. Protein Chem. 63 (2003) 131-149]. Here, a comparison is presented of W. succinogenes QFR to the recently determined structure of the mono-haem containing succinate:quinone reductase from Escherichia coli [Yankovskaya et al., Science 299 (2003) 700-704]. In spite of differences in polypeptide and haem composition, the overall topology of the membrane anchors and their relative orientation to the conserved hydrophilic subunits is strikingly similar. A major difference is the lack of any evidence for a 'proximal' quinone site, close to the hydrophilic subunits, in W. succinogenes QFR.     

Some physiological and biochemical considerations of larval development in the American lobster,Homarus Americanus Milne Edwards

The weight-specific respiration rates of fed and starved lobsters and the ammonia excretion rates of fed lobsters increased with each larval stage (I through IV) and decreased with the first postlarval stage (V). The rate of change in metabolic rates was greater than the rate of change of body size of the larval stages, indicating an increased energy demand of the later larval stages. There was no significant difference in the O: N ratio for the first three larval stages but a reduction was observed in stage IV and V lobsters, reflecting an increased dependence on protein catabolism for energy.Protein was the principal biochemical constituent of all lobster stages. Significant decreases in lipid content and increases in ash and chitin content of the last larval (IV) and first postlarval (V) stages were detected.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism.

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.     

Site-exposure model for proton-lactose symport in Escherichia coli.

A model is presented for the lactose-proton co-transporter of E. coli. Either proton translocation inwards or galactoside translocation outwards brings about the exposure of galactoside binding sites externally. This alternation in the exposure of the galactoside binding site to either side of the membrane is viewed as the fundamental event in coupled uptake, rather than affinity changes for galactoside.The transporter is proposed to function as a dimer, exhibiting two forms corresponding to the “cis” and the “trans” orientation of the two galactosyl binding sites. A galactoside or a proton gradient brings about conversion of the sites from the “trans” to the “cis” configuration. The two forms can be experimentally differentiated by the accessibility of non-transportable substrate analogs to the galactosyl binding sites.     

Studies of the beta-galactoside transporter in inverted membrane vesicles of Escherichia coli. I. Symmetrical facilitated diffusion and proton gradient-coupled transport.

Facilitated diffusion of [14C]lactose into inverted membrane vesicles of Escherichia coli was measured using HgCl2 as a stopping reagent and polylysine to flocculate the vesicles for filtration. Equilibration of lactose between the internal and external volumes required expression of the y gene of the lac operon and was inhibited by thiodigalactoside or by prior incubation with N-ethylmaleimde or HgCl2. The initial rate of uptake was saturable, with a Kt of 0.95 mM. Counterflow of [14C]lactose was demonstrated in either direction. ATP hydrolysis or respiration drove the efflux of internal lactose. The effect of ATP required addition of F1 coupling factor (ATPase) from E. coli when lactose transport was studied in F1-deficient inverted vesicles. Accumulation of lactose against a concentration gradient was achieved by forming an artificial electrochemical proton gradient consisting of a membrane potential negative inside or a pH gradient basic inside. Addition of ATP inhibited this proton driven uptake showing that it occurred in inverted vesicles. It was concluded that the lactose-proton co-transport protein (M protein) is qualitatively symmetrical with respect to the facilitated diffusion of lactose and the coupling of proton and lactose transport.     

The emerging biology of the nitrite anion

Nitrite has now been proposed to play an important physiological role in signaling, blood flow regulation and hypoxic nitric oxide homeostasis. A recent two-day symposium at the US National Institutes of Health highlighted recent advances in the understanding of nitrite biochemistry, physiology and therapeutics.     

Herbicide-induced changes in charge recombination and redox potential of Q(A) in the T4 mutant of Blastochloris viridis

To gain new insights into the function of photosystem II (PSII) herbicides DCMU (a urea herbicide) and bromoxynil (a phenolic herbicide), we have studied their effects in a better understood system, the bacterial photosynthetic reaction center of the terbutryn-resistant mutant T4 of Blastochloris (Bl.) viridis. This mutant is uniquely sensitive to these herbicides. We have used redox potentiometry and time-resolved absorption spectroscopy in the nanosecond and microsecond time scale. At room temperature the P(+)(*)Q(A)(-)(*) charge recombination in the presence of bromoxynil was faster than in the presence of DCMU. Two phases of P(+)(*)Q(A)(-)(*) recombination were observed. In accordance with the literature, the two phases were attributed to two different populations of reaction centers. Although the herbicides did induce small differences in the activation barriers of the charge recombination reactions, these did not explain the large herbicide-induced differences in the kinetics at ambient temperature. Instead, these were attributed to a change in the relative amplitude of the phases, with the fast:slow ratio being approximately 3:1 with bromoxynil and approximately 1:2 with DCMU at 300 K. Redox titrations of Q(A) were performed with and without herbicides at pH 6.5. The E(m) was shifted by approximately -75 mV by bromoxynil and by approximately +55 mV by DCMU. As the titrations were done over a time range that is assumed to be much longer than that for the transition between the two different populations, the potentials measured are considered to be a weighted average of two potentials for Q(A). The influence of the herbicides can thus be considered to be on the equilibrium of the two reaction center forms. This may also be the case in photosystem II.