Pathways,pathway tubes,pathway docking,and propagators in electron transfer proteins

The simplest views of long-range electron transfer utilize flat one-dimensional barrier tunneling models, neglecting structural details of the protein medium. The pathway model of protein electron transfer reintroduces structure by distinguishing between covalent bonds, hydrogen bonds, and van der Waals contacts. These three kinds of interactions in a tunneling pathway each have distinctive decay factors associated with them. The distribution and arrangement of these bonded and nonbonded contacts in a folded protein varies tremendously between structures, adding a richness to the tunneling problem that is absent in simpler views. We review the pathway model and the predictions that it makes for protein electron transfer rates in small proteins, docked proteins, and the photosynthetic reactions center. We also review the formulation of the protein electron transfer problem as an effective two-level system. New multi-pathway approaches and improved electronic Hamiltonians are described briefly as well.     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.     

Prenatal and postnatal transfer of fatty acids from mother to pup in the hooded seal

Unlike most mammals, hooded seal (Cystophora cristata) pups are born with a substantial layer of adipose tissue. Subsequently, during the brief lactation period of only 4 days, fasting mothers mobilize enormous amounts of lipid from blubber and secrete milk (60% fat) at rates of 10 kg·day^-1. Pups gain 7 kg·day^-1 due primarily to the deposition of fat in blubber. We measured blubber content and fatty acid composition of blubber and milk in hooded seal mother-pup pairs at birth and over the 4-day lactation period to examine the nature and source of fetal lipids, the incorporation of maternal blubber fatty acids into milk lipid, and patterns of fatty acid deposition in suckling young. The fatty acid composition of the blubber of the newborn was notably different from that of its mother. Fetal deposition was likely due to a combination of both fetal synthesis and direct placental transfer of maternal circulating fatty acids. The blubber of the newborn was characterized by high levels (>90% of total fatty acids) of saturated and monounsaturated fatty acids of primarily endogenous origin. In particular, the fetus appeared to have high Δ-9 desaturase activity as evidenced by the large amounts of 14:1n-5 (4.2%) and 16:1n-7 (37.0%) in newborn blubber compared to maternal blubber (0.2% and 14.1%, respectively). Nevertheless, essential and long-chain polyunsaturated fatty acids of the n-3 and n-6 families, which could only have originated by direct transfer from the mother, comprised>7% of pup blubber fatty acids and indicated greater rates of placental transfer than found in humans. In hooded seal mothers, rapid lipid transfer during the brief lactation period appeared to be facilitated by direct incorporation of mobilized fatty acids into milk. Although some differences in proportions of specific fatty acids were found between milk and maternal blubber, most of these differences declined over the course of lactation. However, selective mobilization of 20:5n-3 from maternal blubber into milk was apparent throughout lactation and resulted in elevated levels in pup blubber at weaning compared to maternal blubber. Ingested fatty acids were deposited directly and without modification into the blubber of pups, and by 4 days the fatty acid composition of pup blubber was virtually identical to that of the milk consumed.     

Light and electron microscopic studies on the seminal secretions and the vas deferens of the penaeiodean shrimp,Sicyonia ingentis

Unlike the other penaeiodean shrimp, the ridge back shrimp, Sicyoniaingentis does not produce a spermatophore, but transfers sperm suspended in seminal plasm. This paper reports on the histomorphology and ultrastructure of the vas deferens with reference to its functional role in secreting the sperm bearing materials. The vas deferens is divisible into proximal secretory, mid storage and distal ejaculatory regions. The epithelial cells lining the proximal vas deferens are comprised of secretory and absorptive cell types. The loose sperm cells found in the lumen of this region are in an immature condition, and are agglutinated into a compact mass with signs of spermiogenesis in the mid vas deferens. The epithelial cells lining the mid vas deferens are short flattened cells. The distal vas deferens is ensheathed by muscular fibres. The inner epithelial cells are highly secretory and contain numerous microvilli at the luminal end. The sperm cord gets liquefied in this region facilitating the transfer of sperm in liquid form to the female during mating.     

Microbiological aspects of surfactant use for biological soil remediation

Biodegradation of hydrophobic organic compounds in polluted soil is a process involving interactions among soil particles, pollutants, water, and micro-organisms. Surface-active agents or surfactants are compounds that may affect these interactions, and the use of these compounds may be a means of overcoming the problem of limited bioavailability of hydrophobic organic pollutants in biological soil remediation. The effects of surfactants on the physiology of micro-organisms range from inhibition of growth due to surfactant toxicity to stimulation of growth caused by the use of surfactants as a co-substrate. The most important effect of surfactants on the interactions among soil and pollutant is stimulation of mass transport of the pollutant from the soil to the aqueous phase. This can be caused by three different mechanisms: emulsification of liquid pollutant, micellar solubilisation, and facilitated transport. The importance of these mechanisms with respect to the effect of surfactants on bioavailability is reviewed for hydrophobic organic pollutants present in different physical states. The complexity of the effect of surfactants on pollutant bioavailability is reflected by the results in the literature, which range from stimulation to inhibition of desorption and biodegradation of polluting compounds. No general trends can be found in these results. Therefore, more research is necessary to make the application of surfactants a standard tool in biological soil remediation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Structural features of the metal binding site and dynamics of gallium putidaredoxin, a diamagnetic derivative of a Cys4Fe2S2ferredoxin

The first reconstitution of an Fe₂S₂ferredoxin with a diamagnetic prosthetic group was recently described[Kazanis et al. (1995) J. Am. Chem. Soc., 117, 6625–6626]. Thereplacement of the iron–sulfur cluster of the bacterial ferredoxinputidaredoxin (Pdx) by gallium (Ga³⁺) renders the proteindiamagnetic and permits the use of high-resolution NMR methods to identifyresonances near the metal binding site. We now describe structural featuresof the metal binding site that are not observable by standard NMR methods innative Pdx due to paramagnetic line broadening. These results provide thefirst example of high-resolution NMR-derived structural data concerning themetal binding domain of an Fe₂S₂ ferredoxin, andthe first structural information of any sort for the metal binding site of aferredoxin from this class, which includes adrenodoxin, placental ferredoxinand terpredoxin. Assignments were obtained by applying multidimensional NMRmethods to a series of selectively and nonselectively ¹⁵N- and¹³C/¹⁵N-labeled GaPdx samples. For mostexperiments, a mutant of Pdx was used in which a nonligatingCys⁸⁵ is replaced by serine. All of the major structuralfeatures that were identified in native Pdx are conserved in GaPdx. Theoverall protein dynamics is considerably faster in GaPdx than in the nativeprotein, as reflected by amide proton exchange rates. The C-terminalresidue, Trp¹⁰⁶, also exhibits considerable mobility, asindicated by ¹⁵N{¹H} NOE and ¹⁵NT₁ values of the C-terminal residue of the protein.     

Synthesis of some racemicγ-fluoro-α-amino acids

Summary Versatile three-step procedures for syntheses of seven racemi-fluoro-a-amino acids are described. Alkylation oftert-butyl N-(diphenylmethylene) glycinate with 1-bromo-2-fluoroalkanes gave N-protected aminoacid esters both in anhydrous medium using lithium-diisopropylamide as base at low temperature or in a two phase system of 50% aqueous sodium hydroxide and methylene chloride with triethylbenzylammonium chloride as the phase transfer catalyst at room temperature. Subsequent two-step deprotection with citric acid and hydrochloric acid gave the title compounds in 13–33% overall yields.Dedicated to Professor Dr.mult., Dr.h.c. Alois Haas on the occasion of his 65th birthday     

Characterization of electron transfer reactions of microperoxidase assembled at short-chain thiol-monolayers on gold

In this study thiol-monolayers were used in order to modify gold and provide suitable chemical functionalities for the immobilization of the small redox-active haem-containing peptide, microperoxidase (MP-11). Initially, we assembled a variety of thiol-containing species on the gold electrodes and measured a series of electron transfer reactions, each characteristic of the surface-modifier. Using suitable immobilization strategies, we subsequently covalently bound MP-11 to appropriate monolayers and found two characteristic electrochemical responses (i.e. using MP-11 bound to mercaptopropionic acid, redox peaks were seen at E^0′ = −315 mV and at +179 mV versus Ag|AgCl, with the former being attributed to the haem and the latter with the thiol monolayer). Exposure of the peptide-thiol surface to UV irradiation resulted in cleavage of the Au---S bond, leading to a decrease in the magnitude of both responses. Our work is supported by corroborative evidence showing the immobilization of the peptide, obtained using both X-ray photoelectron and reflectance Fourier transform infra-red spectroscopies. We hypothesize that differences in the ionic charges on the protein backbone account for the shift in E^0′ for MP-11, as observed in our system, when compared to that found for MP-11 immobilized by different strategies.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution.

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.