Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor in Halobacterium halobium. We have obtained resonance Raman vibrational spectra of the native membrane-bound form of SR587 and used these data to determine the structure of its retinal prosthetic group. The similar frequencies and intensities of the skeletal fingerprint modes in SR587, bacteriorhodopsin (BR568), and halorhodopsin (HR578) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm-1) demonstrate that the retinal chromophore has an all-trans configuration. The shift of the C = N stretching mode from 1628 cm-1 in H2O to 1620 cm-1 in D2O demonstrates that the chromophore in SR587 is bound to the protein by a protonated Schiff base linkage. The small shift of the 1195 cm-1 C14-C15 stretching mode in D2O establishes that the protonated Schiff base bond has an anti configuration. The low value of the Schiff base stretching frequency together with its small 8 cm-1 shift in D2O indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion. This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.     

Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism

By elevating the pH to 9.5 in 3 M KCl, the concentration of the N intermediate in the bacteriorhodopsin photocycle has been enhanced, and time-resolved resonance Raman spectra of this intermediate have been obtained. Kinetic Raman measurements show that N appears with a half-time of 4 +/- 2 ms, which agrees satisfactorily with our measured decay time of the M412 intermediate (2 +/- 1 ms). This argues that M412 decays directly to N in the light-adapted photocycle. The configuration of the chromophore about the C13 = C14 bond was examined by regenerating the protein with [12,14-2H]retinal. The coupled C12-2H + C14-2H rock at 946 cm-1 demonstrates that the chromophore in N is 13-cis. The shift of the 1642-cm-1 Schiff base stretching mode to 1618 cm-1 in D2O indicates that the Schiff base linkage to the protein is protonated. The insensitivity of the 1168-cm-1 C14-C15 stretching mode to N-deuteriation establishes a C = N anti (trans) Schiff base configuration. The high frequency of the C14-C15 stretching mode as well as the frequency of the 966-cm-1 C14-2H-C15-2H rocking mode shows that the chromophore is 14-s-trans. Thus, N contains a 13-cis, 14-s-trans, 15-anti protonated retinal Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids.

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2',3'-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2',3'-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) AND G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloracetate, lactate, and cytosine (N-3) were obtained from 1H nuclear magnetic resonance line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: At neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole, and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7 to 9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. This leads to the conclusion that protonation of the nucleobase effects a qualitative increase in the ability of the amino protons to form hydrogen bonds: from very poor in the neutral base to "normal" in the conjugate acid.     

Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes

Legume seeds are heterotrophic and dependent on mitochondrial respiration. Due to the limited diffusional gas exchange, embryos grow in an environment of low oxygen. O(2) levels within embryo tissues were measured using microsensors and are lowest in early stages and during night, up to 0.4% of atmospheric O(2) concentration (1.1 micro M). Embryo respiration was more strongly inhibited by low O(2) during earlier than later stages. ATP content and adenylate energy charge were lowest in young embryos, whereas ethanol emission and alcohol dehydrogenase activity were high, indicating restricted ATP synthesis and fermentative metabolism. In vitro and in vivo experiments further revealed that embryo metabolism is O(2) limited. During maturation, ATP levels increased and fermentative metabolism disappeared. This indicates that embryos become adapted to the low O(2) and can adjust its energy state on a higher level. Embryos become green and photosynthetically active during differentiation. Photosynthetic O(2) production elevated the internal level up to approximately 50% of atmospheric O(2) concentration (135 micro M). Upon light conditions, embryos partitioned approximately 3-fold more [(14)C]sucrose into starch. The light-dependent increase of starch synthesis was developmentally regulated. However, steady-state levels of nucleotides, free amino acids, sugars, and glycolytic intermediates did not change upon light or dark conditions. Maturing embryos responded to low O(2) supply by adjusting metabolic fluxes rather than the steady-state levels of metabolites. We conclude that embryogenic photosynthesis increases biosynthetic fluxes probably by providing O(2) and energy that is readily used for biosynthesis and respiration.     

Pathways of inclusion of isotopes 2H and 13C into exometabolites in course of glucose utilization by medusomycete]

The inclusion of 2H and 13C isotopes into the products of glucose utilization by medusomycete during its growth on deuterated media was studied by high-resolution NMR spectroscopy. Both unlabeled and 13C-labeled (in positions 1, 2, 6) glucose was used. It was shown that the glucose utilization proceeds by the classical Embden-Meyerhof-Parnas pathway. The incorporation of deuterium to the methyl group of ethanol can occur only during glucose-fructose-6-phosphate and phosphoenolpyruvate--pyruvate conversion. None of these stages by themselves is responsible for the existing distribution of deuterium atoms. The maximum inclusion of deuterium to the methyl group is no more than two atoms for the first glucose fragment (C1-C2-C3) and no more than one, for the second fragment (C4-C5-C6). The methylene group of ethanol is more accessible for deuterons because the proton surroundings of carbon atoms C2 and C5 completely changes. It was concluded that the maximum proton exchange occurs at positions C2 and C5; at positions C1, the proton exchange is lesser, and at position C6 it is the least. It was also shown that about 10% C1-C3 of triose leave the glycolysis cycle and are used in other processes.     

Limitation of deuterium labelled methoximes as internal standards in the mass spectral analysis of prostaglandins

A reported method for the preparation of d3-methoxime derivatives as internal standards for prostaglandin assays by gas chromatography-mass spectrometry was evaluated. Sample derivatization resulted in 1.5-86% exchange of the d3-methoxime in a series of prostaglandins. Exchange was minimal when the methoxime was on the 5-membered ring; whereas, acyclic methoximes exhibited extensive exchange. Induced strain energy due to the steric interaction of the hydroxyl group and the C13-C20 alkyl side chain with the gem-dimethoxylamine transition state is offered as an explanation for the unusual stability of PGE2. The use of 18O exchange of the carboxylic acid function is presented as an alternative for the preparation of unavailable labelled eicosanoids.     

Structure of the retinal chromophore in 7,9-dicis-rhodopsin

Bovine rhodopsin was bleached and regenerated with 7,9-dicis-retinal to form 7,9-dicis-rhodopsin, which was purified on a concanavalin A affinity column. The absorption maximum of the 7,9-dicis pigment is 453 nm, giving an opsin shift of 1600 cm-1 compared to 2500 cm-1 for 11-cis-rhodopsin and 2400 cm-1 for 9-cis-rhodopsin. Rapid-flow resonance Raman spectra have been obtained of 7,9-dicis-rhodopsin in H2O and D2O at room temperature. The shift of the 1654-cm-1 C = N stretch to 1627 cm-1 in D2O demonstrates that the Schiff base nitrogen is protonated. The absence of any shift in the 1201-cm-1 mode, which is assigned as the C14-C15 stretch, or of any other C-C stretching modes in D2O indicates that the Schiff base C = N configuration is trans (anti). Assuming that the cyclohexenyl ring binds with the same orientation in 7,9-dicis-, 9-cis-, and 11-cis-rhodopsins, the presence of two cis bonds requires that the N-H bond of the 7,9-dicis chromophore points in the opposite direction from that in the 9-cis or 11-cis pigment. However, the Schiff base C = NH+ stretching frequency and its D2O shift in 7,9-dicis-rhodopsin are very similar to those in 11-cis- and 9-cis-rhodopsin, indicating that the Schiff base electrostatic/hydrogen-bonding environments are effectively the same. The C = N trans (anti) Schiff base geometry of 7,9-dicis-rhodopsin and the insensitivity of its Schiff base vibrational properties to orientation are rationalized by examining the binding site specificity with molecular modeling.     

Resonance Raman spectra of the pyridoxal coenzyme in aspartate aminotransferase. Evidence for pyridine protonation and a novel photochemical H/D exchange at the imine carbon atom

Resonance Raman (RR) spectra are reported for aspartate aminotransferase from pig heart cytosol, and for inhibitor complexes. They are interpreted with reference to the previously analyzed spectra of pyridoxal phosphate (PLP) Schiff base adducts. This comparison shows that, as expected, the pyridine N atom is protonated in the native enzyme at pH 5, and in the glutarate complexes at pH 8.5, and that it is also protonated in the alpha-methylaspartate complex; the stabilization of the pyridine proton at high pH must be due to the interaction with aspartate 222 seen in the x-ray crystal structure. RR spectra of the erythro-beta-hydroxy-DL-aspartate complex, representing the p-quinoid enzyme intermediate, as well as of AlIII complexes of PLP Schiff bases with phenylalanine and tyrosine ethyl ester have been obtained via the coherent anti-Stokes Raman scattering technique, and partially assigned. A novel H/D exchange at the coenzyme C4' atom has been observed for the native enzyme in D2O, and has been determined, by a combination of NMR and RR measurements, to be due to the Raman laser irradiation. This photoprocess, which is not observed for PLP Schiff bases in aqueous solution, is attributed to a photoexcited p-quinoid intermediate, similar to that implicated in the enzyme mechanism. It is suggested that this intermediate is stabilized by protein interactions which localize charge on the phenolate O atom, plausibly a hydrogen bond from the nearby tyrosine 225. H/D exchange would then follow via the aldimine-ketimine interconversion known to take place in the enzyme reaction.     

Variations in the natural abundance of oxygen-18 and deuterium in plant carbohydrates

Variations in the natural abundance of ¹⁸O and ²H in plant cellulose are influenced by the isotopic composition of the water directly involved in metabolism—the metabolic water fraction. The isotopic distinction between the metabolic source water and total tissue water must reflect the formation of isotopic gradients within the tissue that are influenced by the rate of water turnover, by properties of the water conducting system and by environmental conditions. It seems that the ¹⁸O abundance in the metabolic water is conserved in cellulose with a relatively constant isotope effect. The relationship of the ²H abundance between metabolic water and cellulose is more complex. Hydrogen incorporated into photosynthetic products during primary reduction steps is highly depleted in ²H. However, a large proportion of these hydrogens are subsequently replaced by exchange with water, leading to ²H enrichment during heterotrophic metabolism. Deciphering the oxygen isotope ratio of cellulose could help in providing insights into the carbon and oxygen fluxes exchanged between plants and the atmosphere. This is because the ¹⁸O abundance in cellulose records the ¹⁸O abundance in the metabolic water, which in turn, controls the oxygen isotopic signatures of the CO₂ and O₂ released by plants into the atmosphere. The hydrogen isotope effects associated with carbohydrate metabolism provide insights into the autotrophic state of a plant tissue. This is because the hydrogen isotope ratio of carbohydrates must reflect the net effects of the two opposing isotope effects associated with photosynthesis and heterotrophic metabolism.     

Towards an explanation of carboxylation/oxygenation bifunctionality in Rubisco. Transition structure for the carboxylation reaction of 2,3,4-pentanetriol

We construct a theoretical model of the transition structure for the carboxylation reaction of ribulose-1,5-biphosphate catalyzed by Rubisco. This is a first-order saddle point on the energy hypersurface for the nucleophilic attack of carbon dioxide on CH₃-(CHOH)₃-CH₃ at the C2 center.Ab initio analytical gradients methods at a 4-31G basis set level are used.The carbon framework and oxygens of the stationary structure superpose with the corresponding atoms of 2-carboxyarabinitol-1,5-biphosphate, which is a transition state analog that has recently been highly refined with X-ray methods. The hydroxyl group in C3 iscis to the C2 oxygen. The C3 center is somewhat pyramidized, the dienol O2-C2-C3-O3 is not planar.The geometry of the transition state allows for simple explanations of both the enolization of Rubisco's substrate ribulose-1,5-biphosphate, O₃PO-CH2-CO-(CHOH)₂-CH₂-OPO₃ and oxygenation reaction. The former is due to the pyramidal deformation at C3 and out of plane of O2-C2-C3-O3 framework: the enoliation is intramolecular and is probably enhanced by proton tunnelling. The latter is related with the fact that a rotation around an ethylene-like bond brings the triplet state down in energy. The reactive skeleton has a stationary geometry in the triplet state not very different from the one obtained in the global transition structure. There, the triplet is only 9 kcal/mol above the singlet. The spin densities at C2 and C3 centers clearly indicate the place where oxygenation will take place.     

Polarized Fourier transform infrared spectroscopy of bacteriorhodopsin. Transmembrane alpha helices are resistant to hydrogen/deuterium exchange.

The secondary structure of bacteriorhodopsin has been investigated by polarized Fourier transform infrared spectroscopy combined with hydrogen/deuterium exchange, isotope labeling and resolution enhancement methods. Oriented films of purple membrane were measured at low temperature after exposure to H2O or D2O. Resolution enhancement techniques and isotopic labeling of the Schiff base were used to assign peaks in the amide I region of the spectrum. alpha-helical structure, which exhibits strong infrared dichroism, undergoes little H/D exchange, even after 48 h of D2O exposure. In contrast, non-alpha-helical structure, which exhibits little dichroism, undergoes rapid H/D exchange. A band at 1,640 cm-1, which has previously been assigned to beta-sheet structure, is found to be due in part to the C = N stretching vibration of protonated Schiff base of the retinylidene chromophore. We conclude that the membrane spanning regions of bR consist predominantly of alpha-helical structure whereas most beta-type structure is located in surface regions directly accessible to water.     

Metabolic physiology of alcohol degradation and adaptation in Drosophila larvae as studied by means of carbon-13 nuclear magnetic resonance spectroscopy

Alcohol dehydrogenase-mediated degradation of [2-¹³C]ethanol was followed in third instar larvae of Drosophila by means of ¹³C NMR. The tricarboxylic acid (TCA) cycle intermediates, citrate-C(2),4 and succinate-C2,3; the amino acids, glutamate-C4,3,2, glutamine-C4,3,2, proline-C4, alanine-C2,3 and the carbon nuclei of the glucosyl units of the disaccharide, α,α-trehalose, were intensely labeled in perchloric acid extracts of whole larvae. A model of the intermediary metabolism of ethanol degradation in larvae was formulated from these observations. The C2 atom of ethanol enters the mitochondrial TCA cycle as C2-acetyl-CoA and is converted into the TCA cycle intermediates. The TCA cycle intermediate 2-oxoglutarate(-C4) apparently is readily converted into glutamate(-C4) and subsequently to glutamine(-C4) and proline(-C4). Dietary ethanol is also a substrate for trehalose synthesis. This may occur by an exchange of malate(-C2,3) between its mitochondrial and cytosolic pools. Part of the cytosolic malate(-C2,3) may be diverted into pyruvate then generating alanine(-C2,3) as another product. The other part may be converted into glucose and subsequently into α,α-trehalose by the gluconeogenic pathway. ¹³C natural abundance signals of stored fatty acids and glycerol were readily detectable in chloroform extracts of control larvae. De novo synthesis of fatty acids from labeled ethanol also occurred after a lag period. Our findings show the coordinated nature of metabolic pathways, and we point to its consequences in understanding the dynamics in evolutionary processes.     

X-ray crystallographic conformational study of 5'-O-[N-(L-alanyl)-sulfamoyl]adenosine, a substrate analogue for alanyl-tRNA synthetase

In order to elucidate the substrate specificity of alanyl-tRNA synthetase, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (Ala-SA), an analogue of alanyl-AMP, was chemically synthesized. Its binding ability is similar to that of the substrate based on the inhibitory activity for the aminoacylation of alanyl-tRNA synthetase. Taking advantage of the stable sulfamoyl bond of Ala-Sa, compared with the highly labile aminoacyl bond of alanyl-AMP, the molecular conformation of the former inhibitor was studied by X-ray single crystal analysis. Crystal data are as follows: C13H19N7O7S.2H2O, space group C2, a = 39.620(6), b = 5.757(1), c = 20.040(3) A, beta = 117.2(1) degrees, V = 4065(9) A3, Z = 8, and final R = 0.065 for 2785 independent reflections of F(2)0 greater than or equal to 2 sigma (F0)2. In the crystal, the molecule is in a zwitterionic state with the terminal amino group protonated and sulfamoyl group deprotonated, and takes an open conformation, where the L-alanine moiety is located far from the adenosine moiety with gauche/trans and trans orientations about the exocyclic C(4')-C(5') and C(5')-O(5') bonds, respectively. The conformation of the adenosine moiety is anti for the glycosyl bond and C(3')-endo for the ribose puckering, and alanine is in the usually observed trans region for the psi torsion angle. The molecular dimensions of the sulfamoyl group are nearly the same as those of the phosphate group. The biological significance of the observed Ala-SA conformation is discussed in relation with the molecular conformation of tyrosyl-AMP complexed with tyrosyl-tRNA synthetase.     

Quantitation of positional isomers of deuterium-labeled glucose by gas chromatography/mass spectrometry.

A method for determining the site and extent of deuterium (D) labeling of glucose by GC/MS and mass fragmentography was developed. Under chemical and electron impact ionization, ion clusters m/z 328, 242, 217, 212, and 187 of glucose aldonitrile pentaacetate and m/z 331 and 169 of pentaacetate derivative were produced. From the mass spectra of 13C- and D-labeled reference compounds, glucose carbon and hydrogen (C-H) positions included in these fragments were deduced to be m/z 328 = C1-C6, 2,3,4,5,6,6-H6; m/z 331 = C1-C6, 1,2,3,4,5,6,6-H7; m/z 169 = C1-C6, 1,3,4,5,6,6-H6; m/z 187 = C3-C6, 3,4,5,6,6-H5; m/z 212 = C1-C5, 2,3,4,5-H4; m/z 217 = C4-C6, 4,5,6,6-H4; and m/z 242 = C1-C4, 2,3,4-H3. After correction for isotope discrimination and deuterium-hydrogen exchange, the D enrichment of these fragments can be quantitated using selective ion monitoring, and the D enrichment of all C-H positions can be obtained by the difference in enrichment of the corresponding ion pairs. The validity of this approach was tested by examining D enrichment of known mixtures of 1-d1-, 2-d1-, 3-d1-, and 5,6,6-d3-glucose with unlabeled glucose and D enrichment of perdeuterated glucose using these fragments. This method was used to determine deuterium incorporation in C1 through C6 of blood glucose in fasted (24 h) rats infused with deuterated water. The distribution of deuterium was similar to that found by Postle and Bloxham (1980, Biochem. J. 192, 65-73). Approximately one deuterium atom was incorporated into C5 and only 75% deuterium atom was incorporated into C2. The enrichment of C2 and C6 of glucose relative to that of water indicated that 74 +/- 9% of plasma glucose was newly formed 4 h after the onset of deuterium infusion, and gluconeogenesis accounted for about 76 +/- 7% of the glucose 6-phosphate flux.     

Rates of oxygen and hydrogen exchange as indicators of TPQ cofactor orientation in amine oxidases.

This study presents the first detailed examination by resonance Raman (RR) spectroscopy of the rates of solvent exchange for the C5 and C3 positions of the TPQ cofactor in several wild-type copper-containing amine oxidases and mutants of the amine oxidase from Hansenula polymorpha (HPAO). On the basis of crystal structure analysis and differing rates of C5 [double bond] O and C3 [bond] H exchange within the enzyme systems, but equally rapid rates of C5 [double bond] O and C3 [bond] H exchange in a TPQ model compound, it is proposed that these data can be used to determine the TPQ cofactor orientation within the active site of the resting enzyme. A rapid rate of C5 [double bond] O exchange (t(1/2) < 30 min) and a slow (t(1/2) = 6 h) to nonexistent rate of C3 [bond] H exchange was observed for wild-type HPAO, the amine oxidase from Arthrobacter globiformis, pea seedling amine oxidase at pH 7.1, and the E406Q mutant of HPAO. This pattern is ascribed to a productive TPQ orientation, with the C5 [double bond] O near the substrate-binding site and the C3 [bond] H near the Cu. In contrast, a slow rate of C5 [double bond] O exchange (t(1/2) = 1.6-3.3 h) coupled with a fast rate of C3 [bond] H exchange (t(1/2) < 30 min) was observed for the D319E and D319N catalytic base mutants of HPAO and for PSAO at pH 4.6 (t(1/2) = 4.5 h for C5 [double bond] O exchange). This pattern identifies a flipped orientation, involving 180 degrees rotation about the C alpha-C beta bond, which locates the C3 [bond] H near the substrate-binding site and the C5 double bond] O near the Cu. Finally, fast rates of both C5 [double bond] O and C3 [bond] H exchange (t(1/2) < 30 min) were observed for the amine oxidase from Escherichia coli and the N404A mutant of HPAO, suggesting a mobile cofactor, with multiple TPQ orientations between productive and flipped. These results demonstrate that opposing sides of the TPQ ring possess different degrees of solvent accessibility and that the rates of C5 [double bond] O and C3 [bond] H exchange can be used to predict the TPQ cofactor orientation in the resting forms of these enzymes.     

Pharmacological uses and perspectives of heavy water and deuterated compounds.

Since the discovery of D20 (heavy water) and its use as a moderator in nuclear reactors, its biological effects have been extensively, although seldom deeply, studied. This article reviews these effects on whole animals, animal cells, and microorganisms. Both "solvent isotope effects," those due to the special properties of D20 as a solvent, and "deuterium isotope effects" (DIE), which result when D replaces H in many biological molecules, are considered. The low toxicity of D20 toward mammals is reflected in its widespread use for measuring water spaces in humans and other animals. Higher concentrations (usually >20% of body weight) can be toxic to animals and animal cells. Effects on the nervous system and the liver and on formation of different blood cells have been noted. At the cellular level, D20 may affect mitosis and membrane function. Protozoa are able to withstand up to 70% D20. Algae and bacteria can adapt to grow in 100% D2O and can serve as sources of a large number of deuterated molecules. D2O increases heat stability of macromolecules but may decrease cellular heat stability, possibly as a result of inhibition of chaperonin formation. High D2O concentrations can reduce salt- and ethanol-induced hypertension in rats and protect mice from gamma irradation. Such concentrations are also used in boron neutron capture therapy to increase neutron penetration to boron compounds bound to malignant cells. D2O is more toxic to malignant than normal animal cells, but at concentrations too high for regular therapeutic use. D2O and deuterated drugs are widely used in studies of metabolism of drugs and toxic substances in humans and other animals. The deuterated forms of drugs often have different actions than the protonated forms. Some deuterated drugs show different transport processes. Most are more resistant to metabolic changes, especially those changes mediated by cytochrome P450 systems. Deuteration may also change the pathway of drug metabolism (metabolic switching). Changed metabolism may lead to increased duration of action and lower toxicity. It may also lead to lower activity, if the drug is normally changed to the active form in vivo. Deuteration can also lower the genotoxicity of the anticancer drug tamoxifen and other compounds. Deuteration increases effectiveness of long-chain fatty acids and fluoro-D-phenylalanine by preventing their breakdown by target microorganisms. A few deuterated antibiotics have been prepared, and their antimicrobial activity was found to be little changed. Their action on resistant bacteria has not been studied, but there is no reason to believe that they would be more effective against such bacteria. Insect resistance to insecticides is very often due to insecticide destruction through the cytochrome P450 system. Deuterated insecticides might well be more effective against resistant insects, but this potentially valuable possibility has not yet been studied.     

Resonance Raman study on intact pea phytochrome and its model compounds: evidence for proton migration during the phototransformation.

Resonance Raman (RR) scattering from intact pea phytochrome was observed in resonance with the blue band at ambient temperature. The relative populations of the red-light-absorbing form (Pr) and far-red-light-absorbing form (Pfr) under laser illumination were estimated from the absorption spectra. The most prominent RR band of Pr obtained by 364-nm excitation under 740-nm pumping exhibited a frequency shift between H2O and D2O solutions, but that of Pfr obtained by 407-nm excitation under 633-nm pumping did not, indicating a distinct difference in a protonation state of their chromophores. Since the protonation level of a whole molecule of intact phytochrome remains unchanged between Pr and Pfr, this observation indicates migration of a proton from the chromophore of Pr to the protein moiety of Pfr. As model compounds, octaethylbiliverdin (OEBV-h3), its deuterated and 15N derivatives, and their protonated forms were also studied with both RR and 1H and 15N NMR spectroscopies. The RR spectrum of the protonated form, for which the protonation site was determined to be C-ring pyrrole nitrogen by NMR, displayed a deuteration shift corresponding to that of Pr, suggesting a similar protonated structure for the pyrrolic rings of Pr. The RR spectral difference between OEBV-h3 and OEBV-d3 and that between H2O and D2O solutions of Pfr suggested that the N-H protons of the A-, B-, and D-rings of intact phytochrome are replaced with deuterons in D2O. A role of the 7-kDa segment of phytochrome is discussed on the basis of RR spectral differences between the intact and large phytochromes.     

Direct evidence for the protonation of aspartate-75, proposed to be at a quinol binding site, upon reduction of cytochrome bo3 from Escherichia coli

Aspartate-75 (D75) was recently suggested to participate in a ubiquinone-binding site in subunit I of cytochrome bo(3) from Escherichia coli on the basis of a structural model [Abramson, J., Riistama, S., Larsson, G., Jasaitis, A., Svensson-Ek, M., Laakkonen, L., Puustinen, A., Iwata, S., and Wikstr?m, M. (2000) Nat. Struct. Biol. 7 (10), 910-917]. We studied the protonation state of D75 for the reduced and oxidized forms of the enzyme, using a combined site-directed mutagenesis, electrochemical, and FTIR spectroscopic approach. The D75H mutant is catalytically inactive, whereas the more conservative D75E substitution has quinol oxidase activity equal to that of the wild-type enzyme. Electrochemically induced FTIR difference spectra of the inactive D75H mutant enzyme show a clear decrease in the spectroscopic region characteristic of protonated aspartates and glutamates. Strong variations in the amide I region of the FTIR difference spectrum, however, reflect a more general perturbation due to this mutation of both the protein and the bound quinone. Electrochemically induced FTIR difference spectra on the highly conservative D75E mutant enzyme show a shift from 1734 to 1750 cm(-1) in direct comparison to wild type. After H/D exchange, the mode at 1750 cm(-1) shifts to 1735 cm(-1). These modes, concomitant with the reduced state of the enzyme, can be assigned to the nu(C=O) vibrational mode of protonated D75 and E75, respectively. In the spectroscopic region where signals for deprotonated acidic groups are expected, band shifts for the nu(COO(-))(s/as) modes from 1563 to 1554-1539 cm(-1) and from 1315 to 1336 cm(-1), respectively, are found for the oxidized enzyme. These signals indicate that D75 (or E75 in the mutant) is deprotonated in the oxidized form of cytochrome bo(3) and is protonated upon full reduction of the enzyme. It is suggested that upon reduction of the bound ubiquinone at the high affinity site, D75 takes up a proton, possibly sharing it with ubiquinol.     

Synthesis of chitosan-based polymeric surfactants and their adsorption properties for heavy metals and fatty acids

Chitosan-based polymeric surfactants (CBPSs) were prepared by N-acylation of chitosans (chitosan 10 and 500) with several acid anhydrides such as hexanoic (C6), lauric (C12), and palmitic (C16) anhydrides. Among the CBPS samples, CBPSs having a good solubility at pH 4.0 were selected and observed for viscosity, surface tension, and adsorption of heavy metals (Cd2+, Co2+, Cr2O7(2-), and Pb2+) as well as the fatty acid (n-octanoic acid). The 1H NMR spectrum of chitosan 10 modified with C16 at the substitution ratio of 0.4 (CBPS10-C16,0.4) showed 85% of acylation in 1% DCl/D2O solutions. CBPS10 with the substitution ratio less than 0.4 showed a good solubility because of shorter repeating units and lesser amounts of hydrophobic substituents. The intrinsic viscosity of CBPS10 was slightly increased, while that of CBPS500 was decreased. As the substitution ratio and length of the carbon chain increased, the surface tension of CBPS10 tended to decrease. CBPS10-C16,0.2 had high adsorption ability for cationic metal ions such as Cd2+, Co2+, and Pb2+ comparable to chitosan. Interestingly, CBPS(10)-C(16,0.2) showed a unique pH optimum for the anionic metal ion such as Cr2O7(2-). In addition, CBPS10-C16,0.2 exhibited the highest adsorption ability for n-octanoic acid among the tested CBPS10 with different carbon chains.     

Effects of hyperoxia on the metabolic response to cold of the newborn rat.

We asked what effects hyperoxia may have on the metabolic response to cold of the newborn rat. Whole body gaseous metabolism (VO2 and VCO2) was measured in 2-day old rats by open flow respirometry at ambient temperatures (Tamb) between 40 and 20 degrees C, changed at a rate of 0.5 degrees C/min during normoxia and hyperoxia (100% O2 breathing). In normoxia, the thermoneutral range was very narrow, at Tamb = 33-35 degrees C. A decrease in Tamb at first stimulated VO2; a further drop in Tamb below 28 degrees C reduced metabolic rate. The metabolic response to cold was not sufficient to maintain body temperature (Tb). In hyperoxia average values of VO2 were above the normoxic values at all Tamb, but the difference was mostly apparent at low Tamb; at 20 degrees C, hyperoxic VO2 averaged 73% more than in normoxia. This metabolic increase determined a significant but small rise of Tb. We conclude that in the 2-days-old rat hyperoxia has a stimulatory effect on metabolism which is Tamb-dependent, being much more apparent in the cold. This supports the concept that the normoxic VO2 of the newborn is limited by the supply of O2. However, the fact that in the cold, even in hyperoxia, VO2 did not reach very high values, and Tb was not maintained, suggests that not only O2 availability, but also the rate of O2 utilization limits the aerobic metabolic response of the newborn.