Binding of imidazole and 2-methylimidazole by hemes in organic solvents. Evidence for five-coordination

By means of absorption spectroscopy we show that in benzene solutions, only one molecule of 2-methylimidazole is bound with a great affinity by deuteroheme (K = 1.25 10⁴ M^?1) and mesotetraphenylheme (K = 2.4 10⁴ M^?1). Besides, two overlapping steps may be distinguished when hemes bind imidazole molecules. The equilibrium constants are K₁ = 4.5 10³ M^?1 and 8.8 10³ M^?1, K₂ = 6.8 10⁴ M^?1 and 7.9 10⁴ M^?1 for deuteroheme and mesotetraphenylheme respectively.     

Micelle formation of diacylglycerophosphocholines in organic solvents I. effects of the solvents on krafft points

Krafft points of diacylglycerophosphocholines (PC) were measured in alkanes-cyclohexane solutions by differential scanning calorimetry, and it was found that they were regularly increased following the increase in alkane content in the solutions and the chain length of the alkanes. From these results it was deduced that the mixing of PC with alkanes occurred in the gel state of the PC, but not in micelles at higher temperatures above the Krafft points. where micellar solutions are provided. The penetration of alkanes into gel state PC was found to be dominated by Langmuir type interaction, and the affinity of alkanes increases with increasing in chain lengths. Above the Krafft points, the micelle formation was confirmed by using the fluorescence probe technique.     

Evidence for a nickel-containing carbon monoxide dehydrogenase in Methanobrevibacter arboriphilicus

In growing cultures of Methanobrevibacter arboriphilicus (Methanobrevibacter arboriphilus), the synthesis of active carbon monoxide dehydrogenase required nickel. The 21-fold-purified enzyme from 63Ni-labeled cells of M. arboriphilicus comigrated with 63Ni during gel filtration. These results provide evidence that the carbon monoxide dehydrogenase of methanogens is a nickel protein.     

Evidence for carbon monoxide binding to sickle cell polymers during melting.

The melting of sickle cell hemoglobin (HbS) polymers was induced by rapid dilution using a stopped-flow apparatus. The kinetics of polymer melting were monitored using light scattering. Polymer melting in the absence of any hemoglobin ligand was compared to melting when the diluting buffer was saturated with carbon monoxide (CO). In this way the role of CO in polymer melting could be assessed. The data were analyzed using models that assumed that melting occurs only at the ends of polymers. It was further assumed that CO could only bind to HbS in the solution phase. However, our data could not be fitted to this model, where CO cannot bind directly to the polymer. Thus, CO probably binds directly to the polymers during our melting experiments. This result is discussed in terms of oxygen induced polymer melting and polymerization processes in sickle cell disease     

Effects of 2,4-substituents of deuteroheme upon peroxidase functions. Reactions of peroxidase and myoglobin with oxygen, carbon monoxide and alkylisocyanides

The oxygen affinity of myoglobins increased in the order diacetyl- < chlorocruoro- < proto- < meso-myoglobins, which was a decreasing order of electron-withdrawing capacities of 2,4-substituents of deuteroheme. The results led to a conclusion that the π bonding predominates over the σ bonding in the myoglobin-oxygen interaction.On the contrary, the affinities of myoglobins and reduced peroxidases for ethylisocyanide decreased as the electron-withdrawing capacities of the substituents were decreased and it was therefore concluded that the σ bonding plays a dominant role in the ethylisocyanide binding to these hemoproteins.No simple relationship was observed in the cases of reduced peroxidase-CO complexes and myoglobin-isopropylisocyanide and -tertiarybutylisocyanide complexes. Among the hemoproteins tested, protohemoproteins appeared to have the lowest affinities for these ligands.As was the case with ligands such as azide and cyanide, the affinities of a deuterohemo-protein for oxygen, CO, and isocyanides were invariably much higher than the value expected from the Hammett-type relationship. The linear relationship between enthalpy and entropy was found to be applied for the reactions of ligands and hemoproteins including deutero derivatives and the irregularly high affinities of deuterohemoproteins for ligands could not be explained on the basis of thermodynamic analyses.     

Nickel requirement for carbon monoxide dehydrogenase formation in Clostridium pasteurianum

Formation of carbon monoxide dehydrogenase in growing Clostridium pasteurianum was found to be dependent on trace nickel present as contaminant in the growth medium. The evidence is: i) Synthesis of the enzyme was increased, when NiCl₂ (0.1 M) was added to the medium; ii) Synthesis of the enzyme was almost completely inhibited when the cells were grown in the presence of nitrilotriacetate (0.1 mM) or of other chelating agents, which inhibited the uptake of trace nickel from the medium; iii) Inhibition of enzyme synthesis by the chelators could be specifically overcome by supplementing the medium with nickel (1M).     

Binding of carbon monoxide to alpha-hemocyanin and beta-hemocyanin from Helix pomatia.

The binding of carbon monoxide to alpha and beta-hemocyanin from the snail Helix pomatia was studied under equilibrium conditions. Homotropic interactions upon carbon monoxide binding were much weaker than upon the binding of oxygen. Heterotropic interactions (Bohr effect and calcium-ion effect), however, were just as strong as in the case of the binding of oxygen. For alpha-hemocyanin a linkage has been observed between the binding of carbon monoxide and a change in quaternary structure of the protein.     

Large acceleration of alpha-chymotrypsin-catalyzed dipeptide formation by 18-crown-6 in organic solvents

The effects of 18-crown-6 on the synthesis of peptides catalyzed by alpha-chymotrypsin are reported. Lyophilization of the enzyme in the presence of 50 equivalents of 18-crown-6 results in a 425-fold enhanced activity when the reaction between the 2-chloroethylester of N-acetyl-L-phenylalanine and L-phenylalaninamide is carried out in acetonitrile. Addition of crown ether renders the dipeptide synthesis in nonaqueous solvents catalyzed by alpha-chymotrypsin possible on a preparative scale. The acceleration is observed in different solvents and for various peptide precursors. Copyright 1998 John Wiley & Sons, Inc.     

Synthesis of organic compounds from carbon monoxide and water by UV photolysis

The photolysis of water vapor with carbon monoxide at 1849 Å yields alcohols, aldehydes and organic acids, with an overall quantum yield of 3.3×10^–2. This rather high quantum yield could have led to a contribution of 10¹¹ organic molecules cm^–2 sec^–1 to the pool of organic material on the primitive Earth. The reactions are initiated by the photolysis of water molecules and the resulting hydrogen atoms reduce the carbon monoxide to a variety of one and two carbon compounds. The organic molecules are dissolved in water and thus escape destruction by photolysis. Photolysis of water vapor with carbon dioxide did not yield organic compounds under these conditions.     

Designing enzymes for use in organic solvents.

Enzymes are routinely used in organic solvents where numerous reactions of interest to synthetic and polymer chemists can be performed with high selectivity. Recently, it has become apparent that the catalytic properties of an enzyme can be tailored to a specific catalytic requirement by the use of solvent and protein engineering. The former involves altering the polarity, hydrophobicity, water content, etc., of the organic milieu, while the later applies site-directed mutagenesis to alter the physicochemical properties of the biocatalyst. The dominant effects of organic solvents on enzyme structure and function, and the potential of solvent and protein engineering to design enzymes to function optimally in organic media, are the major foci of this review.     

Rate of formation of carboxyhemoglobin in exercising humans exposed to carbon monoxide.

The purpose of this study was to test the CFK equation for its prediction of the rate of formation of carboxyhemoglobin (HbCO) in exercising humans by use of measured values of the respiratory variables and to characterize the rate of appearance of HbCO with frequent blood sampling. Ten nonsmoking male subjects were exposed to carbon monoxide (CO) on two separate occasions distinguished by the level of activity. Steady-state exercise was conducted on a cycle ergometer at either a low (approximately 45 W) or moderate (approximately 90 W) power output. Each experiment began with an exposure of 3,000 ppm CO for 3 min during a rest period followed by three intermittent exposures ranging from 3,000 ppm CO for 1 min at low exercise to 667 ppm CO for 3 min at moderate exercise. Increases in HbCO were normalized against predicted values to account for individual differences in the variables that govern CO uptake. No difference in the normalized uptake of CO was found between the low- and moderate-exercise trials. However, the CFK equation underpredicted the increase in HbCO for the exposures at rest and the first exposure at exercise, whereas it overpredicted for the latter two exposures at exercise. The net increase in HbCO after all exposures (approximately 10% HbCO) deviated by less than 1% HbCO between the measured and predicted values. The rate of appearance of HbCO fits a sigmoidal shape with considerable overshoot at the end of exposure. This can be explained by delays in the delivery of CO to the blood sampling point (dorsal hand vein) and by a relatively small blood circulation time compared with other regions of the body. A simple circulation model is used to demonstrate the overshoot phenomenon.     

Intracellular metabolite profiling and the evaluation of metabolite extraction solvents for Clostridium carboxidivorans fermenting carbon monoxide

Clostridium carboxidivorans ferments CO, CO₂, and H₂ via the Wood-Ljungdahl pathway. CO, CO_2, and H₂ are unique substrates, unlike other carbon sources like glucose, so it is necessary to analyze intracellular metabolite profiles for gas fermentation by C. carboxidivorans for metabolic engineering. Moreover, it is necessary to optimize the metabolite extraction solvent specifically for C. carboxidivorans fermenting syngas. In comparison with glucose media, the gas media allowed significant abundance changes of 38 and 34 metabolites in the exponential and stationary phases, respectively. Especially, C. carboxidivorans cultivated in the gas media showed changes of fatty acid metabolism and higher levels of intracellular fatty acid synthesis possibly due to cofactor imbalance and slow metabolism. Meanwhile, the evaluation of extraction solvents revealed the mixture of water-isopropanol-methanol (2:2:5, v/v/v) to be the best extraction solvent, which showed a higher extraction capability and reproducibility than pure methanol, the conventional extraction solvent. This is the first metabolomic study to demonstrate the unique intracellular metabolite profiles of the gas fermentation compared to glucose fermentation, and to evaluate water-isopropanol-methanol as the optimal metabolite extraction solvent for C. carboxidivorans on gas fermentation.     

A physiological model for predicting carboxyhemoglobin formation from exposure to carbon monoxide in rats.

A time-dependent simulation model, based on the Coburn-Forster-Kane equation, was written in Advanced Continuous Simulation Language to predict carboxyhemoglobin (HbCO) formation and dissociation in F-344 rats during and after exposure to 500 parts/million CO for 1 h. Blood-gas analysis and CO-oximetry were performed on samples collected during exposure and off-gassing of CO. Volume displacement plethysmography was used to measure minute ventilation (VE) during exposure. CO diffusing capacity in the lung (DLCO) was also measured. Other model parameters measured in the animals included blood pH, total blood volume, and Hb concentration. Comparisons between model predictions using values for VE, DLCO, and the Haldane coefficient cited in the literature and predictions using measured VE, DLCO, and calculated Haldane coefficient for individual animals were made. General model predictions using values for model parameters derived from the literature agreed with published HbCO values by a factor of 0.987 but failed to simulate experimental data. On average, the general model overpredicted measured HbCO level by nearly 9%. A specific model using the means of measured variables predicted HbCO concentration within a factor of 0.993. When experimentally observed parameter fluctuations were included, the specific model predictions reflected experimental effects on HbCO formation.     

Binding of oxygen and carbon monoxide to the hemocyanin from the spiny lobster

A high precision, two-dimensional study of oxygen and carbon monoxide binding to Panulirus interruptus hemocyanin has been carried out. Global data analysis of three types of experiments, probing the molecule in its various states of CO and O2 ligation, revealed the entire hexamer to be the basic allosteric unit involved in a two-state mechanism. The co-operativity and linkage of the two ligands are presented in terms of derivative Hill plot surfaces extended along co-ordinates of CO and O2 activities giving a detailed and comprehensive view of the binding behavior. Among the findings is an apparent high co-operativity of carbon monoxide binding at high oxygen activity. The results are discussed in view of a general mechanism for co-operative behavior found in larger hemocyanin aggregates concerning "nested" allosteric interactions.     

Enzyme function in organic solvents.

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non-aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water less than 0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface. The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits. Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering. Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry.     

Influence of carbon monoxide on metabolite formation in Methanosarcina acetivorans

Methanogenic archaea conserve energy for growth by reducing some one- and two-carbon compounds to methane and concomitantly generating an ion motive force. Growth of Methanosarcina acetivorans on carbon monoxide (CO) is peculiar as it involves formation of, besides methane, formate, acetate and methylated thiols. It has been argued that methane formation is partially inhibited under carboxidotrophic conditions and that the other products result from either detoxification of CO or from bypassing methanogenesis with other pathways for energy conservation. To gain a deeper understanding of the CO-dependent physiology of M. acetivorans we analyzed metabolite formation in resting cells. The initial rates of methane, acetate, formate, and dimethylsulfide formation increased differentially with increasing CO concentrations but were maximal already at the same moderate CO partial pressure. Strikingly, further increase of the amount of CO was not inhibitory. The maximal rate of methane formation from CO was approximately fivefold lower than that from methanol, consistent with the previously observed significant downregulation of the energy converting sodium-dependent methyltransferase. The rate of dimethylsulfide formation from CO was only 1–2% of that of methane formation under any conditions tested. Implications of the data presented for previously proposed pathways of CO utilization are discussed.     

Tyrosinase inactivation in organic solvents.

The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 x 10(3) mol product/((mol enzyme)(min)) in toluene and 8.7 x 10(3) mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 x 10(3) mol product/mol enzyme in toluene and 7.2 x 10(4) mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction.