Strong ligand-protein interactions revealed by ultrafast infrared spectroscopy of CO in the heme pocket of the oxygen sensor FixL

In heme-based sensor proteins, ligand binding to heme in a sensor domain induces conformational changes that eventually lead to changes in enzymatic activity of an associated catalytic domain. The bacterial oxygen sensor FixL is the best-studied example of these proteins and displays marked differences in dynamic behavior with respect to model globin proteins. We report a mid-IR study of the configuration and ultrafast dynamics of CO in the distal heme pocket site of the sensor PAS domain FixLH, employing a recently developed method that provides a unique combination of high spectral resolution and range and high sensitivity. Anisotropy measurements indicate that CO rotates toward the heme plane upon dissociation, as is the case in globins. Remarkably, CO bound to the heme iron is tilted by ~30° with respect to the heme normal, which contrasts to the situation in myoglobin and in present FixLH-CO X-ray crystal structure models. This implies protein-environment-induced strain on the ligand, which is possibly at the origin of a very rapid docking-site population in a single conformation. Our observations likely explain the unusually low affinity of FixL for CO that is at the origin of the weak ligand discrimination between CO and O(2). Moreover, we observe orders of magnitude faster vibrational relaxation of dissociated CO in FixL than in globins, implying strong interactions of the ligand with the distal heme pocket environment. Finally, in the R220H FixLH mutant protein, where CO is H-bonded to a distal histidine, we demonstrate that the H-bond is maintained during photolysis. Comparison with extensively studied globin proteins unveils a surprisingly rich variety in both structural and dynamic properties of the interaction of a diatomic ligand with the ubiquitous b-type heme-proximal histidine system in different distal pockets.     

Studies on heterologous expression of Rhizobium meliloti nifA gene and oxygen sensitivity of its product

When the R. meliloti (Rm) nifA'-lacZ fusion-carrying plasmids were introduced into the strains of Agrobacterium tumefaciens, R. trifolii and R. astragalus, beta-galactosidase activity was demonstrated. However, activity was not induced by microaerobiosis. Furthermore, R. meliloti nifA'-lacZ fusion was also not expressed in the nodule bacteroids of R. trifolii and R. astragalus. We speculate that some factor(s) important for the induction of Rm nifA presumed to be the fixLJ regulatory system would not be operative in these bacteria. Experiments using R. meliloti nifH'-lacZ/K. Pneumoniae nifH'-lacZ fusion and the constitutive Rm nifA system to test the nifA-dependent expression of nifH'-lacZ under aerobic and microaerobic conditions in E. coli were performed. The inhibition of the Rm nifA activation of nifH'-lacZ expression in the bacteria grown in the aerobic condition was shown. Assays on the Rm nifA-m RNA produced by the constitutive Rm nifA in E. coli under aerobic and microaerobic conditions with the cloned nifA as a probe for dot blot hybridization showed a marked decrease of Rm nifA mRNA when the bacteria were grown under aeration.     

Assignment of the 1H and 13C NMR signals of some benzofurocoumarins

The complete 1H and 13C NMR assignment of four 6,7-benzo-fused furocoumarins (1-4) and three 3,4-benzo-fused furocoumarins (5-7) has been performed using 1D and 2D NMR techniques, including COSY, HMQC and HMBC experiments.     

Assignment of the 1H and 13C NMR signals of some hydroxyphenylcoumarins

The complete 1H and 13C NMR assignments of six hydroxyphenylcoumarins have been made using 1D and 2D NMR techniques, including COSY, HMQC and HMBC experiments.     

Quantitative analysis of cannabinoids from Cannabis sativa using 1H-NMR

A (1)H-NMR method has been developed for the quantitative analysis of pure cannabinoids and for cannabinoids present in Cannabis sativa plant material without any chromatographic purification. The experiment was performed by the analysis of singlets in the range of delta 4.0-7.0 in the (1)H-NMR spectrum, in which distinguishable signals of each cannabinoid are shown. Quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds to the known amount of the internal standard, anthracene. For this method no reference compounds are needed. It allows rapid and simple quantitation of cannabinoids with a final analysis time of only 5 min without the need for a pre-purification step.     

A role for highly conserved carboxylate, aspartate-140, in oxygen activation and heme degradation by heme oxygenase-1.

Heme oxygenase (HO) catalyzes the oxygen-dependent degradation of heme to biliverdinIXalpha, CO, and free iron ion via three sequential monooxygenase reactions. Although the distinct active-site structure of HO from cytochrome P450 families suggests unique distal protein machinery to activate molecular oxygen, the mechanism and the key amino acid for the oxygen activation have not been clear. To investigate the functionality of highly conserved polar amino acids in the distal helix of HO-1, we have prepared alanine mutants: T135A, R136A, D140A, and S142A, and found drastic changes in the heme degradation reactions of D140A. In this paper, we report the first evidence that D140 is involved in the oxygen activation mechanism in HO-1. The heme complexes of HO mutants examined in this study fold and bind heme normally. The pK(a) values of the iron-bound water and autoxidation rates of the oxy-form are increased with R136A, D140A, and S142A mutations, but are not changed with T135A mutation. As the wild-type, T135A, R136A, and S142A degrade heme to verdohemeIXalpha with H(2)O(2) and to biliverdinIXalpha with the NADPH reductase system. On the other hand, D140A heme complex forms compound II with H(2)O(2), and no heme degradation occurs. For the NADPH reductase system, the oxy-form of D140A heme complex is accumulated in the reaction, and only 50% of heme is degraded. The stopped flow experiments suggest that D140A cannot activate iron-bound dioxygen and hydroperoxide properly. To investigate the carboxylate functionality of D140, we further replaced D140 with glutamic acid (D140E), phenylalanine (D140F), and asparagine (D140N). D140E degrades heme normally, but D140N shows reactivity similar to that of D140A. D140F loses heme degradation activity completely. All of these results indicate that the carboxylate at position 140 is essential to activate the iron-bound dioxygen and hydroperoxide. On the basis of the present findings, we propose an oxygen activation mechanism involving the hydrogen-bonding network through the bridging water and D140 side chain.     

Quantitative analysis of ephedrine analogues from ephedra species using 1H-NMR

Four ephedrine analogues such as ephedrine, pseudoephedrine, methylephedrine, and methylpseudoephedrine were determined by (1)H-NMR from Ephedra species. In the region of delta 5.0-4.0, the signals of H-1 attached to the same carbon with a hydroxyl, were well separated from each other in CDCl(3). The amount of each alkaloid was calculated by the relative ratio of the intensity of H-1 signal to the known amount of internal standard, 200 microg of anthracene. This method allows rapid determination of the quantity of four ephedrine alkaloids from Ephedra species. The amount of these alkaloids was in the range of 1.0-2.0% of dry weight depending on the plant materials.     

Angular dependence of 1H-NMR and 2H-NMR spectra of water absorbed in cellulose film

¹H-NMR and ²H-NMR spectra of water (H₂O and D₂O) absorbed in Visking cellulose tubing have been observed as a function of the angle Θ between the film surface and the magnetic field. ¹H-NMR spectra show broad lines, and the chemical shift and the linewidth depend on Θ. From the angular dependence of the chemical shift, the anisotropic volume diamagnetic susceptibilities of the film are determined, i.e., χ_∥ in the direction longitudinal to the tubing (stretched direction) is 0.43 ppm, and χ_⊥ in the transverse direction is 0.57 ppm. The different values of χ_∥ and χ_⊥ afford an evidence of the anisotropy of the film. ²H-NMR spectra of D₂O absorbed in the film show quadrupole splitting which also depends on Θ. The angular dependences of the linewidth (¹H-NMR) and the quadrupole splitting (²H-NMR) indicate that the H? H axes of the water molecules have a tendency to orient in the direction longitudinal to the film surface.     

Assignment of 1H signals in complex overlapping spectral regions using proton-detected 2D heteronuclear shift correlation spectroscopy

The application of a heteronuclear 2D pulse sequence is demonstrated which correlates chemical shifts of carbons with directly bound protons and with remote protons belonging to the same coupled spin system by using the effect of Hartmann—Hahn cross polarization among protons. The enhanced spectral resolution obtained by spreading individual proton subsystems into the carbon domain and the option for multiplicity discrimination makes these kind of experiments most suitable for proton and carbon signal assignments of natural products as alkaloids and steroids with strongly overlapping proton resonances. With the application to a steroid we want to demonstrate that such unambiguous signal assignments are the prerequisite and the basis for detailed structural investigations using additional, more conventional NMR experiments.     

X-Ray and 1H-NMR Configurational Assignment of Valofan (α-Allphanoyl-α-allyl-γ-valerolactone) Diastereoisomers

The two diastereoisomers of valofan were separated by semi-preparative HPLC. The preferred solution conformation of the two diastereoisomer was established by ¹H-NMR spectroscopy. The spectra could be interpreted in confirgurational terms. The configuration of the more lipophilic isomer was ascertained bty X-ray crystallography as r-2-allophanoyl-2-allyl-t-4-methylbutyrolactone.     

Changes in the heme ligation during folding of a Geobacter sulfurreducens sensor GSU0935

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H(2)O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.     

Multiple quantum1H-NMR of ferrocene in isotropic solution

Summary Excitation of multiple quantum coherence in the C₅ symmetrical A₅-spin system of ferrocene was possible by multiexponential relaxation of degenerated NMR transitions. The mechanism of the excitation was analyzed by means of a tensor operator formalism. Symmetry selection rules were postulated for the relaxation pathways.Dedicated to Professor Dr. Karl Schlögl on occasion of his 65th birthday