Metallobleomycins: electron spin resonance study on nitrosyl complex of iron(II)-Bleomycin

The ESR spectrum of the bleomycin-Fe(II)NO complex shows rhombic symmetry with a triplet hyperfine interaction in the g_z signal, and its ESR parameters have been compared with those of the ferrous-NO complexes of hemoproteins. The substitution of ₁₄NO by ₁₅NO gives the transition from a triplet to a doublet in the g_z absorption with a concomitant change in the nitrogen hyperfine constant. The addition of DNA to the ferrous-NO complex of bleomycin induces the greater separation of the g_x and g_y absorptions in comparison with the original ESR spectrum. The present three-line g_z signal for the bleomycin-Fe(II)-NO complex is indicative of weakened fifth axial nitrogen ligand-to-iron bonding with concomitantly stronger NO-to-iron bonding. On the other hand, the ESR feature of the bleomycin-Fe(III) complex is typical of the rhombic low-spin type, and no stable ferric-NO complex of bleomycin is formed.     

Multinuclear nuclear magnetic resonance and X-ray crystallographic investigation of some mixed ligand alkylisocyanide platinum(II) complexes

Single crystal X-ray diffraction structure determination of tetra-n-butylammonium tricyanomethylisocyanideplatinate(II) (1) show that the complex does not feature stacking of the anions or significant Pt-Pt orbital interactions. The cis-dicyanobismethylisocyanideplatinum(II) (2) and cis-dicyanobisethylisocyanideplatinum(II) (3) complexes do crystallize with the platinum atoms collinear with one another but with a Pt-Pt separation distance on the order of 3.5 Å, which is too great for significant orbital overlap. In each of the complexes studied, the Pt-CNR bond lengths of the isocyanides are shorter than the Pt-CN bond lengths of the cyanide ligands. Additionally, each of these complexes have Pt-CNR bond distances marginally shorter than in the parent complex, [Pt(CNR)₄][BF₄]₂ (5). The shortened Pt-CNR distances in the mixed complexes are consistent with the isocyanide ligand being a stronger π-acid than the cyanide ligand, resulting in a preferred cis configuration of the mixed ligand complexes. In solution, the NMR spectra of these complexes are unusual because they display ¹⁹⁵Pt-¹⁴N and ¹H-¹⁴N coupling with high resolution. The NMR parameters of these complexes are compared with those of and (R = CH₃ or C₂H₅).     

Construction of hydrodynamic bead models from high-resolution X-ray crystallographic or nuclear magnetic resonance data.

Computer software such as HYDRO, based upon a comprehensive body of theoretical work, permits the hydrodynamic modeling of macromolecules in solution, which are represented to the computer interface as an assembly of spheres. The uniqueness of any satisfactory resultant model is optimized by incorporating into the modeling procedure the maximal possible number of criteria to which the bead model must conform. An algorithm (AtoB, for atoms to beads) that permits the direct construction of bead models from high resolution x-ray crystallographic or nuclear magnetic resonance data has now been formulated and tested. Models so generated then act as informed starting estimates for the subsequent iterative modeling procedure, thereby hastening the convergence to reasonable representations of solution conformation. Successful application of this algorithm to several proteins shows that predictions of hydrodynamic parameters, including those concerning solvation, can be confirmed.     

Hydrogen-1 nuclear magnetic resonance of the nitrogenase iron protein (Cp2) from Clostridium pasteurianum

Proton NMR spectra (250 MHz) of the nitrogenase iron protein from Clostridium pasteurianum (Cp2) were found to display 9 or 10 paramagnetically shifted resonances in the 15-50 ppm range. The most shifted resonances belonged to two approximately equal subsets having temperature dependences of opposite sign. The latter occurrence is consistent with the interaction of the corresponding protons with an antiferromagnetically coupled metal center. The number of proton resonances of Cp2, their positions, and their temperature dependences were similar to those observed in spectra of (4Fe-4S)+ ferredoxins, particularly those of the latter that contain a single tetranuclear cluster, such as the ferredoxin from Bacillus stearothermophilus. The effects of several adenine nucleotides on the paramagnetically shifted proton resonances of Cp2 have been investigated. Whereas MgAMP had no effect at all, MgADP and MgATP were found to induce different modifications, which in both cases involved approximately half only of the shifted proton resonances. These data suggest that nucleotide binding affects mainly one part of the iron-sulfur cluster. A remarkable feature of the spectra of Cp2 in the presence of MgATP is the grouping of the shifted proton resonances in sets of two or four having identical chemical shifts and temperature dependences. A nearly perfect 2-fold symmetry is thus suggested for the arrangement of the cysteine protons around the active site. These observations lend support to the proposal that the (4Fe-4S) cluster is held symmetrically between the two identical subunits and are consistent with the existence of two MgATP binding sites on nitrogenase iron proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance and spectrophotometric studies of nickel(II)-iron(II) hybrid hemoglobins

Ni(II)-Fe(II) hybrid hemoglobins, alpha(Fe)2 beta(Ni)2 and alpha(Ni)2 beta(Fe)2 have been characterized by proton nuclear magnetic resonance with Ni(II) protoporphyrin IX (Ni-PP) incorporated in apoprotein, which serves as a permanent deoxyheme. alpha(Fe)2 beta(Ni)2, alpha(Ni)2 beta(Fe)2, and NiHb commonly show exchangeable proton resonances at 11 and 14 ppm, due to hydrogen-bonded protons in a deoxy-like structure. Upon binding of carbon monoxide (CO) to alpha(Fe)2 beta(Ni)2, these resonances disappear at pH 6.5 to pH 8.5. On the other hand, the complementary hybrid alpha(Ni)2 beta(Fe-CO)2 showed the 11 and 14 ppm resonances at low pH. Upon raising pH, the intensities of both resonances are reduced, although these changes are not synchronized. Electronic absorption spectra and hyperfine-shifted proton resonances indicate that the ligation of CO in the beta(Fe) subunits induced changes in the coordination and spin states of Ni-PP in the alpha subunits. In a deoxy-like structure, the coordination of Ni-PP in the alpha subunits is predominantly in a low-spin (S = 0) four-coordination state, whereas in an oxy-like structure the contribution of a high-spin (S = 1) five-coordination state markedly increased. Ni-PP in the beta subunits always takes a high-spin five-coordination state regardless of solution conditions and the state of ligation in the partner alpha(Fe) subunits. In the beta(Ni) subunits, a significant downfield shift of the proximal histidyl N delta H resonance and a change in the absorption spectrum of Ni-PP were detected, upon changing the quaternary structure of the hybrid.(ABSTRACT TRUNCATED AT 250 WORDS)     

113Cd nuclear magnetic resonance of Cd(II) alkaline phosphatases

113Cd NMR spectra of 113Cd(II)-substituted Escherichia coli alkaline phosphatase have been recorded over a range of pH values, levels of metal site occupancy, and states of phosphorylation. Under all conditions resonances attributable to cadmium specifically bound at one or more of the three pairs of metal-binding sites (A, B, and C sites) are detected. By following changes in both the 113Cd and 31P NMR spectra of 113Cd(II)2 alkaline phosphatase during and after phosphorylation, it has been possible to assign the cadmium resonance that occurs between 140 and 170 ppm to Cd(II) bound to the A or catalytic site of the enzyme and the resonance occurring between 51 and 76 ppm to Cd(II) bound to B site, which from x-ray data is located 3.9 A from the A site. The kinetics of phosphorylation show that cadmium migration from the A site of one subunit to the B site of the second subunit follows and is a consequence of phosphate binding, thus precluding the migration as a sufficient explanation for half-of-the-sites reactivity. Rather, there is evidence for subunit-subunit interaction rendering the phosphate binding sites inequivalent. Although one metal ion, at A site, is sufficient for phosphate binding and phosphorylation, the presence of a second metal ion at B site greatly enhances the rate of phosphorylation. In the absence of phosphate, occupation of the lower affinity B and C sites produces exchange broadening of the cadmium resonances. Phosphorylation abolishes this exchange modulation. Magnesium at high concentration broadens the resonances to the point of undetectability. The chemical shift of 113Cd(II) in both A and B sites (but not C site) is different depending on the state of the bound phosphate (whether covalently or noncovalently bound) and gives separate resonances for each form. Care must be taken in attributing the initial distribution of cadmium or phosphate in the reconstituted enzyme to that of the equilibrium species in samples reconstituted from apoenzyme. Both 113Cd NMR and 31P NMR show that some conformational changes consequent to metal ion or phosphate binding require several days before the final equilibrium species is formed.     

Studies on nitrosyl hemes in Ni(II)–Fe(II) hybrid hemoglobins

Subunit heterogeneity within a particular subunit in hemoglobin A have been explored with electron paramagnetic resonance spectroscopy using the nitrosyl hemes in Ni-Fe hybrid Hb under various solution conditions. Our previous studies on the crystal structure of NiHb demonstrated the presence of subunit heterogeneity within alpha-subunit. To further cross check this hypothesis, we made a hybrid Hb in which either the alpha- or beta-subunit contains iron, which alone can bind to NO. By this way dynamic exchange between penta- and hexa-coordinated forms within a subunit was confirmed. Upon the addition of inositol hexa phosphate (IHP) to these hybrids, R to T state transition is observed for [alpha(2)(Fe-NO)beta(2)(Ni)] but such a direct transformation is less marked in [alpha(2)(Ni)beta(2)(Fe-NO)]. Hence the bond between N(epsilon) and Fe is fundamental to the structure-function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) porphyrin compounds

1. There was no apparent correlation between the rate of respiration and rate of accumulation of proline in Candida albicans cells. 2. In contrast to normal cells, the respiration in the starved cells became completely cyanide insensitive. The starvation of cells in the presence of cycloheximide prevented the cells from becoming cyanide insensitive. The addition of Fe(III), however, accelerated the process. 3. Oxidizable substrates e.g. NADH, acetate and glucose, when added to cyanide-insensitive starved cells, exhibited 40--280% stimulation in respiration rate. However, this enhancement in oxidation by various substrates was not coupled to a simultaneous increase in the proline uptake or in intracellular ATP levels. 4. There was 6-fold stimulation in proline uptake when cyanide-insensitive cells were preincubated with 50 mM glucose. The preincubation of starved cells resulted in a partial restoration of cyanide sensitivity and increased intracellular ATP levels. The preincubation of starved cells with other oxidizable substrates resulted in a partial restoration of cyanide sensitivity but had no stimulatory effect on intracellular ATP levels and proline accumulation. 5. Both the enhanced uptake and ATP levels in glucose preincubated cells were found to be completely abolished by iodoacetate. 6. It is proposed that the increased proline uptake in cells preincubated with glucose was mainly due to the production of glycolytic energy.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

¹³C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing ¹³C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of ¹³C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH₂ resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.     

The reaction of ruthenium(II) octaethylporphyrin and ruthenium(II) tetraphenylporphyrin complexes with carbon monoxide

The reactions of Ru^IIOEP(L)₂ and Ru^IITPP(L)₂ with carbon monoxide, where OEP and TPP are the dianions of octaethylporphyrin and tetraphenylporphyrin, respectively, are reported for various ligands (L=dimethyl formamide, acetonitrile, aniline and substituted benzonitriles). The first-order rate constants for the loss of XC₆H₄CN from Ru^II OEP(XC₆H₄CN)₂ increase with increasing electron-withdrawing ability of X. The best Hammett σ/ϱ correlation is obtained when both σ⁺ and σ⁻values are employed. It is concluded that sigma donation from ligand-to-metal is the major mode of bonding in ruthenium-porphyrin-benzonitrile complexes.     

A high-resolution structure of a DNA-chromomycin-Co(II) complex determined from pseudocontact shifts in nuclear magnetic resonance

BACKGROUND: The drug chromomycin-A(3) binds to the minor groove of DNA and requires a divalent metal ion for complex formation. (1)H, (31)P and (13)C pseudocontact shifts occurring in the presence of a tightly bound divalent cobalt ion in the complex between d(TTGGCCAA)(2) and chromomycin-A(3) have been used to determine the structure of the complex. The accuracy of the structure was verified by validation with nuclear Overhauser enhancements (NOEs) and J-coupling constants not used in the structure calculation. RESULTS: The final structure was determined to 0.7 A resolution. The structure was compared with a structure obtained in an earlier study using NOEs, in order to assess the accuracy of NOEs in giving global structural information for a DNA complex. Although some basic features of the structures agreed, they differed substantially in the fine structural details and in the DNA axis curvature generated by the drug. The distortion of base-pair planarity that was observed in the NOE structure was not seen in our structure. Differences in drug orientation and hydrogen bonding also occurred. The curvature and elongation of the DNA that was obtained previously was not found to occur in our study. CONCLUSIONS: The use of pseudocontact shifts has enabled us to obtain a high-precision global structure of the chromomycin-DNA complex, which provides an accurate template on which to consider targeting minor groove binding drugs. The effect of such binding is not propagated far along the helix but is restricted to a local kink in the axis that reverts to its original direction within four base pairs.     

Alkylation of manganese(II) tetraphenylporphyrin by antimalarial fluorinated artemisinin derivatives

The alkylating properties of two artemisinin derivatives bearing a trifluoromethyl substituent at C10 were evaluated toward manganese(II) tetraphenylporphyrin, considered as a heme model. Chlorin-type covalent adducts were obtained by alkylation of the porphyrin ring by C-centered radicals derived from reductive activation of the peroxide function of the drugs.     

Assessing and refining molecular dynamics simulations of proteins with nuclear magnetic resonance data

The sophistication of the force fields, algorithms and hardware used for molecular dynamics (MD) simulations of proteins is continuously increasing. No matter how advanced the methodology, however, it is essential to evaluate the appropriateness of the structures sampled in a simulation by comparison with quantitative experimental data. Solution nuclear magnetic resonance (NMR) data are particularly useful for checking the quality of protein simulations, as they provide both structural and dynamic information on a variety of temporal and spatial scales. Here, various features and implications of using NMR data to validate and bias MD simulations are outlined, including an overview of the different types of NMR data that report directly on structural properties and of relevant simulation techniques. The focus throughout is on how to properly account for conformational averaging, particularly within the context of the assumptions inherent in the relationships that link NMR data to structural properties.     

Iron nitrosyl complexes are formed from nitrite in the human placenta

Placental nitric oxide (NO) is critical for maintaining perfusion in the maternal-fetal-placental circulation during normal pregnancy. NO and its many metabolites are also increased in pregnancies complicated by maternal inflammation such as preeclampsia, fetal growth restriction, gestational diabetes, and bacterial infection. However, it is unclear how increased levels of NO or its metabolites affect placental function or how the placenta deals with excessive levels of NO or its metabolites. Since there is uncertainty over the direction of change in plasma levels of NO metabolites in preeclampsia, we measured the levels of these metabolites at the placental tissue level. We found that NO metabolites are increased in placentas from patients with preeclampsia compared to healthy controls. We also discovered by ozone-based chemiluminescence and electron paramagnetic resonance that nitrite is efficiently converted into iron nitrosyl complexes (FeNOs) within the human placenta and also observed the existence of endogenous FeNOs within placentas from sheep and rats. We show these nitrite-derived FeNOs are relatively short-lived, predominantly protein-bound, heme-FeNOs. The efficient formation of FeNOs from nitrite in the human placenta hints toward the importance of both nitrite and FeNOs in placental physiology or pathology. As iron nitrosylation is an important posttranslational modification that affects the activity of multiple iron-containing proteins such as those in the electron transport chain, or those involved in epigenetic regulation, we conclude that FeNOs merit increased study in pregnancy complications.     

Synthesis and characterization of tetraphenylporphyrin iron(III) complexes with substituted phenylcyanamide ligands

Several five coordinate complexes of [(TPP)Fe^III(L)] in which TPP is the dianion of tetraphenylporphyrin and L is the monoanion of phenylcyanamide (pcyd) (1), 2,5-dichlorophenylcyanamide (2,5-Cl₂pcyd) (2), 2,6-dichlorophenylcyanamide (2,6-Cl₂pcyd) (3), and 2,3,4,6-tetrachlorophenylcyanamide (2,3,4,6-Cl₄pcyd) (4) have been prepared by the reaction of [(TPP)Fe^IIICl] with appropriate thallium salt of phenylcyanamide. Each of the complexes has been characterized by IR, UV-Vis and ¹H NMR spectroscopic data. Dark red-brown needles of [(TPP)Fe^III(2,6-Cl₂pcyd)] (C₅₁H₃₁Cl₂FeN₆ · CHCl₃) crystallize in the triclinic system. The crystal structure of Fe(III) compound shows a slight distortion from square pyramidal coordination with the 2,6-dichlorophenylcyanamide anion in the axial position through nitrile nitrogen atom. Iron atom is 0.47(1) Å out of plane of the porphyrin toward phenylcyanamide ligand. In non-coordinating solvents, such as benzene or chloroform, these complexes exhibit ¹H NMR spectra that are characteristic of high-spin (S = 5/2) species. The X-ray crystal structure parameters are also consistent with high-spin iron(III) complexes. The iron(III) phenylcyanamide complexes are not reactive toward molecular oxygen; however, these complexes react with HCl and produce TPPFe^IIICl.     

X-ray structure and magnetic properties of dinuclear and polymer iron(II) complexes

Five new octahedral iron(II) complexes [FeL2(4-dpa)]_n(EtOH) (1), [FeL2(bipy)]_n(DMF) (2), [FeL1(bpee)]_n (3), [Fe₂L3(1-meim)₄](1-meim)₄ (4) and [FeL1(DMAP)₂] (5), with L1 and L2 being tetradentate coordinating Schiff base like ligands (L1 = (E,E)-[{diethyl-2,2′-[1,2-phenylenebis(iminomethylidyne)]bis[3-oxobutanato](2-)-N,N′,O³,O³′}, L2 = (3,3′)-[{1,2-phenylenebis(iminomethylidyne)]bis(2,4-pentane-dionato)(2-)-N,N′,O²,O²′}) and L3 being a octadentate dinucleating coordinating Schiff base like ligand ({tetraethyl-(E,E,E,E)-2,2′,2′′,2′′′-[1,2,4,5-phenylentetra(iminomethylidine)]tetra[3-oxobutanoato](2-)-N,N′,N′′,N′′′,O³,O³′,O³′′,O³′′′}); 4-dpa = di(4-picolyl)-amine, bipy = 4,4′-bipyridine, bpee = trans-1,2-bis(4-pyridyl)ethylene, 1-meim = 1-methylimidazole and DMAP = 4-dimethylaminopyridine, have been synthesized and characterised using X-ray structure analysis and T-dependent susceptibility measurements. Both methods indicate that all iron(II) centres are in the paramagnetic high-spin state over the whole temperature range investigated. The O-Fe-O angle, the so called bit of the equatorial ligand, is with an average of 111° in the region typical for high-spin iron(II) complexes of this ligand type. In the case of compound 1 an infinite two-dimensional hydrogen bond network can be found, for the compounds 2-4 no hydrogen bond interactions are observed between the complex molecules. A comparison of the curve progression obtained from the magnetic measurements of the mononuclear complex 5 and the polymeric complexes 1-3 leads to the conclusion that no magnetic interactions are mediated over the bridging axial ligands. For the dinuclear complex 4 weak antiferromagnetic interactions between the two iron centres are found.