Q-band electron paramagnetic resonance study of nitrosylprotoheme dimethyl ester complexes with N-,O-, and S-donor ligand as model systems for nitrosylhemoproteins

Electron paramagnetic resonance (epr) spectra (at X- and Q band frequency) of nitrosyl(proto-porphyrin IX dimethyl ester) iron( II) complexes with a trans axial ligand of nitrogen-, oxygen-, and sulfur-donor ligand, in the trozen glass state at 77°K, have been investigated in order to understand the epr spectra of nitrosylhemoproteins. The Q-band spectra resolved the spectral features more clearly than the X-band spectra and distinctly exhibited two groups of absorptions, which were attributable to two molecular species. Significant relations were found between two g values (e.g., g_x-g_z, g_x-g_y) and between the g value and the degree of the hyperfine splitting in central absorption. The epr parameters were not very sensitive to the π-bonding ability of the axial ligand, but registered the steric interaction of the axial ligand with porphynnato core. These findings can be utilized in the characterization of an axial ligand trans to the nitrosyl group in nitrosylhemoproteins.     

The characterization and magnetic properties of the azide and imidazole derivatives of Pseudomonas nitrite reductase

Optical absorption, mcd, and epr spectroscopy have been used to characterize the azide and imidazole derivatives of oxidized Pseudomonas nitrite reductase. At pH 7.0 azide binds solely to heme d1 with an affinity constant, Kaff = 360 M-1, whereas imidazole binds to both hemes c and d1 with kaff = 35 and 55 M-1, respectively. Low-temperature mcd and epr spectroscopy indicate that c and d1 are low-spin ferrihemes in both derivatives, although the epr of the heme d1-azide component is very weak and requires explanation. Attempts to obtain a high-spin heme d1 in the intact enzyme using the weak field ligands fluoride and thiocyanate have proved unsuccessful. Electron paramagnetic resonance experiments involving an oxidized enzyme derivatives in which heme d1 is complexed by NO, and hence epr silent, have enabled unambiguous assignment of the epr spectrum of Pseudomonas nitrite reductase.     

The reactivity of affinity labels: A kinetic study of the reaction of alkyl halides with thiolate anions—a model reaction for protein alkylation

Factors have been investigated which govern the electrophilic reactivity of alkyl halides with thiolate anions in aqueous solution. In the series of alkyl halides studied, some are potential metal-directed affinity labels, while others are frequently used in protein modification. Previous data on the kinetics of this type of alkylation are compared with the present results. The influence of electronic, polar, and steric factors on alkyl halide reactivity is seen. The following order of reactivity for alkyl halides bearing different α substituents was observed: RCH₂CH(X)COOCH₃ > RCH₂CH(X)CONH₂ > RCH₂CH(X)COOH > RCH₂CH₂X > RCH₂CH(X)CH₂OH. The metal-directed affinity labels are imidazole derivatives, some of which have substituents in their imidazole ring. The effect of the imidazole ring and of ring substitution on reactivity is seen. The nucleophilic reactivity of thiols is highly pH dependent since the thiolate anion (RS^?) is the reactive species, but only minor differences emerged between different free thiolates.     

Studies on heme d1 extracted from Pseudomonas aeruginosa nitrite reductase

Heme d1 has been extracted from Pseudomonas nitrite reductase. Imidazole, cyanide, and chloride-ferroheme, and CO, NO, cyanide, imidazole, and pyridine-ferroheme complexes have been prepared for study by UV/vis spectroscopy, and in some cass by epr and low-temperature mcd as well. Iron determinations have been carried out to assess extinction coefficients. Absorption spectra were used to monitor the transition of chloride-ferriheme d1 to an alkaline form of ferriheme d1 and a pka of 6.5 was determined for the process. The epr spectrum of chloride-ferriheme possessed the characteristic g = 6 signal of high spin (S = 5/2) iron, but the alkaline-ferriheme form gave no detectable epr signals. Electron paramagnetic resonance spectra were also obtained for cyanide and imidazole-ferriheme d1 and for NO-ferroheme d1. The imidazole complex gave signals that were very weak in comparison with the cyanide complex, but mcd measurements of imidazole-ferriheme d1 were consistent with it being a low-spin (S = 1/2) system. The epr signals of NO-ferroheme d1 were similar to those of the corresponding holo-enzyme complex. Reduction of alkaline-ferriheme d1 was found to be affected by the presence of oxygen, but under N2 give the same result with ascorbate and dithionite. Autoreduction of alkaline-ferriheme d1 was observed when placed under CO, and NO, atmospheres, or when treated with pyridine.     

Electronic spectra for nitrosyl(protoporphyrin IX dimethyl ester)iron(II) and its complexes with nitrogenous bases as model systems for nitrosylhemoproteins

Soret and visible absorption spectra for nitrosyl(protoporphyrin IX dimethyl ester)iron(II) (Fe(PPIXDME)(NO] and its complexes with nitrogenous bases (imidazoles, pyridines, aliphatic amines, and cyclic secondary amines) as model systems for nitrosylhemoproteins have been measured in various solvents. As the solvent polarity increases, the Soret and visible absorption bands for the five-coordinate Fe(PPIXDME) (NO) were shifted to shorter wavelengths. Accompanying the coordination of a nitrogenous base to the vacant axial position of Fe(PPIXDME)(NO), the Soret band becomes sharp and the band maximum is shifted to longer wavelengths. The band positions for the six-coordinate Fe(PPIXDME)(NO)(Base) complex are not sensitive to the pi-bonding ability of the axial ligand trans to NO group. The electronic spectra of five-coordinate Fe(PPIXDME)(NO) and six-coordinate Fe(PPIXDME)(NO)(Base) complexes are interpreted in relation to the structural information. The comparison of the spectra for model systems with those for nitrosylhemoproteins is discussed.     

EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

The epr spectra of the inhibitor derivatives of cobalt carbonic anhydrase

The epr spectra at 4.2 K of inhibitor derivatives of cobalt carbonic anhydrase have been recorded. The spectra can be grouped into two classes according to whether the low-field signal is broad or sharp and with g ranges of 6.1–6.8, 2.3–2.9, 1.6–1.8, and 5.8–6.2, 2.2–2.8, 1.5–1.8, respectively. The two kinds of spectra have been empirically related to the features of the room-temperature solution electronic spectra. A third kind of epr spectrum with a single broad signal is obtained when the inhibitor is in large excess.The possibility of using the epr spectra for deducing the geometry of cobalt enzymes is discussed.     

Metal phthalocyanine substituted hemoglobins. Complexes of manganese and zinc tetrasulfonated phthalocyanines with apohemoglobin

Artificial hemoglobins have been prepared with Mn(III) and Zn(II) tetrasulfonated phthalocyanines in place of heme. Their structure and properties have been investigated by difference spectroscopy, CD, epr, electrophoresis, and molecular weight estimation.Spectrophotometric titration data indicate the ratio of the reagents in this process to be 1:1. The visible absorption spectra show the main peak at 625 nm for the manganese compound and 681 nm for the zinc one. It is evident from CD experiments that incorporation of Mn(III)L into apohemoglobin increases helical content of the protein whereas that of Zn(II)L increases its unfolding due to the change of electronic configuration of Zn(II) ion on coordination with the protein.On the basis of spectroscopic and epr data, the formula of the manganese complex is suggested to be (O)Mn(IV)L-globin, whereas that of the zinc complex Zn(II)L-globin. Electrophoresis and molecular weight estimation indicate both complexes to be dimers.Manganese complex binds additional ligands as CN^?, imidazole, CO, and NO. Spectroscopic and epr data indicate reduction of the manganese complex and formation of the NO adduct with probable formula (NO)⁺Mn(II)L-globin. Mechanism of this process is suggested.Both phthalocyanine globins are not able to combine reversibly with oxygen and cannot act as physiological oxygen carriers.     

Structural change in the one-electron oxidation-reduction at the copper site in nitrite reductase. Evidence from EXAFS

The structural change of the nitrite reductase in the one-electron reduction at the copper center has been studied using the EXAFS technique. In the one electron reduction, the Cu(cystein) distance is elongated by about 0.1 Å, and CuN(imidazole) distance are slightly changed.     

The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus.

Staphylococcus simulans biovar staphylolyticus produces an extracellular glycylglycine endopeptidase (lysostaphin) that lyses other staphylococci by hydrolyzing the cross bridges in their cell wall peptidoglycans. The genes for endopeptidase (end) and endopeptidase resistance (epr) reside on plasmid pACK1. An 8.4-kb fragment containing end was cloned into shuttle vector pL150 and was then introduced into Staphylococcus aureus RN4220. The recombinant S. aureus cells produced endopeptidase and were resistant to lysis by the enzyme, which indicated that the cloned fragment also contained epr. Treatments to remove accessory wall polymers (proteins, teichoic acids, and lipoteichoic acids) did not change the endopeptidase sensitivity of walls from strains of S. simulans biovar staphylolyticus or of S. aureus with and without epr. Immunological analyses of various wall fractions showed that there were epitopes associated with endopeptidase resistance and that these epitopes were found only on the peptidoglycans of epr+ strains of both species. Treatment of purified peptidoglycans with endopeptidase confirmed that resistance or susceptibility of both species was a property of the peptidoglycan itself. A comparison of the chemical compositions of these peptidoglycans revealed that cross bridges in the epr+ cells contained more serine and fewer glycine residues than those of cells without epr. The presence of the 8.4-kb fragment from pACK1 also increased the susceptibility of both species to methicillin.     

Caffeine complexes of platinum(II): crystal structure of cis-[Pt(C8H10N4O2)2Cl2]·0.4H2O

The preparations are reported of cis[Pt(caffeine)₂Cl₂]·0.4H₂O, Pd(caffeine)₂Cl₂, the methanol adduct of the previously known compound K[Pt(caffeine)Cl₃], and Pt(caffeine)(cytidine)Cl₂. Crystals of [Pt(caffeine)₂Cl₂]·0.4H₂O are tetragonal P4₂/n with a = 13.156(2) 0?, c = 12.734(2) 0?, Z = 4. The structure was refined on 945 reflections to R = 0.025. The molecule is cis with a crystallographic two-fold axis bisecting the ClPtCl and NPtN angles. The Pt is square planar with PtN and PtCl bonds of 2.029(5) Å and 2.271(2) Å respectively. There is a 5.4° dihedral angle between the imidazole and pyrimidine rings, and the imidazole ring is rotated away from the coordination plane by 86.4°. Symmetry related caffeine units pack parallel to each other with an inter-ring separation of 3.45 Å.     

Rat intestinal brush border membrane dipeptidyl-aminopeptidase IV: kinetic properties and substrate specificities of the purified enzyme

The kinetic properties and substrate specificities of dipeptidyl-aminopeptidase IV (EC 3.4.14.—) detergent-solubilized and purified from the brush border membrane of rat small intestinal mucosal cells were investigated. Kinetic analysis of purified dipeptidyl-aminopeptidase IV was carried out with a variety of oligopeptides and β-napthylamide derivatives as substrates. In general, peptides with proline penultimate to the amino terminus (XPro, X= amino acid) are more favored substrates while those with alanine (XAla) are hydrolyzed at a slower rate. There is some activity toward substrates having leucine at both the penultimate position and amino terminus (LeuLeu). The activity of the purified enzyme toward GlylProβ-napthylamide derivative is maximal at pH 8.4 in Tris-HCl buffer, with an activation energy of 7.98 kcal/mol. There is no requirement for metal ion. The ability of various dipeptides to inhibit Gly-l-Pro-β-napthylamide derivative hydrolysis was used to determine the binding specificity of the enzyme for the amino-terminal amino acid. These data show that a free amino acid group is necessary for enzymatic activity and increased hydrophobicity of the amino acid at the amino terminus enhances binding.     

Resonance Raman and EPR of nitrosyl human hemoglobin and chains, carp hemoglobin, and model compounds. Implications for the nitrosyl heme coordination state.

We report the joint resonance Raman (RR) and electron paramagnetic resonance (epr) study of five- and six-coordinate nitrosyl heme model compounds and of the titled nitrosyl hemoproteins. Both epr and RR spectra fall into two types which, in the models, correspond to five- and six-coordinate nitrosyl hemes. However, neither RR nor epr spectroscopy is highly sensitive to the nature of the bond between a nitrosyl heme and a coordinated nitrogenous base, nor do the results of one technique uniformly correlate with those of the other. It is not possible to use epr spectroscopy as a test for the coordination state of a nitrosyl heme. The position of the highest frequency (depolarized) RR band possibly provides such a test. Any breaking of the very weak bond between nitrosyl heme and proximal histidine in T state human HbNO is more a consequence of tertiary structural features unique to the human alphaNO chains than it is of properties of the T quaternary conformation.     

Interaction of human serum albumin with novel imidazole derivatives studied by spectroscopy and molecular docking

This study was a detailed characterization of the interaction of a series of imidazole derivatives with a model transport protein, human serum albumin (HSA). Fluorescence and time‐resolved fluorescence results showed the existence of a static quenching mode for the HSA–imidazole derivative interaction. The binding constant at 296 K was in the order of 10⁴ M^–1, showing high affinity between the imidazole derivatives and HSA. A site marker competition study combined with molecular docking revealed that the imidazole derivatives bound to subdomain IIA of HSA (Sudlow's site I). Furthermore, the results of synchronous, 3D, Fourier transform infrared, circular dichroism and UV–vis spectroscopy demonstrated that the secondary structure of HSA was altered in the presence of the imidazole derivatives. The specific binding distance, r, between the donor and acceptor was obtained according to fluorescence resonance energy transfer. Copyright © 2015 John Wiley & Sons, Ltd.     

X-band electron paramagnetic resonance spectra of bovine serum albumin-copper(II) and bovine serum albumin-copper(II)-aminoacid systems.

X-band electron paramagnetic resonance (epr) spectra of the binary systems, BSA-copper(II) (1:1 and 2:1), and the ternary systems, BSA-Cu(II)-aminoacid (1:1:1), are described. In the binary system, two distinct epr features have been observed. One of the features (towards the low pH), showing broad and overlapping epr signals, has been attributed to non-specific bonding of copper(II) to the albumin and other feature (towards higher pH), showing sharp intense epr signals, has been attributed to the specific bonding. The change from non-specific to specific binding is favoured by increase in pH as well as by increase in protein concentration. Specific binding of copper(II) in BSA-Cu(II) has been suggested to be similar to that in HSA-Cu(II). Spectra of BSA-Cu(II)-aminoacid (1:1:1) show simultaneous presence of binary BSA-Cu(II) and ternary BSA-Cu(II)-aminoacid.     

H93G myoglobin cavity mutant as versatile template for modeling heme proteins: magnetic circular dichroism studies of thiolate- and imidazole-ligated complexes

Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450.     

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.     

Optical properties and a new epr signal from type 3 copper in metal-depleted Rhus vernicifera laccase

Due to conflicting reports on the properties of Rhus laccase depleted in type 2 copper a further investigation of this protein derivative has been undertaken. In contrast to most other reports it is shown that the type 3 copper site retains its absorbance at 330 nm when type 2 copper is removed. The type 3 copper ions are oxidized in the resting protein and part of the type 3 Cu(II) can be made electron paramagnetic resonance (epr) detectable on reduction by ascorbate. This new epr signal is highly rhombic and the epr parameters are comparable to those found in other metalloproteins containing Cu(II) in binuclear sites. Certain preparations of type 2 deficient protein exhibit lower extinction coefficients at 330 nm. Since these protein derivatives have lost some type 3 copper, it is inferred that the absorbance at 330 nm is dependent on a native type 3 copper site. Also in contrast to other reports, it is found that the extinction coefficient at 614 nm of the type 1 Cu(II) decreases from 5700 to 4700 M^?1cm^?1 when type 2 copper is removed. The oxidized-reduced difference spectrum also shows a substantial decrease in the absorbance between 700 and 800 nm. The changes in absorbance above 600 nm are probably due to a modification of the type 1 Cu(II) site on removal of type 2 copper. The present results also suggest some explanations to the apparent discrepancies among the earlier reports.     

The reaction of cytochrome oxidase with cyanide. Preparation of the rapidly reacting form and its conversion to the slowly reacting form

The magnitude of the slow phase of reaction of cytochrome oxidase with cyanide has been correlated with the size of the epr signal at g' = 12. This epr signal was not found in submitochondrial particles, and significant g' = 12 epr was only observed late in the purification of solubilized enzyme. The Hartzell-Beinert procedure for the purification of cytochrome oxidase (Hartzell, C.R., and Beinert, H. (1974) Biochim. Biophys. Acta 368, 318-338) has been modified so that the purified enzyme reacts in a single rapid phase with potassium cyanide and lacks the g' = 12 epr signal. This enzyme could be converted to the slowly reacting form upon incubation at low pH and/or low enzyme concentration. No procedure for the stable reversal of the process could be found. Some physical and chemical properties of the two forms of the enzyme are compared.     

Effects of imidazole derivatives on cytochromes P450 from human hepatocytes in primary culture.

The expression of several forms of cytochrome P450 including P450 1A2, 2D6, 2E1, and 3A was investigated in human hepatocytes maintained in primary culture for 96 h in the absence or presence of 50 microM of various imidazole derivatives. These included ketoconazole, clotrimazole, miconazole, fluconazole, secnidazole and metronidazole. In addition, the typical inducers rifampicin and beta-naphthoflavone were used for comparison. Western and Northern blot analysis of microsomes and RNA prepared from these cultures as well as de novo synthesis experiments revealed that, among the imidazole derivatives tested, only clotrimazole was a strong rifampicin-like inducer of P450 3A. The expression of the other forms of P450 tested was not affected by the treatments. Analysis of the inhibition of 13 monoxygenase activities, including ethoxyresorufin and phenacetin O-deethylases, coumarin 7 alpha-, lauric acid 11- and 12-, mephenytoin 4-, debrisoquin 4-, and aniline hydroxylases, benzphetamine, aminopyrine, mephenytoin and erythromycin demethylases, and cyclosporin oxidase (representative of 10 different forms of P450 in human liver microsomes) revealed that ketoconazole was a strong and selective in vitro inhibitor of P450 3A (cyclosporin oxidase) with a Ki less than 1 microM. Clotrimazole and miconazole were also strong inhibitors of P450 3A-mediated activities in contrast to the other imidazole derivatives.