A comparative study of O2, CO and CN binding to heme IX protein models

Parametrization of a molecular-mechanics program to include terms specific for five- and six-coordinate transition metal complexes results in computer-simulated structures of heme complexes. The principal new feature peculiar to five and six coordination is a term that measures the effect of electron-pair repulsion modified by the ligand electronegativity and takes into account the different structural possibilities. The model system takes into account the structural differences of the fixing centre in the haemoglobin subunits. The customary proximal histidine is added. The prosthetic group heme IX is wholly considered in our model. The calculations show clearly that certain conformations are much more favourable that others for fixing O2. From the O2 binding in haemoglobin, myoglobin and simple Fe porphyrin models it is concluded that the bent O2 ligand is best viewed as bound superoxide O2-. Axial ligands are practically free-rotating. A small modification of the model in both crystal and protein matrix affects the orientation of the ligands in experimental systems.     

CHEMILUMINESCENCE and EPR STUDIES ON THE EXCITATION SITE OF FERRIC-HEME-OXO COMPLEXES OF NATURAL and MODEL HEME SYSTEMS

Chemiluminescence was detected both in the reaction system of H₂O₂ plus heme proteins such as methemo- and metmyoglobin and ferric-protoheme complexes used as a model system. The intensity of chemiluminescence was found to be mediated by ligand binding to the sixth coordination site of the ferric-protoheme compounds, e.g. chemiluminescence was not observed with the bisimidazole ferric-protoheme complex. On the other hand the pentacoordinated histidine ferric-protoheme complex exhibited strong light emission. Comparative studies with various ligand-heme compounds elucidated that light emission was inversely correlated with the binding strength of the respective ligand at the sixth coordination site. The basic reaction mechanism causing the establishment of an excited state was studied by monitoring chemiluminescence and EPR signal formation of ligand-modified heme proteins in the presence of different electron donors. External electron donors such as Trolox C, TMPD and ascorbic acid affected a strong reduction in the development of chemiluminescence suggesting the essential involvement of an inner-molecular electron transfer process. Our results allow the conclusion that chemiluminescence is generated from the decay of an excited state of oxo-heme compounds established as a result of a one electron transfer step from a ligand group to heme iron.     

Heme Binding in Gas-Phase Holo-Myoglobin Cations: Distal Becomes Proximal?

His64 and His93 are the two well-known sites of heme binding in water-dissolved holo-myoglobin, with His93 being a proximal, strongly binding partner, while the distal His64 weakly coordinates to the heme through a small-molecule ligand, e.g., water or O₂. The heme bonding scheme in a water-free environment is as yet unclear. Here we employed electron transfer dissociation tandem mass spectrometry to study the preferential attachment site of the ferri-heme (Fe³⁺) in electrospray-produced 12+, 14+, and 16+ holo-myoglobin ions. Contrary to expectations, in lower-charge complexes that should have a structure resembling that in solution, the heme seems to be preferentially attached to the “distal” histidine. In contrast, in the highest studied charge state, the “proximal” histidine is the site of preferential attachment; the 14+ charge state is an intermediate case. This surprising finding raises a question of heme coordination in proteins transferred to water-free environment, as well as the effect of the protonation sites on heme bonding.     

Alkylamine-ligated H93G myoglobin cavity mutant: a model system for endogenous lysine and terminal amine ligation in heme proteins such as nitrite reductase and cytochrome f

His93Gly sperm whale myoglobin (H93G Mb) has the proximal histidine ligand removed to create a cavity for exogenous ligand binding, providing a remarkably versatile template for the preparation of model heme complexes. The investigation of model heme adducts is an important way to probe the relationship between coordination structure and catalytic function in heme enzymes. In this study, we have successfully generated and spectroscopically characterized the H93G Mb cavity mutant ligated with less common alkylamine ligands (models for Lys or the amine group of N-terminal amino acids) in numerous heme iron states. All complexes have been characterized by electronic absorption and magnetic circular dichroism spectroscopy in comparison with data for parallel imidazole-ligated H93G heme iron moieties. This is the first systematic spectral study of models for alkylamine- or terminal amine-ligated heme centers in proteins. High-spin mono- and low-spin bis-amine-ligated ferrous and ferric H93G Mb adducts have been prepared together with mixed-ligand ferric heme complexes with alkylamine trans to nitrite or imidazole as heme coordination models for cytochrome c nitrite reductase or cytochrome f, respectively. Six-coordinate ferrous H93G Mb derivatives with CO, NO, and O(2) trans to the alkylamine have also been successfully formed, the latter for the first time. Finally, a novel high-valent ferryl species has been generated. The data in this study represent the first thorough investigation of the spectroscopic properties of alkylamine-ligated heme iron systems as models for naturally occurring heme proteins ligated by Lys or terminal amines.     

Fine Tuning of Heme Reactivity: Hydrogen-Bonding and Dipole Interactions Affecting Ligand Binding to Hemoproteins

Heme reactivity in hemoproteins is governed by the microenvironment near the ligand binding site. In order to quantify polarity effects on heme ligand binding, the kinetics of O₂ and CO binding have been measured for a series of synthetic heme models equipped with a range of groups of varying dipole moments positioned near the heme coordination site. For hemes with polar aprotic groups, both O₂ on (k′) and off rates (k) are found to be dependent on the dipole moment. For model systems containing protic groups, the O₂ off rate is substantially reduced due to hydrogen bonding with the coordinated O₂. The hydrogen-bonding stabilization is estimated to be 0.7 and 1.6 kcal/mol for an alcohol and a secondary amide, respectively. CO binding displays little correlation with a polarity effect; instead it seems to depend upon the size and position of the polar group.     

Mixed Ligand Complexes of Oxovanadium (IV) Oxalate

The oxovanadium(IV) complexes VOC₂O₄ · 2DMF, VOC₂O₄ · 2 formamide, VOC₂O₄ · 2 ethylene urea, VOC₂O₄ · 2 urea and VOC₂O₄ · en have been prepared and characterized. The oxalato group functions as a bidentate ligand in all these complexes. Except ethylene diamine the remaining ligands – DMF. formamide, ethylene urea and urea – act as monodentate ligand coordinating through their carbonyl oxygen. Ethylene diamine is bound to the metal through nitrogen. In all the complexes vanadium is five coordinated in distorted tetragonal-pyramidal structures.     

Heme Pocket Architecture in Human Serum Albumin: Regulation of O2 Binding Affinity of a Prosthetic Heme Group by Site-Directed Mutagenesis

Summary : We present the O₂ binding properties of recombinant human serum albumin (rHSA) mutants complexed with an iron(II) protoporphyrin IX as a prosthetic heme group. Iron(III) protoporphyrin IX (hemin) is bound within subdomain IB of HSA with weak axial coordination by Tyr-161. In order to confer O₂ binding capability to this naturally occurring hemoprotein: (i) a proximal histidine was introduced into position Ile-142; and (ii) the coordinated Tyr-161 was replaced with hydrophobic Leu using site-directed mutagenesis. It provided a recombinant HSA double-mutant [rHSA(I142H/Y161L) = rHSA(HL)]. The rHSA(HL)–heme formed a ferrous five-coordinate high-spin complex with axial ligation of His-142 under an Ar atmosphere. This artificial hemoprotein binds O₂ at room temperature. Laser flash photolysis experiments demonstrated that O₂ rebinidng to rHSA(HL)–heme displays monophasic kinetics, whereas the CO recombination process obeyed a double-exponential pattern. This might be attributable to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O₂ binding affinity of rHSA(HL)–heme was considerably lower than those of R-state hemoglobin (Hb) and myoglobin (Mb), principally because of the high O₂ dissociation rate constant. The third mutations have been introduced into the distal side of the heme (at position Leu-185 or Arg-186) to increase the O₂ binidng affinity. The rHSA(HL/L185N)–heme showed high O₂ binding affinity ( : 1 Torr), which is 18-fold greater than that of the original double mutant rHSA(HL)–heme and which is rather close to those of Hb (R-state) and Mb. Furthermore, replacement of polar Arg-186 with Leu or Phe adjusted the O₂ binding affinity ( ) to 10 Torr, which is almost equivalent to value for human red blood cells.     

Non-heme iron catalysts for the benzylic oxidation: a parallel ligand screening approach

Ethylbenzene and 4-ethylanisole were used as model substrates for benzylic oxidation with H₂O₂ or O₂ using a range of non-heme iron catalysts following a parallel ligand screening approach. Effective oxidation was found for Fe complexes based on tetra- and pentadentate nitrogen ligands affording the corresponding benzylic alcohol and ketone.     

Aminopyridinate–FI Hybrids,Their Hafnium and Titanium Complexes,and Their Application in the Living Polymerization of 1‐Hexene

Based on two well‐established ligand systems, the aminopyridinato (Ap) and the phenoxyimine (FI) ligand systems, new Ap‐FI hybrid ligands were developed. Four different Ap‐FI hybrid ligands were synthesized through a simple condensation reaction and fully characterized. The reaction of hafnium tetrabenzyl with all four Ap‐FI hybrid ligands exclusively led to mono(Ap‐FI) complexes of the type [(Ap‐FI)HfBn₂]. The ligands acted as tetradentate dianionic chelates. Upon activation with tris(pentafluorophenyl)borane, the hafnium‐dibenzyl complexes led to highly active catalysts for the polymerization of 1‐hexene. Ultrahigh molecular weights and extremely narrow polydispersities support the living nature of this polymerization process. A possible deactivation product of the hafnium catalysts was characterized by single‐crystal X‐ray analysis and is discussed. The coordination modes of these new ligands were studied with the help of model titanium complexes. The reaction of titanium(IV) isopropoxide with ligand 1 led to a mono(Ap‐FI) complex, which showed the desired fac‐mer coordination mode. Titanium (IV) isopropoxide reacted with ligand 4 to give a complex of the type [(ApH‐FI)₂Ti(OiPr)₂], which featured the ligand in its monoanionic form. The two titanium complexes were characterized by X‐ray crystal‐structure analysis.     

A bis-histidine-ligated unfolded cytochrome c immobilized on anionic SAM shows pseudo-peroxidase activity

Urea-unfolded yeast iso-1-cytochrome c electrostatically adsorbed on a gold electrode coated with an anionic self-assembled monolayer yields a heme-mediated electrocatalytic reduction of H₂O₂ (pseudo-peroxidase activity). Under the same conditions, native cytochrome c is inactive. In the unfolded protein, the Met80 heme iron ligand is replaced by a histidine residue yielding a bis-His-ligated form. H₂O₂ electrocatalysis occurs with an efficient mechanism likely involving direct H₂O₂ interaction with the iron(II) center and formation of a transient ferryl group. Comparison of the catalytic activity of a few urea-unfolded single and double Lys-to-Ala variants shows that the kinetic affinity of H₂O₂ for the heme iron and k_cat of the bis-His-ligated form are strongly affected by the geometry of protein adsorption, controlled by specific surface lysine residues.     

Structural characterization of dinuclear Ti(IV) complexes of rigid tetradentate dianionic diamine bis(phenolato) ligands; effect of steric bulk on coordination features

A small difference in diamine bis(phenolato) ligands, namely an additional single methylene unit, directs formation of dinuclear Ti(IV) complexes rather than mononuclear ones as characterized by X-ray crystallography. Varying steric bulk of the ligand affects the coordination number in the dinuclear complexes and the ligand to metal ratio. A ligand with reduced steric bulk leads to a L₂Ti₂(OiPr)₄ type complex featuring two octahedral metal centers bridged only by the two phenolato ligands, whereas a bulky ligand leads to a Ti₂(μ-L¹)(μ-OiPr)₂(OiPr)₄ type complex with a single chelating ligand, two bridging isopropoxo ligands, and two terminal isopropoxo groups on each of the two metal centers, which are of trigonal bi-pyramidal geometry.     

Structure of the Sulfoxide Synthase EgtB from the Ergothioneine Biosynthetic Pathway

The non‐heme iron enzyme EgtB catalyzes O₂‐dependent C? S bond formation between γ‐glutamyl cysteine and N‐α‐trimethyl histidine as the central step in ergothioneine biosynthesis. Both, the catalytic activity and the architecture of EgtB are distinct from known sulfur transferases or thiol dioxygenases. The crystal structure of EgtB from Mycobacterium thermoresistibile in complex with γ‐glutamyl cysteine and N‐α‐trimethyl histidine reveals that the two substrates and three histidine residues serve as ligands in an octahedral iron binding site. This active site geometry is consistent with a catalytic mechanism in which C? S bond formation is initiated by an iron(III)‐complexed thiyl radical attacking the imidazole ring of N‐α‐trimethyl histidine.     

The Mechanism of Stereospecific C?H Oxidation by Fe(Pytacn) Complexes: Bioinspired Non‐Heme Iron Catalysts Containing cis‐Labile Exchangeable Sites

A detailed mechanistic study of the hydroxylation of alkane C? H bonds using H₂O₂ by a family of mononuclear non heme iron catalysts with the formula [Fe^II(CF₃SO₃)₂(L)] is described, in which L is a tetradentate ligand containing a triazacyclononane tripod and a pyridine ring bearing different substituents at the α and γ positions, which tune the electronic or steric properties of the corresponding iron complexes. Two inequivalent cis‐labile exchangeable sites, occupied by triflate ions, complete the octahedral iron coordination sphere. The C? H hydroxylation mediated by this family of complexes takes place with retention of configuration. Oxygen atoms from water are incorporated into hydroxylated products and the extent of this incorporation depends in a systematic manner on the nature of the catalyst, and the substrate. Mechanistic probes and isotopic analyses, in combination with detailed density functional theory (DFT) calculations, provide strong evidence that C? H hydroxylation is performed by highly electrophilic [Fe^V(O)(OH)L] species through a concerted asynchronous mechanism, involving homolytic breakage of the C? H bond, followed by rebound of the hydroxyl ligand. The [Fe^V(O)(OH)L] species can exist in two tautomeric forms, differing in the position of oxo and hydroxide ligands. Isotopic‐labeling analysis shows that the relative reactivities of the two tautomeric forms are sensitively affected by the α substituent of the pyridine, and this reactivity behavior is rationalized by computational methods.     

基于糖苷芳香酸酯的金属有机材料的制备与研究

选用甲基葡萄糖苷和邻苯二甲酸酐为原料,在三乙胺的催化作用下合成了具有配位能力的齿状有机酯配体甲基葡萄糖苷芳香酸酯(MGAE)(C15H18O9,C15H16O8,C22H30O14,C23H22O12,C31H26O15或C39H30O18)配体;然后,将该新型有机酯配体与金属盐溶液在模板剂三乙胺的作用下经配位制备出一系列MGAE金属有机化合物,并采用FT-IR,XRD,TGA,FSEM,EDS和BET等表征手段对所得的MGAE金属有机化合物进行表征。结果表明:配位过程中主要是MGAE中的羰基参与反应,且影响配合物性能的主要是MGAE中的有机配体和金属阳离子。     

Stability of HIV-1 integrase–ligand complexes: the role of coordinating bonds

It is known that the HIV-1 integrase (IN) strand transfer inhibitors include the chelating fragments forming the coordinating bonds with two Mg²⁺ ions placed in the IN active site. The subject of the article is the role of these coordination bonds on stability of ligand–IN complexes. For this purpose, a set of ligand–IN complexes was investigated theoretically and experimentally. The theoretical model is based on the quantum-chemistry calculations of coordinating bonds geometry and energy. Solvent effects were taking into account using the implicit water model and the two-stage calculation scheme developed previously. For the experimental part of our study a set of the ligands was synthesized, and their IC₅₀ values of IN inhibiting have been measured. It is shown that the main contribution to ligand–IN complexes stability is caused by the substitution of water molecules by the ligand in the first coordination sphere of two Mg²⁺ ions, and the change in the polarization energy of the bulk water. It is shown, that acid–base equilibrium and tautomeric forms of the ligands should be taken into account to improve the prediction ability of the theoretical estimations. All these factors are controlled by the chelating fragments of the ligands. It is demonstrated that our theoretical approach based on the consideration of the coordinating bonds allows to separate active ligands (inhibitors) from inactive ones.     

Effect of histidine on the sensitized generation of singlet oxygen in the complexes with chlorophyll

The energies of the intermolecular interactions of an O₂ molecule in the ground and excited states with the electron-excited and non-excited model complexes of chlorophyll were calculated using the DFT, CASSCF, SA-CASSCF, MCQDPT2, and XMCQDPT2 methods. The activation energies of formation and dissociation of the oxygen complexes were estimated. The radiative electric dipole moments of (0 → 0) spin-allowed S → S, T → T, and spin-forbidden S → T transitions were calculated taking into account the spin-orbit coupling, and rate constants of nonradiative transitions that determine the generation and deactivation of the O₂ molecule (¹Δ_g) were evaluated. The effect of histidine on the probability of singlet oxygen generation sensitized by the model chlorophyll complex was considered in detail.     

Some organoperoxo complexes of molybdenum and tungsten

Several new peroxo complexes of molybdenum and tungsten containing different organic ligands have been prepared. The complexes have the compositions [Mo(O)(O₂)L₂], [Mo(O)₂(O₂)L(H₃O)]⁺, [Mo(O)(O₂)L′] and [W(O)(O₂)L₂] [L = oxoquinolino, aniline-2- carboxylate, 2-aminophenoxide, picolinato or 2-carboxylatoquinolino ligand; L′ = N-(2- oxophenyl)salicylidenimino ligand], respectively. The complexes were found to oxidize allyl alcohol, and also PPh₃ and AsPh₃, to their oxides. The IR spectra of the complexes indicate that the frequency of the v₁-mode of the M(O₂) grouping, which is essentially an OO stretch, decreases with the increase in atomic number of metals in a particular group.     

Peroxo Complexes of Molybdenum(VI) Containing Mannich Base Ligands

The molybdenum(VI)-peroxo complexes containing Mannich base ligands having a formula as [MoO(O2)2(L-L)] [where L-L＝morpholinobenzyl benzamide (MBB), piperidinobenzyl benzamide (PBB), morpholinobenzyl urea (MBU), piperidinobenzyl urea (PBU), morpholinobenzyl thiourea (MBTU), piperdinobenzyl thiourea (PBTU)] have been synthesized and characterized by physico-chemical, electrochemical techniques and TGA/DTA studies. The complexes have been prepared by stirring ammonium molybdate and excess of 30% aqueous-H2O2 and then treatment with ethanolic solution of the ligand. Studies revealed that these complexes were non-electrolytes and diamagnetic in nature. The ligands are bound to metal in a bidentate mode through carbonyl oxygen/thiocarbonyl sulphur and the ring nitrogen. The cyclic voltammograms of the complexes show two quasi-reversible steps involving complexes. The complexes have also been tested for antibacterial activity against Salmonella and Kleibsella. The antibacterial study of the ligands and complexes indicate that the complexes exhibit higher activity than the free ligands.     

基于糖苷芳香酸酯的金属有机材料的制备与研究

选用甲基葡萄糖苷和邻苯二甲酸酐为原料,在三乙胺的催化作用下合成了具有配位能力的齿状有机酯配体甲基葡萄糖苷芳香酸酯（MGAE）（C₁₅H₁₈O₉,C₁₅H₁₆O₈,C₂₂H₃₀O₁₄,C₂₃H₂₂O₁₂,C₃₁H₂₆O₁₅或C₃₉H₃₀O₁₈）配体;然后,将该新型有机酯配体与金属盐溶液在模板剂三乙胺的作用下经配位制备出一系列MGAE金属有机化合物,并采用FT-IR,XRD,TGA,FSEM,EDS和BET等表征手段对所得的MGAE金属有机化合物进行表征。结果表明：配位过程中主要是MGAE中的羰基参与反应,且影响配合物性能的主要是MGAE中的有机配体和金属阳离子。