Genetic engineering of the heme pocket in human serum albumin: modulation of O2 binding of iron protoporphyrin IX by variation of distal amino acids

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O2 in much the same way as hemoglobin (Hb). We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O2 reversibly: (i) Ile-142-->His, which is axially coordinated to the central Fe2+ ion of the heme, and (ii) Tyr-161-->Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/Y161F (HF) or I142H/Y161L (HL)]. Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme (at position Leu-185, Leu-182, or Arg-186) can modulate the O2 binding equilibrium. The coordination structures and ligand (O2 and CO) binding properties of nine rHSA(triple mutant)-heme complexes have been physicochemically and kinetically characterized. Several substitutions were severely detrimental to O2 binding: for example, Gln-185, His-185, and His-182 all generated a weak six-coordinate heme, while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O2. In marked contrast, HSA(HL/L185N)-heme showed very high O2 binding affinity (P1/2O2 1 Torr, 22 degrees C), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O2 binding primarily by reducing the O2 dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O2 binding affinity (P1/2O2 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport.     

O2 and CO binding properties of artificial hemoproteins formed by complexing iron protoporphyrin IX with human serum albumin mutants

The binding properties of O2 and CO to recombinant human serum albumin (rHSA) mutants with a prosthetic heme group have been physicochemically and kinetically characterized. Iron(III) protoporphyrin IX (hemin) is bound in subdomain IB of wild-type rHSA [rHSA(wt)] with weak axial coordination by Tyr-161. The reduced ferrous rHSA(wt)-heme under an Ar atmosphere exists in an unusual mixture of four- and five-coordinate complexes and is immediately autoxidized by O2. To confer O2 binding capability on this naturally occurring hemoprotein, a proximal histidine was introduced into position Ile-142 or Leu-185 by site-directed mutagenesis. A single mutant (I142H) and three double mutants (I142H/Y161L, I142H/Y161F, and Y161L/L185H) were prepared. Both rHSA(I142H/Y161L)-heme and rHSA(I142H/Y161F)-heme formed ferrous five-N-coordinate high-spin complexes with axial ligation of His-142 under an Ar atmosphere. These artificial hemoproteins bind O2 at room temperature. Mutation at the other side of the porphyrin, Y161L/L185H, also allowed O2 binding to the heme. In contrast, the single mutant rHSA(I142H)-heme could not bind O2, suggesting that removal of Y161 is necessary to confer reversible O2 binding. Laser flash photolysis experiments showed that the kinetics of CO recombination with the rHSA(mutant)-heme were biphasic, whereas O2 rebinding exhibited monophasic kinetics. This could be due 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 affinities of the rHSA(mutant)-heme were significantly lower than those of hemoglobin and myoglobin, principally due to the high O2 dissociation rates. Changing Leu-161 to Phe-161 at the distal side increased the association rates of both O2 and CO, which resulted in enhanced binding affinity.     

Microcalorimetry investigation of synthetic hemoprotein (albumin‐heme)

Recombinant human serum albumin (rHSA) incorporating the iron(II) complex of the tetraphenylporphyrin derivative (FepivP or FecycP) is a synthetic O₂‐carrying hemoprotein [albumin‐heme (rHSA‐FepivP or rHSA‐FecycP)], which acts as a red blood cell substitute. The association and dissociation behavior of FepivP and FecycP with rHSA has been initially investigated by isothermal titration calorimetry. A strong heat release appeared after the injection of albumin‐heme into a large molar excess of rHSA. This exothermic enthalpy change was due to the transference of hemes to the other free albumins. The difference in the heme binding affinity to rHSA can be manifested in the enthalpy term. Copyright © 2003 John Wiley & Sons, Ltd.     

Insights Into How Heme Reduction Potentials Modulate Enzymatic Activities of a Myoglobin-based Functional Oxidase

Heme-copper oxidase (HCO) is a class of respiratory enzymes that use a heme-copper center to catalyze O₂ reduction to H₂O. While heme reduction potential (E°′) of different HCO types has been found to vary >500 mV, its impact on HCO activity remains poorly understood. Here, we use a set of myoglobin-based functional HCO models to investigate the mechanism by which heme E°′ modulates oxidase activity. Rapid stopped-flow kinetic measurements show that increasing heme E°′ by ca. 210 mV results in increases in electron transfer (ET) rates by 30-fold, rate of O₂ binding by 12-fold, O₂ dissociation by 35-fold, while decreasing O₂ affinity by 3-fold. Theoretical calculations reveal that E°′ modulation has significant implications on electronic charge of both heme iron and O₂, resulting in increased O₂ dissociation and reduced O₂ affinity at high E°′ values. Overall, this work suggests that fine-tuning E°′ in HCOs and other heme enzymes can modulate their substrate affinity, ET rate and enzymatic activity.     

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.     

Photosensitized reduction of water to hydrogen using human serum albumin complexed with zinc-protoporphyrin IX

We present the photophysical properties of complexes of recombinant human serum albumin (rHSA) with Zn(II)-protoporphyrin IX (ZnPP) and their activities in the photosensitized reduction of water to hydrogen (H2) using methyl viologen (MV2+) as an electron relay. The ZnPP is bound in subdomain IB of wild-type rHSA [rHSA(wt] by an axial coordination of Tyr-161 and, in the rHSA(I142H/Y161L) mutant [rHSA(His], by a His-142 coordination. Both the rHSA(wt)-ZnPP and rHSA(His)-ZnPP complexes showed a long-lived photoexcited triplet state with lifetimes (tauT) of 11 and 2.5 ms, respectively. The accommodation of ZnPP into the protein matrix efficiently eliminated the collisional triplet self-quenching process. The addition of a water-soluble electron acceptor, MV2+, resulted in a significant decrease in the triplet lifetime. The transition absorption spectrum revealed the oxidative quenching of rHSA-3ZnPP* by MV2+. The quenching rate constant (kq) and backward electron transfer rate constant (kb) were determined to be 1.4 x 10(7) and 4.7 x 10(8) M(-1) s(-1) for rHSA(wt)-ZnPP. In the presence of the colloidal PVA-Pt as a catalyst and triethanolamine (TEOA) as a sacrificial electron donor, the photosensitized reduction of water to H2 takes place. The efficiency of the photoproduction of H2 was greater than that of the system using the well-known organic chromophore, tetrakis(1-methylpyridinium-4-yl)porphinatozinc(II) (ZnTMPyP4+), under the same conditions.     

Human Serum Albumin Hybrid Incorporating Synthetic Hemes:A Novel O_2-Carrying Hemoprotein

It has long been known that homologous blood transfusion will result in a lot ofserious problems such as viral infections,for example AIDS,hepatitis,antigenicsensitization and GVHD;therefore aggressive testing of donor blood has beenadopted[1 ,2 ] .Even after this introduction,which is time-consuming and expensive,wecould not eliminate all the risks. In the wake of these kinds of pitfalls,production andclinical use of the blood substitutes have emerged.The essential aim of blood substitutei…     

Inside Cover: Self‐Assembly of One‐ and Two‐Dimensional Hemoprotein Systems by Polymerization through Heme–Heme Pocket Interactions (Angew. Chem. Int. Ed. 7/2009)

Supramolecular protein polymers consisting of cytochrome b₅₆₂ monomers with heme covalently attached to the protein surface are presented by T. Hayashi and co‐workers in their Communication on page 1271 ff. Not only one‐dimensional hemoprotein fibers with submicrometer lengths have been prepared, but when a heme triad was added as a pivot molecule, two‐dimensional protein assembly networks resulted, which cover over 100 square micrometers.

     

O2 Binding to Heme is Strongly Facilitated by Near‐Degeneracy of Electronic States

This paper reports the computed O₂ binding to heme, which for the first time explains experimental enthalpies for this process of central importance to bioinorganic chemistry. All four spin states along the relaxed Fe? O₂‐binding curves were optimized using the full heme system with dispersion, thermodynamic, and scalar‐relativistic corrections, applying several density functionals. When including all these physical terms, the experimental enthalpy of O₂ binding (?59 kJ mol^?1) is closely reproduced by TPSSh‐D3 (?66 kJ mol^?1). Dispersion changes the potential energy surfaces and leads to the correct electronic singlet and heptet states for bound and dissociated O₂. The experimental activation enthalpy of dissociation (～82 kJ mol^?1) was also accurately computed (～75 kJ mol^?1) with an actual barrier height of ～60 kJ mol^?1 plus a vibrational component of ～10 and ～5 kJ mol^?1 due to the spin‐forbidden nature of the process, explaining the experimentally observed difference of ～20 kJ mol^?1 in enthalpies of binding and activation. Most importantly, the work shows how the nearly degenerate singlet and triplet states increase crossover probability up to ～0.5 and accelerate binding by ～100 times, explaining why the spin‐forbidden binding of O₂ to heme, so fundamental to higher life forms, is fast and reversible.     

The Quest for Single-Source Precursors for BaTiO3 and SrTiO3

The reactions between titanium alkoxides Ti(OR)₄ (R = Et,i Pr) and strontium -diketonates Sr(-dik)₂ (-dik = thd, acac) were investigated. The various Sr-Ti species, Sr₂Ti₂(-dik)₄ (OR)₈, have a 1:1 Sr:Ti stoichiometry and were characterized by elemental analysis, FT-IR and by single-crystal X-ray diffraction for Sr₂Ti₂(₃-OiPr)₂ (-OiPr)₄ (OiPr)₂(thd)₄ (1). The hydrolysis-polycondensation reactions of the various species were investigated and the resulting powders analyzed by light scattering and XRD. While acetone was found to have little influence on the hydrolysis reactions of the Sr-Ti species, polycondensation of Ti(OiPr)₄ in neat acetone offers a trinuclear enolate Ti(₃-O)₂(OCMe=CH₂)₃ (OiPr)₅(iPrOH) (4). Comparisons between the Ba-Ti and Sr-Ti systems are given.     

Self‐Assembly of One‐ and Two‐Dimensional Hemoprotein Systems by Polymerization through Heme–Heme Pocket Interactions

Supramolecular protein polymers : When a heme moiety was introduced to the surface of an apo‐cytochrome b₅₆₂(H63C) mutant, supramolecular polymers formed through noncovalent heme–heme pocket interactions. The incorporation of a heme triad as a pivot molecule in the protein polymer further led to a two‐dimensional protein network structure, which was visualized by tapping‐mode atomic force microscopy (see picture).

     

Adapting Synthetic Models of Heme/Cu Sites to Energy-Efficient Electrocatalytic Oxygen Reduction Reaction

In nature, cytochrome c oxidases catalyze the 4e⁻ oxygen reduction reaction (ORR) at the heme/Cu site, in which Cu^I is used to assist O₂ activation. Because of the thermodynamic barrier to generate Cu^I, synthetic Fe-porphyrin/Cu complexes usually show moderate electrocatalytic ORR activity. We herein report on a Co-corrole/Co complex 1-Co for energy-efficient electrocatalytic ORR. By hanging a Co^II ion over Co corrole, 1-Co realizes electrocatalytic 4e⁻ ORR with a half-wave potential of 0.89 V versus RHE, which is outstanding among corrole-based electrocatalysts. Notably, 1-Co outperforms Co corrole hanged with Cu^II or Zn^II. We revealed that the hanging Co^II ion can provide an electron to improve O₂ binding thermodynamically and dynamically, a function represented by the biological Cu^I ion of the heme/Cu site. This work is significant to present a remarkable ORR electrocatalyst and to show the vital role of a second-sphere redox-active metal ion in promoting O₂ binding and activation.     

Hydrothermal syntheses and structural characterizations of three polyoxomolybdates frameworks linked by M(HL)2 units (M = Co, Ni, Zn; L = 3-(2-pyridyl)pyrazole)

Three polyoxomolybdate compounds, namely {[M^II(HL)₂]₂(Mo₈O₂₆)} _n (M = Co (1), Ni (2), Zn (3)) (HL, 3-(2-pyridyl)pyrazole), were designed and synthesized under the hydrothermal conditions and characterized by elemental analysis, IR spectroscopy, and TGA. Single-crystal X-ray diffraction analysis results reveal that compounds 1–3 own the isostructural chain structure consisting of the β-[Mo₈O₂₆]^4? anions, which are linked by M(HL) ₂ ²⁺ units via the terminal oxygen atoms. TGA curves show that the organic ligands separate from the related compounds above ca. 673 K.     

Coordination structure of active site in synthetic hemoprotein (albumin-heme) with dioxygen and carbon monoxide

Recombinant human serum albumin (rHSA) incorporating the tetraphenylporphinatoiron(II) derivative with a covalently linked proximal base (FeP) [albumin-heme (rHSA-FeP)] is a synthetic hemoprotein, which can bind and release dioxygen (O₂) reversibly under physiological conditions. The coordination structure and spin-state of the active site in rHSA-FeP with O₂ and carbon monoxide (CO) were revealed by magnetic circular dichroism (MCD), resonance Raman (RR), and infrared (IR) spectroscopy. Under an N₂ atmosphere, the MCD spectrum of rHSA-FeP showed the formation of the five-coordinate ferrous high-spin complex of FeP. Upon exposure of this solution to O₂ or CO, the spectral pattern immediately changed to that of a six-coordinate ferrous low-spin species. The vibration stretching frequencies of the coordinated O₂ (ν_O2) and CO (ν_CO) were observed at 1158 cm⁻¹ and 1964 cm⁻¹, respectively. The electronic structures of the O₂- and CO-adduct complexes of FeP in the hydrophobic pocket of albumin are both identical to those for FeP itself in toluene solution.     

Coordination compounds of cobalt,nickel, copper,and zinc with 2-bromo-3-phenylpropenal benzoylhydrazone and thiosemicarbazone

By X-ray structural analysis the crystal structure of 2-bromo-3-phenylpropenal benzoylhydrazone (HL) was determined. The molecule is not flat. In the crystal the HL molecules form infinite chains with reciprocal van der Waals interaction. 2-Bromo-3-phenylpropenal hydrazone (HL) and thiosemicarbazone (HL′) react with cobalt, nickel, copper and zinc chlorides, nitrates and acetates to form coordination compounds of the composition Cu(HL)(L)₂ [HL = C₆H₅-CH=CBr-CH=N-NH-C(O)-C₆H₅], MX₂·2 HL′·nH₂O [M = Co, Ni, Cu, Zn; X = Cl, NO₃, HL′ = C₆H₅-CH=CBr-CH=N-NH-C(S)-NH₂; n = 0–3], MX₂·HL·n H₂O [M = Ni, Cu; n = 0, 1], and ML′₂·nH₂O [M = Co, Ni, Zn; n = 0–3]. The same reactions in the presence of amines (A = C₅H₅N, 2-CH₃C₅H₄N, 3-CH₃C₅H₄N, 4-CH₃C₅H₄N) afford complexes of the composition CuALCl and MALX·n H₂O [M = Cu, Ni; X = Cl, NO₃; n = 0–2]. Structure of the coordination node in the amine-containing copper derivatives is polynuclear, in complexes Cu(HL)(L)₂ is octahedral, in other compounds it is tetrahedral. The azomethines (HL and HL′) in these complexes behave as bidentate N,O and N,S ligands. Thermolysis of the complexes includes a step of dehydration (60–90°C) and complete thermal decomposition (430–590°C).     

Dioxygenation of human serum albumin having a prosthetic heme group in a tailor-made heme pocket

Human serum albumin (HSA) is the most abundant plasma protein in our bloodstream and serves as a transporter for small hydrophobic molecules such as fatty acids, bilirubin, and steroids. Hemin dissociated from methemoglobin is also bound within a narrow D-shaped cavity in subdomain IB of HSA. In terms of the general hydrophobicity of the alpha-helical pocket, HSA potentially has features similar to the heme-binding site of myoglobin (Mb) or hemoglobin (Hb). However, the reduced ferrous HSA-heme complex is immediately oxidized by O2, because HSA lacks the proximal histidine that enables the heme group to bind O2. In this paper, we report the introduction of a proximal histidine into the subdomain IB of HSA by site-directed mutagenesis to construct a tailor-made heme pocket (I142H/Y161L), which allows a reversible O2 binding to the prosthetic heme group. Laser flash photolysis experiments revealed that this artificial hemoprotein appears to have two different geometries of the axial-imidazole coordination, and these two species (I and II) showed rather low O2 binding affinities (P1/2O2 = 18 and 134 Torr) relative to those of Mb and Hb.     

Oxygen-carrying plasma hemoprotein “albumin-heme”: nitric oxide binding and physiological responses after administration in vivo

Recombinant human serum albumin complexed with tetraphenylporphinatoiron(II) derivative, “albumin-heme (rHSA-FeP)”, is a synthetic oxygen (O₂)-carrying plasma hemoprotein, which becomes a new class of red blood cell substitute. The UV-vis. absorption and ESR spectroscopy revealed that rHSA-FeP formed six-coordinate nitrosyl complex after exposure of nitric oxide (NO) gas. Although the NO-binding affinity of rHSA-FeP (P_1/2^NO: 1.7 × 10⁻⁶ Torr, pH 7.3, 25°C) is 9-fold higher compared to that of hemoglobin (Hb), the administration of this artificial hemoprotein solution into anesthetized rat does not induce an acute increase in blood pressure (hypertension), which is often observed in Hb-based O₂-carriers due to the depletion of NO (endothelial derived relaxing factor).     

Oxygen-Exchange Reaction Between Artificial Lung Device: The Heme Embedded in Polymerized Lipo Liposome as an Artificial Oxygen Carrier

An oxygen-supplying medium (OSM), which was prepared by embedding a synthetic heme complex in a bilayer of polymerized lipid liposome (polylipid liposome/heme), could bind molecular oxygen reversibly under physiological conditions. The oxygen-exchange reaction of OSM with blood was examined by using a liquid/liquid artificial lung device. For example, deoxy-blood (p _s(O₂) = 0 torr) was passed countercurrent to oxy-OSM (p _s(O₂) = 154 torr) through hollow fibers of the device to provide oxy-blood (p _b(O₂) = 57 torr). The OSM was physicochemically and mechanically stable under strong shear stress during the passage through the hollow fibers and acted as the oxygen mediator to blood.     

Cu(II), Ni(II), Zn(II) and Fe(III) complexes containing a N2O2 donor ligand: Synthesis, characterization, DNA cleavage studies and crystal structure of [Cu(HL)Cl]

A polydentate ligand, H₂L “[1-(5-isopropyl-2-methyl phenoxy)-3-(N-2-hydroxy benzyl-N-((pyridine-2-yl)amino) propan-2-ol]”, containing a N₂O₂ donor moiety was synthesized by refluxing 2-((5-isopropyl-2-methylphenoxy)methyl)oxirane and HBPA (N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)amine). This synthesized ligand was used for the preparation of complexes with different metal ions, viz. [Cu(HL)Cl] (1), [Ni(HL)Cl] (2), [Zn(HL)Cl] (3) and [Fe(HL)Cl₂] (4). The ligand and metal complexes were characterized by ¹H NMR, mass, ESI-MS, elemental analysis, IR, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy. The crystal structure for one of the complexes, [Cu(HL)Cl], was solved from the X-ray crystallography data. The structure of the complex, based on the trigonality index tau, suggests an intermediate geometry between square pyramidal (sp) and trigonal bipyramidal (tb). Both the ligand and the metal complexes show oxidative cleavage of plasmid DNA (pBR322) without addition of any exogenous agent, even at a concentration of 5 μM. The binding constants for these compounds were found to be in the range 5.33-0.065 × 10⁵ M⁻¹.     

Azo derivative of 2-thenoyltrifluoroacetone as a reagent for the photometric determination of copper(II)

1-(2-Thenoyl)-4-trifluoro-2-[2-hydroxy-3-sulfo-5-nitrophenylazo]butadione-1,3 (H₂L) was synthesized from 2-thenoyltrifluoroacetone. The formation of a copper(II) complex of the synthesized reagent was studied in the presence and absence of 8-hydroxyquinoline (HR). Monoligand [Cu(HL)₂] and mixed-ligand [Cu(HL)₂HR] compounds were obtained at pH 4 and 3, respectively. The ratio of components in the monoligand and mixed-ligand compounds were 1 : 2 and 1 : 2 : 1, respectively. Beers law was obeyed in the ranges of copper concentrations from 0.20 to 2.56 and from 0.25 to 2.56 µg/mL, respectively. The dissociation constants of the reagent were .K₁ = 4.25µ0.01 and .K₂ = 8.20µ0.01 . The stability constants of [Cu(HL)₂] and [Cu(HL)₂HR] complexes were K₁ = 4.96µ0.03 and 4.92µ0.01, respectively. A procedure was developed for the photometric determination of copper(II) in rocks.Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 2, 2005, pp. 157–161.Original Russian Text Copyright © 2005 by Alieva, Chyragov, Makhmudov.This revised version was published online in April 2005 with corrections to the author names and book review format.