Artificial Hemoprotein Research Articles

An Artificial Hemoprotein with Inducible Peroxidase- and Monooxygenase-Like Activities.

A novel inducible artificial metalloenzyme obtained by covalent attachment of a manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial bidomain repeat protein, (A3A3')Y26C, is reported. The protein is part of the αRep family. The biohybrid was fully characterized by MALDI-ToF mass spectrometry, circular dichroism and UV/Vis spectroscopies. The peroxidase and monooxygenase activities were evaluated on the original and modified scaffolds including those that have a) an additional imidazole, b) a specific αRep bA3-2 that is known to induce the opening of the (A3A3') interdomain region and c) a derivative of the αRep bA3-2 inducer extended with a His6 -Tag (His6 -bA3-2). Catalytic profiles are highly dependent on the presence of co-catalysts with the best activity obtained with His6 -bA3-2. The entire mechanism was rationalized by an integrative molecular modeling study that includes protein-ligand docking and large-scale molecular dynamics. This constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.

Recent advances in the field of artificial hemoproteins: New efficient eco-compatible biocatalysts for nitrene-, oxene- and carbene-transfer reactions

In the last few years, the field of artificial hemoproteins has been expanding through two main strategies involving either the incorporation of synthetic metalloporphyrin derivatives into the chiral cavity of a protein or the directed evolution of natural hemoproteins such as myoglobin and cytochromes P450. First, various synthetic water-soluble porphyrins including ions of transition metals such as iron and manganese have been inserted covalently or by supramolecular anchoring into non-specifically designed native proteins or into proteins modified by a minimum number of mutations. The obtained artificial hemoproteins were able to catalyze oxene transfer reactions such as epoxidation of alkenes or sulfoxidation of sulfides and cyclopropanation reactions with good activities and moderate enantioselectivities. Recently, a second approach, based on the design of the active site of already existing native hemoproteins such as myoglobin and cytochromes P450 by directed evolution, has led to new artificial hemoproteins that are able to catalyze oxene transfer reactions with improved activities as well as with abiological reactions. This approach thus provided promising tools for the catalysis of reactions such as intramolecular or intermolecular carbene and nitrene transfer reactions with high efficiencies. In addition, in all cases, after a few rounds of mutagenesis, mutants that were able to catalyze those reactions with a high enantioselectivity could be obtained. Finally, several groups showed that these new artificial metalloenzymes could also be used for the preparative scale-production of compounds with an excellent enantioselectivity, opening new pathways for the industrial synthesis of compounds of pharmaceutical interest.

Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme-Heme Exciton Coupling.

A supramolecular assembly of units of cytochrome b562 with externally attached heme having intermolecular linkages formed via the heme-heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome b562 provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme-heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme-heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly.

From Hemoabzymes to Hemozymes: The Long but Fascinating Story of the Elaboration of New Biocatalysts for Selective Oxidation Reactions

The design of artificial hemoproteins that could lead to new biocatalysts for selective oxidation reactions constitutes a really promising challenge for a wide range of industrial applications. The main objective stands in the generation of new artificial materials that are able to work under a wide range of conditions and to use clean oxidants such as O2 or H2O2, which fits the current concept of “green chemistry” in a global context of “sustainable growth”. In vivo, such reactions are performed by a wide class of heme-thiolate proteins, cytochromes P450, that catalyze the oxidation of drugs at their heme-active site, using dioxygen as oxidant, in the presence of electrons delivered from NADPH by cytochrome P450 reductase. Several strategies were used to design new artificial hemoproteins to mimic these enzymes, that associate a synthetic iron(III)-tetraarylporphyrin, to mimic the heme of P450s, to a protein that is supposed to mimic the apoprotein of P450s, and thus, that is supposed to selectively position the substrate with respect to the iron atom and to induce a selectivity in the catalyzed reaction. A first generation of artificial hemoproteins or “Hemoabzymes”, was obtained by the non-covalent association of a synthetic Fe(III)-a3b-tetra-o-carboxyphenylporphyrin (FeToCPP) or of a microperoxidase 8 (MP8) with monoclonal antibodies raised against these cofactors. The later MP8-antibody complexes were shown to catalyze the regio-selective nitration of phenol derivatives by H2O2/NO2 and the stereo-selective oxidation of organic compounds such as sulphides by H2O2. A second generation of artificial hemoproteins or “Hemozymes”, was obtained by the non-covalent association of a non-relevant protein with a tetraarylporphyrin. Several strategies were used, the most successful of which, named “host-guest” strategy involved the non-covalent incorporation of water-soluble anionic iron-porphyrins into xylanase A from Streptomyces Lividans, that was low-cost, available in large quantities, and, in addition, heat-resistant. The artificial hemoproteins obtained were found able to perform efficiently the stereoselective oxidation of organic compounds such as sulphides and alkenes by H2O2 and KHSO5. New strategies aiming at using dioxygen as an oxidant, are currently investigated.

From "hemoabzymes" to "hemozymes": towards new biocatalysts for selective oxidations.

The design of artificial hemoproteins that could catalyze selective oxidations using clean oxidants such as O2 or H2O2 under ecocompatible conditions constitutes a real challenge for a wide range of industrial applications. In vivo, such reactions are performed by heme-thiolate proteins, cytochromes P450, which catalyze the oxidation of substrates by dioxygen in the presence of electrons delivered from NADPH by cytochrome P450 reductase. Several strategies were used to design new artificial hemoproteins that mimic these enzymes. The first one involved the non-covalent association of synthetic hemes with monoclonal antibodies raised against these cofactors. This led to the first generation of artificial hemoproteins or "hemoabzymes" that displayed a peroxidase activity, and in some cases catalyzed the regioselective nitration of phenols by H2O2/NO2 and the stereoselective oxidation of sulfides by H2O2. The second one involved the non-covalent association of easily affordable non-relevant proteins with metalloporphyrin derivatives, using either the "Trojan Horse strategy" or the "host-guest" strategy. This led to a second generation of artificial hemoproteins or "hemozymes", some of which were found able to catalyze the stereoselective oxidation of organic compounds such as sulfides and alkenes by H2O2 and KHSO5.

Various strategies for obtaining oxidative artificial hemoproteins with a catalytic oxidative activity: from "Hemoabzymes" to "Hemozymes"?

The design of artificial hemoproteins that could lead to new biocatalysts for selective oxidation reactions using clean oxidants such as O2or H2O2under ecocompatible conditions constitutes a really promising challenge for a wide range of industrial applications. In vivo, such reactions are performed by heme-thiolate proteins, cytochromes P450, that catalyze the oxidation of drugs by dioxygen in the presence of electrons delivered from NADPH by cytochrome P450 reductase. Several strategies were used to design new artificial hemoproteins to mimic these enzymes, that associate synthetic metalloporphyrin derivatives to a protein that is supposed to induce a selectivity in the catalyzed reaction. A first generation of artificial hemoproteins or "hemoabzymes" was obtained by the non-covalent association of synthetic hemes such as N-methyl-mesoporphyrin IX, Fe(III) -α3β-tetra-o-carboxyphenylporphyrin or microperoxidase 8 with monoclonal antibodies raised against these cofactors. The obtained antibody-metalloporphyrin complexes displayed a peroxidase activity and some of them catalyzed the regio-selective nitration of phenols by H2O2/ NO2and the stereo-selective oxidation of sulphides by H2O2. A second generation of artificial hemoproteins or "hemozymes", was obtained by the non-covalent association of non-relevant proteins with metalloporphyrin derivatives. Several strategies were used, the most successful of which, named "host-guest" strategy involved the non-covalent incorporation of metalloporphyrin derivatives into easily affordable proteins. The artificial hemoproteins obtained were found to be able to perform efficiently the stereoselective oxidation of organic compounds such as sulphides and alkenes by H2O2and KHSO5.

Coordination chemistry studies and peroxidase activity of a new artificial metalloenzyme built by the “Trojan horse” strategy

In the general context of green chemistry, a considerable research effort is devoted to the elaboration of new artificial metalloproteins that catalyze, under mild conditions, the oxidation of a wide range of organic compounds, using cheap and environmentally friendly oxidants. A new artificial hemoprotein was obtained by the so-called “Trojan horse” strategy involving the non-covalent insertion of a cationic iron–porphyrin–estradiol cofactor into an anti-estradiol antibody. UV–vis titrations showed the formation of a 1/2 antibody/cofactor complex with a dissociation constant K D = 4.10 −7 M. UV–vis determination of the Fe-imidazole binding constants showed that the protein provided a weak steric hindrance around the iron–porphyrin cofactor. The antibody–estradiol–iron–porphyrin complex displayed a peroxidase activity and catalyzed the oxidation of ABTS by H 2O 2 with about double the efficiency of the iron–porphyrin–estradiol alone. Kinetic studies revealed that this was due to a faster formation of the intermediate high valent iron–oxo species in the presence of the antibody protein. Consequently, the association of an anti-estradiol antibody with an iron–porphyrin–estradiol cofactor leads to a new artificial hemoprotein with an interesting peroxidase activity and the “Trojan horse” strategy appears as a valuable method to generate artificial metalloenzymes that could act as biocatalysts for selective oxidations.

Various strategies for obtaining artificial hemoproteins: From “hemoabzymes” to “hemozymes”

The design of artificial hemoproteins that could lead to new biocatalysts for selective oxidation reactions of organic compounds presents a huge interest especially in pharmacology, both for a better understanding of the metabolic profile of drugs and for the synthesis of enantiomerically pure molecules that could be involved in the design of drugs. The present results show that the so-called “host-guest strategy” that involves the non-covalent incorporation of anionic water-soluble iron-porphyrins into xylanase A from Streptomyces lividans, a low cost protein, leads to such an artificial hemoprotein that is able to perform the stereoselective oxidation of sulfides.

Selective oxidation of aromatic sulfide catalyzed by an artificial metalloenzyme: new activity of hemozymes

Two new artificial hemoproteins or "hemozymes", obtained by non covalent insertion of Fe(III)-meso-tetra-p-carboxy- and -p-sulfonato-phenylporphyrin into xylanase A from Streptomyces lividans, were characterized by UV-visible spectroscopy and molecular modeling studies, and were found to catalyze the chemo- and stereoselective oxidation of thioanisole into the S sulfoxide, the best yield (85 +/- 4%) and enantiomeric excess (40% +/- 3%) being obtained with Fe(III)-meso-tetra-p-carboxyphenylporphyrin-Xln10A as catalyst in the presence of imidazole as co-catalyst.

The role of an amino acid triad at the entrance of the heme pocket in human serum albumin for O2 and CO binding to iron protoporphyrin IX

Complexation of iron(II) protoporphyrin IX (Fe(2+)PP) into a genetically engineered heme pocket on recombinant human serum albumin (rHSA) creates an artificial hemoprotein which can bind O(2) reversibly at room temperature. Here we highlight a crucial role of a basic amino acid triad the entrance of the heme pocket in rHSA (Arg-114, His-146, Lys-190) for O(2) and CO binding to the prosthetic Fe(2+)PP group. Replacing His-146 and/or Lys-190 with Arg resolved the structured heterogeneity of the possible two complexing modes of the porphyrin and afforded a single O(2) and CO binding affinity. Resonance Raman spectra show only one geometry of the axial His coordination to the central ferrous ion of the Fe(2+)PP.

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

AbstractSummary: 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 ($P_{1/2}^{{\rm O}_2 } $: 1 Torr), which is 18‐fold greater than that of the original double mutant rHSA(HL)–heme and which is rather close to those of Hb (R‐state) and Mb. Furthermore, replacement of polar Arg‐186 with Leu or Phe adjusted the O2 binding affinity ($P_{1/2}^{{\rm O}_2 } $) to 10 Torr, which is almost equivalent to value for human red blood cells.

Hemozymes Peroxidase Activity Of Artificial Hemoproteins Constructed From the Streptomyces lividans Xylanase A and Iron(III)-Carboxy-Substituted Porphyrins

To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-alpha4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 microM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV-visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)-Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.

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.

Artificial hemoprotein nanotubes

Artificial hemoprotein nanotubes have been prepared by a layer-by-layer deposition technique with human serum albumin (HSA) incorporating the synthetic heme (FeP) [HSA-FeP] using an anodic porous alumina template; each of the liberated tubules has a very uniform outer/inner diameter and can reversibly bind dioxygen (O(2)) at 25 degrees C.

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.

Albumin Clusters: Structurally Defined Protein Tetramer and Oxygen Carrier Including Thirty-Two Iron(II) Porphyrins

Recombinant human serum albumin (rHSA) clusters have been synthesized and physicochemically characterized. Cross-linking between the Lys groups of the core albumin and a unique Cys-34 of the shell albumins with an N-succinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate produced the structurally defined rHSA trimer and tetramer. MALDI-TOF-MS showed a single peak with the triple and quadruple masses of rHSA. Their molar ellipticities and the isoelectric points (pI = 4.8) are all identical to those of the monomer, suggesting that the essential structures of the albumin units were intact. TEM observations demonstrated a uniform morphology of the rHSA tetramer with a diameter of 20-30 nm. The circulation half-life (tau1/2) of the 125I-labeled rHSA tetramer in rat (5.5 h) was significantly longer than that of the monomer (2.3 h) due to the low ratio of the distribution phase (alpha-phase). A total of 24 and 32 molecules of the synthetic iron(II) porphyrins (FePs) are incorporated into the hydrophobic cavities of the rHSA trimer and tetramer, respectively, producing huge artificial hemoproteins. These albumin-heme clusters can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) and showed similar O2-binding properties (O2-binding affinity, association and dissociation rate constants) to those of the corresponding monomer. A large volume of O2 can be chemically dissolved into the albumin-heme cluster solutions relative to the monomeric rHSA-FeP when the molar concentration of the albumin scaffold is identical.

Heat-Resistant Oxygen-Carrying Hemoproteins Consist of Recombinant Xylanases and Synthetic Iron(II) Porphyrin

Synthetic iron(II) porphyrin (FeP) is equivalently incorporated into recombinant Thermotoga maritima xylanase B (TMX; family F/10 of glycoside hydrolase), producing a heat-resistant artificial hemoprotein (TMX-FeP) that can bind and release oxygen (O(2)) in aqueous medium (pH 7.3, 25 degrees C) in the same manner as hemoglobin and myoglobin. The oxygenated species was sufficiently stable; the half-lifetime against the ferric state (tau(1/2)) was 5 h. This O(2)-carrying hemoprotein showed a high degree of thermal stability over a wide range of temperatures up to 90 degrees C (tau(1/2) = 5 min at 90 degrees C and 9 min at 75 degrees C). Dictyoglomus thermophilum xylanase B (DTX; family G/11) also incorporates FeP, and DTX-FeP showed identical O(2)-binding parameters and thermostability. TMX-FeP is capable of catalyzing the beta-1,4-d-xylan hydrolysis reaction. Its larger K(m) value compared to that of TMX itself suggested competitive FeP binding to the active site of the host enzyme.

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.

Synthesis of protoheme IX derivatives with a covalently linked proximal base and their human serum albumin hybrids as artificial hemoprotein

The simple one-pot reaction of protoporphyrin IX and omega-(N-imidazolyl)alkylamine or O-methyl-L-histidyl-glycine with benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate at room temperature produced a series of protoporphyrin IX species with a covalently linked proximal base at the propionate side-chain. The central iron was inserted by the general FeCl2 method, converting the free-base porphyrins to the corresponding protoheme IX derivatives. Mesoporphyrin IX and diacetyldeuteroporphyrin IX analogues were also prepared by the same procedure. The Fe(II) complexes formed dioxygen (O2) adducts in dimethylformamide at 25 degrees C. Some of them were incorporated into the hydrophobic domain of recombinant human serum albumin (rHSA), providing albumin-heme hybrids (rHSA-heme), which can bind and release O2 in aqueous media (pH 7.3, 25 degrees C). The oxidation process of converting the dioxygenated heme in rHSA to the inactive Fe(III) state obeyed first-order kinetics, indicating that the mu-oxo dimer formation was prevented by the immobilization of heme in the albumin scaffold. The rHSA-heme, in which the histidylglycil tail coordinates to the Fe(II) center, showed the most stable O2 adduct complexes.

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

AbstractRecombinant 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 (P1/2NO: 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).