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.     

Influence of the distal his in imparting imidazolate character to the proximal his in heme peroxidase: (1)h NMR spectroscopic study of cyanide-inhibited his42-->ala horseradish peroxidase

The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.     

Influences of the Hydrophobicity of the Heme－binding Pocket on the Properties and Functions of Cytochrome b5 Mutants

The mutation sites of the four mutants F35Y, P40V, V45E and V45Y of cytochrome b5 are located at the edge of the heme-binding pocket. The solvent accessible areas of the “pocket inte-rior“ of the four mutants and the wild-type cytochrome b5 have been calculated based on their crystal structures at high resolu-tion. The change in the hydrophobicity of the heme-binding pocket resulting from the mutation can be quantitatively de-scribed using the difference of the solvent accessible area of the “pocket interior“ of each mutant from that of the wild-type cy-tochrome b5. The influences of the hydrophobicity of the heme-binding pocket on the protein stability and redox potential are discussed.     

Study on the Gas Phase Stability of Heme-binding Pocket in Cytochrome Tb_5 and Its Mutants by Electrospray Mass Spectrometry

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Probing the function of Mycobacterium tuberculosis catalase-peroxidase by site-directed mutagenesis

Catalase-peroxidase is a multi-functional heme-dependent enzyme which is well known for its ability to carry out both catalatic and peroxidatic reactions. Catalase-peroxidase from Mycobacterium tuberculosis(mtCP) is of particular interest because this enzyme activates the pro-antitubercular drug isoniazid. It is estimated that 2 billion people are infected with M. tuberculosis, the principal causative agent of tuberculosis, and that 2 million people die from the disease each year. The rise of drug-resistant strains continues to be of critical concern and it is well documented that mutations which reduce activity or inactivate mtCP lead to increased levels of isoniazid resistance in M. tuberculosis. The recent determination of the crystal structure for M. tuberculosis mtCP has aided the understanding of how the enzyme functions and provides a three-dimensional framework for testing hypotheses about the roles of various residues in the active site. Here we report site-directed mutagenesis studies of three conserved residues located near the heme of mtCP, His-108, Trp-107 and Trp-321 including the construction of the double mutant W107F-W321F. Resulting mutants have been purified and their catalatic and peroxidatic activities have been determined. Data are compared in the context of related studies aimed at dissecting the roles of these residues in the different activities of the enzyme. Analyses of single and double mutants studied here emphasise that the hydrogen bonding network surrounding the heme in the active site appears more important for maintenance of catalatic rather than peroxidatic activity in CP enzymes.     

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.     

A distal pocket Leu residue inhibits the binding of O2 and NO at the distal heme site of cytochrome c'

Cytochromes c' are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O(2). Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O(2) binding (K(d) ~49 nM) without apparent H-bond stabilization of the Fe-O(2) moiety, as well as an extremely high distal heme-NO affinity (K(d) ~70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing k(on) as well as increasing k(off). The connection between distal steric constraints, k(off) values, and distal to proximal heme-NO conversion is discussed.     

Microemulsion-controlled reaction sites in biocatalytic films for electrochemical reduction of vicinal dibromides

We report herein the electrochemical dehalogenation of vicinal dibromides in microemulsions using cross-linked films of the redox protein myoglobin (Mb) and poly-l-lysine (PLL) covalently bonded to carbon electrodes. Catalytic reduction of the dibromides to olefins was more efficient in an SDS microemulsion than in a CTAB microemulsion. SDS shifts the Mb redox potential more negative, but a comparison to Mb-SDS films suggests that the activation free energy of the reduction is controlled by an inner-sphere mechanism. SDS also enters the positively charged Mb-PLL films and preconcentrates the dibromide reactants, enhancing catalytic efficiency in SDS microemulsions. Shifts in formal potential and Soret absorbance bands for Mb-PLL films suggested binding of trans-1,2-dibromocyclohexane in the iron heme distal pocket with little catalysis. Results are consistent with active catalytic reduction sites for reactant bound on the protein surface and less-reactive sites in the distal heme pocket. Preconcentration into catalytic PLL films using SDS incorporated from microemulsions may be a general way to improve catalytic efficiency for nonpolar reactants in microemulsions.     

Unusual ligand discrimination by a myoglobin reconstituted with a hydrophobic domain-linked heme

New, reconstituted horse heart myoglobins possessing a hydrophobic domain at the terminal of the two heme propionate side chains were constructed. The O2 and CO bindings for the reconstituted deoxymyoglobins were examined in detail by laser flash photolysis and stopped-flow rapid mixing techniques. The artificially created domain worked as a barrier against exogenous ligand penetration into the heme pocket, whereas the bound O2 was stabilized in the reconstituted myoglobin as well as in the native one. In contrast, the CO dissociation rate for the reconstituted myoglobin increased by 20-fold compared to the native protein, suggesting that the incorporation of the hydrophobic domain onto the heme pocket perturbs the distal-site structure of the reconstituted myoglobin. As a result, the substantial ligand selectivity for the reconstituted myoglobin significantly increases in favor of O2 over CO with the M' value (= KCO/KO2) of 0.88, whereas, to the best of our knowledge, there is no myoglobin mutant in which the O2 affinity exceeds the CO one. The present work concludes that the O2 selectivity of myoglobin over CO is markedly improved by chemically modifying the heme propionates without any mutation of the amino acid residues in the distal site.     

Studies on Cytochrome c Modified by [PtdienNO_3]Cl

For the first time, [PtdienNO_3]Cl was used as a stable reagent to modify ferricytochrome c and the reaction products were separated and purified with the CM-52 cation exchange chromatography. Five components were obtained, corresponding to the native cytochrome c single-labeled, dual-labeled, and triple-labeled derivatives as shown by the analysis of the molar ratio of the two metal atoms (Pt and Fe). The reduction potentials of these proteins were measured by differential pulse voltammetry. His-33 and Trp-59 were identified by~1HNMR as the binding sites of the platinum complex in the modified cytochrome c derivatives. Trp-59 was a conserved amino acid connected with the heme through hydrogen bond, which had not been modified by other transition metal complexes. The platinummodified cytochrome c derivatives might be valuable in exploring the role of the aromatic amino acids, especially Trp-59, in electron transfer.     

Structures of Cytochrome b_5 Mutated at the Charged Surface-Residues and Their Interactions with Cytochrome c

ＩｎｔｒｏｄｕｃｔｉｏｎＭｉｃｒｏｓｏｍａｌｃｙｔｏｃｈｒｏｍｅｂ5(Ｃｙｔｂ5)ｉｓａｍｅｍｂｅｒｏｆｃｙｔｏｃｈｒｏｍｅｂ5ｆａｍｉｌｙ ,ａｎｄｉｔｓｅｒｖｅｓａｓａｎｅｌｅｃｔｒｏｎｃａｒｒｉｅｒｉｎａｓｅｒｉｅｓｏｆｅｌｅｃｔｒｏｎ ｔｒａｎｓｆｅｒｐｒｏｃｅｓｓｅｓｉｎｂｉｏｌｏｇｉｃａｌｓｙｓ ｔｅｍｓ .1 3 Ｃｙｔｂ5ｉｓａｍｅｍｂｒａｎｅｐｒｏｔｅｉｎｗｉｔｈＭｒ～ 16ｋＤａ ,ｃｏｎｓｉｓｔｉｎｇｏｆｔｗｏｄｏｍａｉｎｓ ,ｏｎｅｈｙｄｒｏｐｈｏｂｉｃｄｏｍａｉｎｗｈｉｃｈａｎｃｈｏｒｓｔｈ…     

Clarification of the role of protein in carbonmonoxy myoglobin by investigating electronic states

This article reports the proton tautomerization effects of distal histidine residues in carbonmonoxy myoglobin according to the density functional calculations of the whole protein. The electron eigenstates and electrostatic potential (ESP) distributed around heme and its pocket vary significantly depending on the protonation positions of the distal histidine residues. To investigate the range over which the electronic structures are affected by the proton tautomerization, the quantum mechanics/molecular mechanics (QM/MM) method is applied to probe the QM size to reproduce the atomic partial charges and ESP around the active center. Consequently, we show that these properties converged for the 300 pm QM/MM system in this study. During the analysis, we also find that amino residues such as Phe43, Val68, and Phe138 interact strongly with heme through orbital mixing, indicating that the protein is a medium not only interacting with the reaction center, but also buffering on electrons. © 2013 Wiley Periodicals, Inc.     

Solution 1H NMR of the molecular and electronic structure of the heme cavity and substrate binding pocket of high-spin ferric horseradish peroxidase: effect of His42Ala mutation.

Solution 1H NMR has been used to assign a major portion of the heme environment and the substrate-binding pocket of resting state horseradish peroxidase, HRP, despite the high-spin iron(III) paramagnetism, and a quantitative interpretive basis of the hyperfine shifts is established. The effective assignment protocol included 2D NMR over a wide range of temperatures to locate residues shifted by paramagnetism, relaxation analysis, and use of dipolar shifts predicted from the crystal structure by an axial paramagnetic susceptibility tensor normal to the heme. The most effective use of the dipolar shifts, however, is in the form of their temperature gradients, rather than by their direct estimation as the difference of observed and diamagnetic shifts. The extensive assignments allowed the quantitative determination of the axial magnetic anisotropy, Deltachi(ax) = -2.50 x 10(-8) m(3)/mol, oriented essentially normal to the heme. The value of Deltachi(ax) together with the confirmed T(-2) dependence allow an estimate of the zero-field splitting constant D = 15.3 cm(-1), which is consistent with pentacoordination of HRP. The solution structure was generally indistinguishable from that in the crystal (Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nature Structural Biology 1997, 4, 1032-1038) except for Phe68 of the substrate-binding pocket, which was found turned into the pocket as found in the crystal only upon substrate binding (Henriksen, A.; Schuller, D. J.; Meno, K.; Welinder, K. G.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8054-8060). The reorientation of several rings in the aromatic cluster adjacent to the proximal His170 is found to be slow on the NMR time scale, confirming a dense, closely packed, and dynamically stable proximal side up to 55 degrees C. Similar assignments on the H42A-HRP mutant reveal conserved orientations for the majority of residues, and only a very small decrease in Deltachi(ax) or D, which dictates that five-coordination is retained in the mutant. The two residues adjacent to residue 42, Ile53 and Leu138, reorient slightly in the mutant H42A protein. It is concluded that effective and very informative 1H NMR studies of the effect of either substrate binding or mutation can be carried out on resting state heme peroxidases.     

Strong ligand-protein interactions revealed by ultrafast infrared spectroscopy of CO in the heme pocket of the oxygen sensor FixL

In heme-based sensor proteins, ligand binding to heme in a sensor domain induces conformational changes that eventually lead to changes in enzymatic activity of an associated catalytic domain. The bacterial oxygen sensor FixL is the best-studied example of these proteins and displays marked differences in dynamic behavior with respect to model globin proteins. We report a mid-IR study of the configuration and ultrafast dynamics of CO in the distal heme pocket site of the sensor PAS domain FixLH, employing a recently developed method that provides a unique combination of high spectral resolution and range and high sensitivity. Anisotropy measurements indicate that CO rotates toward the heme plane upon dissociation, as is the case in globins. Remarkably, CO bound to the heme iron is tilted by ~30° with respect to the heme normal, which contrasts to the situation in myoglobin and in present FixLH-CO X-ray crystal structure models. This implies protein-environment-induced strain on the ligand, which is possibly at the origin of a very rapid docking-site population in a single conformation. Our observations likely explain the unusually low affinity of FixL for CO that is at the origin of the weak ligand discrimination between CO and O(2). Moreover, we observe orders of magnitude faster vibrational relaxation of dissociated CO in FixL than in globins, implying strong interactions of the ligand with the distal heme pocket environment. Finally, in the R220H FixLH mutant protein, where CO is H-bonded to a distal histidine, we demonstrate that the H-bond is maintained during photolysis. Comparison with extensively studied globin proteins unveils a surprisingly rich variety in both structural and dynamic properties of the interaction of a diatomic ligand with the ubiquitous b-type heme-proximal histidine system in different distal pockets.     

Serial Femtosecond Zero Dose Crystallography Captures a Water-Free Distal Heme Site in a Dye-Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV=O Formation

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (Fe^IV=O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the Fe^III and Fe^IV=O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.     

鼠脑红蛋白突变体F106L与氰根的结合作用

构建了鼠脑红蛋白(Mouse neuroglobin)的突变体F106L, 以探求近端残基对脑红蛋白血红素口袋结构的贡献. 通过溶液核磁共振方法研究了外来配体氰根离子与NgbF106L蛋白的结合作用, 结果显示, 此结合存在动力学过程, 并且NgbF106LCN 突变蛋白氰根络合物可以可逆地释放氰根离子, 并使原来的第6配体His64(E7)又结合回到血红素铁上. 研究结果揭示, G5(Phe106)残基对脑红蛋白血红素构象而言较为保守; QM/MM结构优化结果表明, 位于G5 和FG5的近端残基对蛋白结构稳定性具有重要作用, 并可调控外来配体与蛋白作用的配位平衡与热动力学性质.     

Regulating the Coordination State of a Heme Protein by a Designed Distal Hydrogen-Bonding Network

Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins.     

Assignment of the hyperfine-shifted 1H-NMR signals of the heme in the oxygen sensor FixL from Rhizobium meliloti

Background: The Rhizobial oxygen sensor FixL is a hemoprotein with kinase activity. On binding of strong-field ligands, a change of the ferrous or ferric heme iron from high to low spin reversibly inactivates the kinase. This spin-state change and other information on the heme pocket have been inferred from enzymatic assays, absorption spectra and mutagenesis studies. We set out to investigate the spin-state of the FixL heme and to identify the hyperfine-shifted heme-proton signals by NMR spectroscopy.Results: Using one-dimensional N MR we directly observed the high- and low-spin nature of the met- and cyanomet-FixL heme domain, respectively. We determined the hyperfine-shifted ¹H-NMR signals of the heme and the proximal histidine by one- and two-dimensional spectroscopy and note the absence of distal histidine signals.Conclusions: These findings support the spin-state mechanism of FixL regulation. They establish that the site of heme coordination is a histidine residue and strongly suggest that a distal histidine is absent. With a majority of the heme resonances identified, one- and two-dimensional NMR techniques can be extended to provide structural and mechanistic information about the residues that line the heme pocket.     

Unique Properties of Heme Binding of the Porphyromonas gingivalis HmuY Hemophore-like Protein Result from the Evolutionary Adaptation of the Protein Structure

To acquire heme, Porphyromonas gingivalis uses a hemophore-like protein (HmuY). HmuY sequesters heme from host hemoproteins or heme-binding proteins produced by cohabiting bacteria, and delivers it to the TonB-dependent outer-membrane receptor (HmuR). Although three-dimensional protein structures of members of the novel HmuY family are overall similar, significant differences exist in their heme-binding pockets. Histidines (H134 and H166) coordinating the heme iron in P. gingivalis HmuY are unique and poorly conserved in the majority of its homologs, which utilize methionines. To examine whether changes observed in the evolution of these proteins in the Bacteroidetes phylum might result in improved heme binding ability of HmuY over its homologs, we substituted histidine residues with methionine residues. Compared to the native HmuY, site-directed mutagenesis variants bound Fe(III)heme with lower ability in a similar manner to Bacteroides vulgatus Bvu and Tannerella forsythia Tfo. However, a mixed histidine-methionine couple in the HmuY was sufficient to bind Fe(II)heme, similarly to T. forsythia Tfo, Prevotella intermedia PinO and PinA. Double substitution resulted in abolished heme binding. The structure of HmuY heme-binding pocket may have been subjected to evolution, allowing for P. gingivalis to gain an advantage in heme acquisition regardless of environmental redox conditions.     

Coupled oxidation vs heme oxygenation: insights from axial ligand mutants of mitochondrial cytochrome b5

Mutation of His-39, one of the axial ligands in rat outer mitochondrial membrane cytochrome b(5) (OM cyt b(5)), to Val produces a mutant (H39V) capable of carrying out the oxidation of heme to biliverdin when incubated with hydrazine and O(2). The reaction proceeds via the formation of an oxyferrous complex (Fe(II)(-)O(2)) that is reduced by hydrazine to a ferric hydroperoxide (Fe(III)(-)OOH) species. The latter adds a hydroxyl group to the porphyrin to form meso-hydroxyheme. The observation that catalase does not inhibit the oxidation of the heme in the H39V mutant is consistent with the formation of a coordinated hydroperoxide (Fe(III)(-)OOH), which in heme oxygenase is the precursor of meso-hydroxyheme. By comparison, mutation of His-63, the other axial ligand in OM cyt b(5), to Val results in a mutant (H63V) capable of oxidizing heme to verdoheme in the absence of catalase. However, the oxidation of heme by H63V is completely inhibited by catalase. Furthermore, whereas the incubation of Fe(III)(-)H63V with H(2)O(2) leads to the nonspecific degradation of heme, the incubation of Fe(II)(-)H63V with H(2)O(2) results in the formation of meso-hydroxyheme, which upon exposure to O(2) is rapidly converted to verdoheme. These findings revealed that although meso-hydroxyheme is formed during the degradation of heme by the enzyme heme oxygenase or by the process of coupled oxidation of model hemes and hemoproteins not involved in heme catabolism, the corresponding mechanisms by which meso-hydroxyheme is generated are different. In the coupled oxidation process O(2) is reduced to noncoordinated H(2)O(2), which reacts with Fe(II)-heme to form meso-hydroxyheme. In the heme oxygenation reaction a coordinated O(2) molecule (Fe(II)(-)O(2)) is reduced to a coordinated peroxide molecule (Fe(III)(-)OOH), which oxidizes heme to meso-hydroxyheme.