Role of histidine in ligand binding ability of hemoglobin gene

The atomic and electronic structures of heme complexes with His, Gly, and Cys residues (Heme-His, Heme-Gly, and Heme-Cys) in the fifth coordination position of the Fe atom and with oxygen and nitrogen oxide molecules in the sixth Fe position were studied by the semiempirical quantum-chemical method PM3. A comparative analysis of internuclear distances showed that the strength of chemical bonding between the ligand molecules (oxygen and nitrogen oxide) is greater for Heme-Cys than for Heme-His and Heme-Gly complexes. Consequently, the strengthening of the chemical bond of the oxygen (or nitrogen oxide) molecule with Heme-Cys substantially weakens the chemical bond in the ligand molecule. The Mulliken population analysis showed that the electronic density of ligand (oxygen or nitrogen oxide) p-orbitals is transferred to the d-orbitals of the Fe atom, whose charge, calculated according to the Mulliken analysis, formally becomes negative. In the Heme-His complex with oxygen, this charge is substantially greater than in the complex with NO, and the oxygen molecule becomes polarized. No oxygen polarization is observed in the Heme-Cys complex, and the electron density (judging from the change in the Fe charge) is transferred to the coordinated sulfur atom. This is also characteristic of Heme-Cys complexes with nitrogen oxide. An analysis of charges on the atoms indicates that the character of chemical bonding of the oxygen molecule in Heme-Cys and Heme-Gly complexes is similar and basically differs from that in the case of the Heme-His complex. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

The catalytic cycle of biosynthetic thiolase: a conformational journey of an acetyl group through four binding modes and two oxyanion holes

Biosynthetic thiolase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA. This is a key step in the synthesis of many biological compounds, including steroid hormones and ketone bodies. The thiolase reaction involves two chemically distinct steps; during acyl transfer, an acetyl group is transferred from acetyl-CoA to Cys89, and in the Claisen condensation step, this acetyl group is further transferred to a second molecule of acetyl-CoA, generating acetoacetyl-CoA. Here, new crystallographic data for Zoogloea ramigera biosynthetic thiolase are presented, covering all intermediates of the thiolase catalytic cycle. The high-resolution structures indicate that the acetyl group goes through four conformations while being transferred from acetyl-CoA via the acetylated enzyme to acetoacetyl-CoA. This transfer is catalyzed in a rigid cavity lined by mostly hydrophobic side chains, in addition to the catalytic residues Cys89, His348, and Cys378. The structures highlight the importance of an oxyanion hole formed by a water molecule and His348 in stabilizing the negative charge on the thioester oxygen atom of acetyl-CoA at two different steps of the reaction cycle. Another oxyanion hole, composed of the main chain nitrogen atoms of Cys89 and Gly380, complements a negative charge of the thioester oxygen anion of the acetylated intermediate, stabilizing the tetrahedral transition state of the Claisen condensation step. The reactivity of the active site may be modulated by hydrogen bonding networks extending from the active site toward the back of the molecule.     

Axial ligand complexes of the<Emphasis Type="Italic"> Rhodnius</Emphasis> nitrophorins: reduction potentials,binding constants,EPR spectra,and structures of the 4-iodopyrazole and imidazole complexes of NP4

Previously, we utilized 4-iodopyrazole (4IPzH) as a heavy atom derivative for the initial solution of the crystal structure of the nitrophorin from Rhodnius prolixus, NP1, where it was found to bind to the heme with the iodo group disordered in two positions. We have now determined the structure of the 4IPzH complex of NP4 at pH 7.5 and find that the geometry and bond lengths at the iron center are extremely similar to those of the imidazole (ImH) complex of the same protein (structure determined at pH 5.6), except that the G–H loop is not in the closed conformation. 4IPzH binds to the heme of NP4 in an ordered manner, with the iodo substituent pointed toward the opening of the heme pocket, near the surface of the protein. In order to understand the solution chemistry in terms of the relative binding abilities of 4IPzH, ImH, and histamine (Hm, a physiological ligand for the nitrophorins), we have also investigated the equilibrium binding constants and reduction potentials of these three ligand complexes of the four Rhodnius nitrophorins as a function of pH. We have found that, unlike the other Lewis bases, 4IPzH forms less stable complexes with the Fe(III) than the Fe(II) oxidation states of NP1 and NP4, and similar stability for the two oxidation states of NP2 and NP3, suggesting that this ligand is a softer base than ImH or Hm, for both of which the Fe(III) complexes are more stable than those of Fe(II) for all four nitrophorins. Surprisingly, in spite of this and the much lower basicity of 4IPzH than imidazole and histamine, the EPR g-values of all three ligand complexes are very similar.Abbreviations NP1–4 nitrophorins 1–4 from Rhodnius prolixus - 4IPzH 4-iodopyrazole - ImH imidazole - Hm histamine - NO nitric oxide - NOS nitric oxide synthase     

Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase

Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks.     

Hydrolysis of the amide bond in methionine-containing peptides catalyzed by various palladium(II) complexes: Dependence of the hydrolysis rate on the steric bulk of the catalyst

¹H NMR spectroscopy was applied to study the reactions of cis-[Pd(L)(H₂O)₂]²⁺ complexes (L is en, pic and dpa) with the N-acetylated tripeptides L-methionylglycylglycine, MeCOMet–Gly–Gly, and glycyl–L-methionyl–glycine, MeCOGly–Met–Gly. All reactions were performed in the pH range 2.0–2.5 with equimolar amounts of the cis-[Pd(L)(H₂O)₂]²⁺ complex and the tripeptide at 60 °C. The hydrolytic reactions of the cis-[Pd(en)(H₂O)₂]²⁺, cis-[Pd(pic)(H₂O)₂]²⁺ and cis-[Pd(dpa)(H₂O)₂]²⁺ complexes with MeCOMet–Gly–Gly were regioselective and only the amide bond involving the carboxylic group of methionine was cleaved. However, in the reactions of these three Pd(II) complexes with MeCOGly–Met–Gly, two amide bonds, Met–Gly and MeCO–Gly, were cleaved. From UV–Vis spectrophotometry studies, it was found that the rate-determining step of these hydrolytic reactions is the monodentate coordination of the corresponding Pd(II) complex to the sulfur atom of the methionine side chain. The rate of the cleavage of these amide bonds is dependent on the nature of the bidentate coordinated diamine ligand L (en > pic > dpa). The hydrolytic reaction of cis-[Pd(L)(H₂O)₂]²⁺-type complexes with MeCOMet–Gly–Gly, containing the methionine side chain in the terminal position of the peptide, is regioselective while in the reaction of these Pd(II) complexes with MeCOGly–Met–Gly, none selective cleavage of the peptide occurs. This study contributes to a better understanding of the selective cleavage of methionine-containing peptides employing palladium(II) complexes as catalysts.     

Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli

Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.     

Biological Investigations and Spectroscopic Studies of New Moxifloxacin/Glycine‐Metal Complexes.

Two novel ligand?metal complexes were prepared through the reaction of Zn(II) and Sn(II) with moxifloxacin (MOX) in the presence of glycine (Gly) to investigate their biological activities. IR, UV/VIS and ¹H‐NMR analysis have been carried out for insuring the chelation process. Results suggested that MOX and Gly react with the metal ions through the carbonyl oxygen atom and the oxygen atom of the carboxylic group of MOX and Gly. The antimicrobial activity was carried out against some common bacterial and fungal pathogens and the radical scavenging activity (RSA%) was evaluated using DPPH and ABTS methods. Phytotoxic effect of the prepared complexes was evaluated in vitro against Raphanus raphanistrum and Lepidium sativum. Hemolytic activity was tested against cell membrane of erythrocytes. Results showed that the two prepared complexes exhibited high antimicrobial activity against all tested phytopathogens and no significant phytotoxic effect has been observed. Only MOX?Zn(II) complex showed moderate hemolysis at 100 % concentration.     

The structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin

Copper K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and (15)N NMR relaxation studies were performed on samples of a variant azurin in which the surface-exposed histidine ligand of the copper atom (His117) has been replaced by glycine. The experiments were performed to probe the structure of the active site and the protein dynamics. The cavity in the protein structure created by the His-->Gly replacement could be filled by external ligands, which can either restore the spectroscopic properties of the original type-1 copper site or create a new type-2 copper site. The binding of external ligands occurs only when the copper atom is in its oxidised state. In the reduced form, the binding is abolished. From the EXAFS experiments, it is concluded that for the oxidised type-1 copper sites the protein plus external ligand (L) provide an NSS*L donor set deriving from His46, Cys112, Met121 and the external ligand. The type-2 copper site features an S(N/O)(3) donor set in which the S-donor derives from Cys112, one N-donor from His46 and the remaining two N or O donors from one or more external ligands. Upon reduction of the type-1 as well as the type-2 site, the external ligand drops out of the copper site and the coordination reduces to 3-fold with an SS*N donor set deriving from His46, Cys112 and Met121. The Cu-S(delta)(Met) distance is reduced from about 3.2 to 2.3 A. Analysis of the NMR data shows that the hydrophobic patch around His117 has gained fluxionality when compared to wild-type azurin, which may explain why the His117Gly variant is able to accommodate a variety of external ligands of different sizes and with different chelating properties. On the other hand, the structure and dynamics of the beta-sandwich, which comprises the main body of the protein, is only slightly affected by the mutation. The unusually high reduction potential of the His117Gly azurin is discussed in light of the present results.     

Coordination between manganese and nitrogen within the ligands in the manganese complexes facilitates the reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations

The water-oxidizing complex (WOC) within photosystem II (PSII) can be reconstituted with synthetic manganese complexes by a process called photoactivation; however, the key factors affecting the efficiency of synthetic manganese complexes in reconstitution of electron transport and oxygen evolution activity in manganese-depleted PSII remain unclear. In the present study, four complexes with different manganese coordination environments were used to reconstitute the WOC, and an interesting relationship was found between the coordination environment of the manganese atom in the complexes and their efficiency in restoring electron transport and oxygen evolution. If Mn(II) is coordinated to nitrogen atoms within the ligand, it can restore significant rates of electron transport and oxygen evolution; however, if the manganese atom is coordinated only to oxygen atoms instead of nitrogen atoms, it has no capability to restore electron transport and oxygen evolution. So, our results demonstrate that the capability of manganese complexes to reconstitute the WOC is mainly determined by the coordination between nitrogen atoms from ligands and the manganese atom. It is suggested from our results that the ligation between the nitrogen atom and the manganese atom within the manganese complex facilitates the photoligation of the manganese atom to histidyl residues on the apo-protein in manganese-depleted PSII during photoactivation.     

Ligand replacement study at the His120 site of purple CuA azurin

The CuA center is a dinuclear Cu2S2(Cys) electron transfer center found in cytochrome c oxidase and nitrous oxide reductase. In a previous investigation of the equatorial histidine ligands' effect on the reduction potential, electron transfer and spectroscopic properties of the CuA center, His120 in the engineered CuA azurin was mutated to Asn, Asp, and Ala. The identical absorption and EPR spectra of these mutants indicate that a common ligand is bound to the copper center. To identify this replacement ligand, the His120Gly CuA azurin mutant was constructed and purified. Absorption and X-band EPR spectra show that His120Gly is similar to the other His120X (X = Asn, Asp, Ala) mutant proteins. Titrations with chloride, imidazole, and azide suggest that the replacement ligand is not exchangeable with exogenous ligands. The possibility of an internal amino acid acting as the replacement ligand for His120 in the His120X mutant proteins was investigated by analyzing the CuA azurin crystal structure and then converting the likely internal ligand, Asn 119, to Asp, Ser, or Ala in the His120Gly mutant. The double mutants H120G/Asn 119X (X = Asp, Ser, or Ala) displayed UV-Vis absorption and EPR spectra that are identical to His120Gly and the other His120X mutants, indicating that Asn119 is not the internal ligand replacing His120 in the His120X mutant proteins. These results demonstrate the remarkable stability of the dinuclear His120 mutants of CuA azurin.     

Synthesis, characterization and antiproliferative activity of metal complexes with the Schiff base derived from the condensation 1:2 of 2,6-diformyl-4-methylphenol and 5,6-diamino-1,3-dimethyluracil

A series of mononuclear complexes with Co(II), Ni(II), Cu(II), Zn(II), Hg(II), Mo(VI) and Pd(II) containing the ligand derived from the 1:2 condensation of 2,6-diformyl-4-methylphenol and 5,6-diamino-1,3-dimethyluracil (hereafter denoted as BDFDAAU) were synthesized. The complexes were characterized by elemental analysis, thermogravimetry (TG) and differential scanning calorimetry (DSC), IR, (1)H, (13)C and (15)N NMR, UV-visible-near IR (UV-VIS-NIR), EPR and magnetic measurements. The deprotonated ligand in the phenolic oxygen shows a symmetric tridentate coordination mode through the two azomethine nitrogen atoms and the phenolic oxygen atom whereas the coordination of the neutral ligand takes place through the phenolic oxygen atom and one azomethine nitrogen atom. In the Mo(VI) complex, the ligand is bideprotonated in the phenolic oxygen and an amino group from one uracil unit; so, the coordination mode changes again into an asymmetric way: phenolic oxygen atom, one azomethine nitrogen atom and the nitrogen atom from the deprotonated amino group. The antiproliferative behaviour against the five human tumor cell lines (human neuroblastoma NB69, human breast cancer MCF-7 and EVSA-T, human glioma H4 and human bladder carcinoma cell line ECV) suggested a modulator behaviour, according to the concentration, of cell growth due to their estrogen-like characteristics.     

Natural abundance 15N NMR assignments delineate structural differences between intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

15N NMR assignments were made to the backbone amide nitrogen atoms at natural isotopic abundance of intact and reactive-site (Arg5-Ile6) hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III and CMTI-III*, respectively) by means of 2D proton-detected heteronuclear single bond chemical shift correlation (HSBC) spectroscopy, utilizing the previously made sequence-specific 1H NMR assignments (Krishnamoorthi et al. (1992) Biochemistry 31, 898-904). Comparison of the 15N chemical shifts of the two forms of the inhibitor molecule revealed significant changes not only for residues located near the reactive-site region, but also for those distantly located. Residues Cys3, Arg5, Leu7, Met8, Cys10, Cys16, Glu19, His25, Tyr27, Cys28 and Gly29 showed significant chemical shift changes ranging from 0.3 to 6.1 ppm, thus indicating structural perturbations that were transmitted throughout the molecule. These findings confirm the earlier conclusions based on 1H NMR investigations.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

Zinc coordination sphere in biochemical zinc sites

Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein inteface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.     

Structural investigations of the CuA centre of nitrous oxide reductase from Pseudomonas stutzeri by site-directed mutagenesis and X-ray absorption spectroscopy.

Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate-reducing catalytic centre, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed-valence metal site. To provide further structural information on the CuA centre of N2O reductase, site directed mutagenesis and Cu K-edge X-ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA centre of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild-type residues. The mutations identified the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu1, and Cys618, Cys622 and His626 as the minimal set of ligands for Cu2 of the CuA centre. Other amino acid substitutions indicated His494 as a likely ligand of CuZ, and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K-edge X-ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N2O reductase. Cu K-edge Extended X-ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at approximately 2.5 A. The Cu K-edge EXAFS of the CuA centre of N2O reductase is very similar to that of the CuA centre of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu-N of 1.92 A, two sulfur atoms at 2.24 A and a Cu atom at 2. 43 A, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 A. The interpretation of the CuA EXAFS is in line with ligands assigned by site-directed mutagenesis. By a difference spectrum approach, using the Cu K-edge EXAFS of the holoenzyme and that of the CuA-only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA centre of N2O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.     

A Comparative Structure–Function Analysis and Molecular Mechanism of Action of Endonucleases from Serratia marcescens and Physarum polycephalum

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water–magnesium complex involved in their catalytic acts. In the enzyme–substrate complexes, the Mg²⁺ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3"-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3"-leaving group. A strained metalloenzyme–substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction.     

Steric interactions and the activity of fentanyl analogs at the mu-opioid receptor

Fentanyl is a highly potent and clinically widely used narcotic analgesic. The synthesis of its analogs remains a challenge in the attempt to develop highly selective mu-opioid receptor agonists with specific pharmacological properties. In this paper, the use of flexible molecular docking in a study of the formation of complexes between a series of active fentanyl analogs and the mu-opioid receptor is described. The optimal position and orientation of fourteen fentanyl analogs in the binding pocket of the mu-receptor were determined. The major receptor amino acids and the ligand functional groups participating in the complex formation were identified. Stereochemical effects on the potency and binding are explained. The proposed model of ligand-receptor binding is in agreement with point mutation experiments explaining the role of the amino acids: Asp147, Tyr148, Asn230, His297, Trp318, His319, Cys321, and Tyr326 in the complex formation. In addition, the following amino acids were identified as being important for ligand binding or receptor activation: Ile322, Gly325, Val300, Met203, Leu200, Val143, and Ile144.     

Nitrosyl-heme structures of Bacillus subtilis nitric oxide synthase have implications for understanding substrate oxidation

The crystal structures of nitrosyl-heme complexes of a prokaryotic nitric oxide synthase (NOS) from Bacillus subtilis (bsNOS) reveal changes in active-site hydrogen bonding in the presence of the intermediate N(omega)-hydroxy-l-arginine (NOHA) compared to the substrate l-arginine (l-Arg). Correlating with a Val-to-Ile residue substitution in the bsNOS heme pocket, the Fe(II)-NO complex with both l-Arg and NOHA is more bent than the Fe(II)-NO, l-Arg complex of mammalian eNOS [Li, H., Raman, C. S., Martasek, P., Masters, B. S. S., and Poulos, T. L. (2001) Biochemistry 40, 5399-5406]. Structures of the Fe(III)-NO complex with NOHA show a nearly linear nitrosyl group, and in one subunit, partial nitrosation of bound NOHA. In the Fe(II)-NO complexes, the protonated NOHA N(omega) atom forms a short hydrogen bond with the heme-coordinated NO nitrogen, but active-site water molecules are out of hydrogen bonding range with the distal NO oxygen. In contrast, the l-Arg guanidinium interacts more weakly and equally with both NO atoms, and an active-site water molecule hydrogen bonds to the distal NO oxygen. This difference in hydrogen bonding to the nitrosyl group by the two substrates indicates that interactions provided by NOHA may preferentially stabilize an electrophilic peroxo-heme intermediate in the second step of NOS catalysis.     

The structure of the iron complex in metaquohemerythrin

The novel binuclear iron complex in metaquohemerythrin is described. One of the two iron atoms is octahedrally coordinated, the other being penta-coordinate. A number of questions concerning the structure of the metal complex in different forms of this nonheme iron oxygen transport protein have been clarified. The structure of the complex presented here differs from that of metazidohemerythrin in that the Fe atom providing the binding locus for azide ion is five-coordinate with no small molecule ligand bound to it. The coordination polyhedron of this Fe is best described as a trigonal bipyramid.