Cataldo Tarricone

Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp.

BACKGROUND The first hemoglobin identified in bacteria was isolated from Vitreoscilla stercoraria (VtHb) as a homodimeric species. The wild-type protein has been reported to display medium oxygen affinity and cooperative ligand-binding properties. Moreover, VtHb can support aerobic growth in Escherichia coli with impaired terminal oxidase function. This ability of VtHb to improve the growth properties of E. coli has important applications in fermentation technology, assisting the overexpression of recombinant proteins and antibiotics. Oxygen binding heme domains have been identified in chimeric proteins from bacteria and yeast, where they are covalently linked to FAD- and NAD(P)H-binding domains. We investigate here the fold, the distal heme site structure and the quaternary assembly of a bacterial hemoglobin which does not bear the typical flavohemoglobin domain organization. RESULTS The VtHb three-dimensional structure conforms to the well known globin fold. Nevertheless, the polypeptide segment connecting helices C and E is disordered, and residues E7-E10 (defined according to the standard globin fold nomenclature) do not adopt the usual alpha-helical conformation, thus locating Gln53(E7) out of the heme pocket. Binding of azide to the heme iron introduces substantial structural perturbations in the heme distal site residues, particularly Tyr29(B10) and Pro54(E8). The quaternary assembly of homodimeric VtHb, not observed before within the globin family, is based on a molecular interface defined by helices F and H of both subunits, the two heme iron atoms being 34 A apart. CONCLUSIONS The unusual heme distal site structure observed shows that previously undescribed molecular mechanisms of ligand stabilization are operative in VtHb. The polypeptide chain disorder observed in the CE region indicates a potential site of interaction with the FAD/NADH reductase partner, in analogy with observations in the chimeric flavohemoglobin from Alcaligenes eutrophus.

Azide, cyanide, fluoride, imidazole and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative: A comparative study

Azide, cyanide, fluoride, imidazole, and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated, from the thermodynamic viewpoint, at pH 7.5 and 25.0 degrees C. Ligand affinity for ferric and ferrous carboxymethylated cytochrome c is higher by about 30- and 400-fold, respectively, than that observed for the native protein. The results here reported: (i) allow the estimation, for the first time, of the ligand-independent free energy associated with the heme-iron sixth coordination bond in ferric and ferrous native cytochrome c, which turns out to be +8.4 kJ mol-1 and +14.6 kJ mol-1, at 25.0 degrees C, respectively, and (ii) suggest an interplay between redox, structural, ligand binding, and recognition properties of cytochrome c.

Overproduction and Characterization of the Bacillus subtilis Anti-sigma Factor FlgM

FlgM is an anti-sigma factor of the flagellar-specific sigma (ς) subunit of RNA polymerase inBacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and characterized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate ςDfactor. The FlgM·ςD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing. ςD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase. The FlgM·ςD complex gave a shift in a DNA mobility shift assay with a probe containing a ςD-dependent promoter sequence. Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively.

Expression, purification, crystallization, and preliminary X‐Ray diffraction analysis of the homodimeric bacterial hemoglobin from Vitreoscilla stercoraria

The recombinant homodimeric hemoglobin from the strictly aerobe gram‐negative bacterium Vitreoscilla stercoraria has been expressed in Escherichia coli, purified to homogeneity, and crystallized by vapor diffusion techniques, using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P21 and diffract to HIGH resolution. The unit cell parameters are a = 62.9, b = 42.5, c = 63.2 Å, β = 106.6°; the asymmetric unit contains the homodimeric hemoglobin, with a volume solvent content of 42%. Proteins 27:154–156

Formate binding to ferric wild type and mutant myoglobins Thermodynamic and X-ray crystallographic study

The X‐ray crystal structure of the formate derivative of ferric loggerhead sea turtle (Caretta caretta) Mb has been determined at 2.0 Å resolution (R = 0.164) by difference Fourier techniques. Formate, sitting in the central part of the heme distal site, is coordinated to the heme iron as unidentate ligand, through the O1 oxygen atom, and is hydrogen bonded to the distal His64(E7) NE2 atom through O2. Thermodynamics for formate binding to ferric loggerhead sea turtle Mb, sperm whale Mb, Aplysia limacina Mb, as well as to the VR and VRS mutants of sperm whale Mb were obtained between pH 4.5 and 8.5, at 20.0°C. These results, representing the first structure of a ferric hemoprotein:formate complex solved by X‐ray crystallography, outline the role of amino acid residues at positions E7, F8 and E10 in modulating ligand binding properties of oxygen carrying proteins.

1H-NMR relaxometric study of pancreatic serine (pro)enzyme inhibition by a Gd(III) chelate bearing boronic functionalities

Binding of the paramagnetic N,N″‐bis(m‐boroxyphenylcarbamoylmethyl)‐diethylenetriamine‐N,N′,N″‐triacetic acid Gd(III) complex (GdBB) to chymotrypsin, chymotrypsinogen, trypsin, trypsinogen and pancreatic elastase has been investigated by 1H‐NMR relaxometry, between pH 6.0 and 8.5, at 25.0°C. Values of Ki for the competitive inhibition of serine proteinases by GdBB are in excellent agreement with values of Kd obtained by 1H‐NMR relaxometry, suggesting that the substrate and the paramagnetic complex bind to the same region. Moreover, 1H‐NMR relaxometry allowed to determine values of Kd for GdBB binding to chymotrypsinogen and trypsinogen, both devoid of catalytic activity. The increase of the water proton relaxation rate upon GdBB binding to serine (pro)enzymes may be useful in the design of novel functional contrast agents for magnetic resonance imaging.