No abstract

An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm(-1) and form 2 with ν(FeC) and ν(CO) at 518 and 1920 cm(-1), respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO.

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2-over-2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O(2) bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

No abstract

A tandem mass spectrometric study of protonated isomeric hydroxypyridine N-oxides was carried out with a hybrid quadrupole/time-of-flight mass spectrometer coupled with different atmospheric pressure ionization sources. The behavior observed in the collision-induced dissociation (CID) mass spectra of the parent cations, was similar irrespective of the source employed. However, there were intrinsic differences in the intensities of the two fragments observed for each isomer. The major fragment because of elimination of a hydroxyl radical, dominated the CID spectra (in contrast with weaker water loss) at different energy thresholds. Therefore, it was possible to differentiate both isomers at collision energies above 13 eV by comparing the ratio of intensities of the major fragment relative to the precursor cation. In addition, quantum chemical calculations at the B3LYP/6-31 + + G(d,p) level of theory were performed for the protonated isomers of hydroxypyridine N-oxide and their radical cation products in order to gain insight into the major routes of dissociation. The results suggest that dissociation from the lowest triplet excited state of the protonated species would provide a reasonable rationalization for the difference in behavior of both isomers.

Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8WF mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O2 adduct of the G8WF mutant, with respect to those of the wild-type protein and the previously studied E7HL and/or B10YF mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues.

A series of hydroxy‐, methoxy‐, and nitrophenylacetamides was synthesized by enzyme catalysis. The 28 new products were obtained through a lipase‐catalyzed two‐step reaction in very good to excellent yield. In the case of nitro derivatives, a one‐pot, two‐step methodology allowed the desired products to be obtained in high yields. The influence of various reaction parameters in the lipase‐catalyzed reactions, such as enzyme source, nucleophile (alcohol or amine)/substrate ratio, enzyme/substrate ratio, solvent and temperature were studied. It was observed that nitro‐substituted phenylacetates were more reactive in the aminolysis reaction than phenylacetates substituted with a hydroxyl group. To study this substituent effect, a Hammett analysis and the determination of the ρ parameter were carried out. Moreover, a computational study was applied to the most representative systems, performing an exploration of the potential energy surface for the catalyzed and noncatalyzed aminolysis reaction for nitro‐ and hydroxyphenylacetates. Both analysis showed that the presence of a strongly electron‐attracting group favors the activity of the enzyme, in complete agreement with the experimental results of the enzymatic catalysis.

Doubly charged microhydrated adducts formed from catechol and calcium(II) were produced in the gas phase using electrospray ionization (ESI) appearing as the most important ions in the mass spectra recorded. The gas phase structures of [Ca(catechol)2(H2O)](2+) and [Ca(catechol)2(H2O)2](2+) have been assayed by IR multiphoton dissociation (IRMPD) spectroscopy, recording their vibrational spectra in the 3450-3750 cm(-1) range (OH stretching region) and in the 900-1700 cm(-1) fingerprint spectral region. The agreement between experimental and calculated IR spectra of the selected cluster ions confirmed the suitability of the proposed geometries. In addition, quantum chemical calculations at the B3LYP/6-311+G(d,p) level of theory were performed for [Ca(catechol)2(H2O)](2+) to gain insight into the major routes of dissociation. The results suggest that loss of the water molecule is the lowest energy fragmentation channel followed by charge separation products and neutral loss of one catechol molecule, in agreement with the product ions observed upon collision-induced dissociation (CID).