Alberto Boffi

The X-ray structure of ferric Escherichia coli flavohemoglobin reveals an unexpected geometry of the distal heme pocket

The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 Å and refined to anR-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophusflavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region. An inspection of the heme environment in E. coli flavohemoglobin reveals an unexpected architecture of the distal pocket. In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species. Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide. Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species.

Pentacoordinate Hemin Derivatives in Sodium Dodecyl Sulfate Micelles: Model Systems for the Assignment of the Fifth Ligand in Ferric Heme Proteins

Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins.

Flavohemoglobin: structure and reactivity.

Flavohemoglobins (flavoHbs) are made of a globin domain fused with a ferredoxin reductaselike FAD‐ and NAD‐binding modules. These proteins are widely represented among bacteria and yeasts and represent a most challenging research subject in view of their high reactivity both as reductases and as oxidases. The functional annotations of flavoHbs are still controversial, and different physiological roles that are linked to cell responses to oxidative and/or nitrosative stress have been proposed. The flavoHb from Escherichia coli (HMP) has been the object of a large number of investigations to unveil its physiological role in the framework of bacterial resistance to nitrosative stress. HMP expression has been demonstrated to respond to the presence of NO in the culture medium, and an explicit mechanism has been proposed that involves NO scavenging and its reduction to N2O under anaerobic conditions. In contrast to (or together with) the anaerobic NO‐reductase activity, HMP has also been shown to be able to catalyze the oxidation of NO to NO3− (NO‐dioxygenase activity) both in vivo and in vitro in the presence of O2 and NADH. HMP has also been shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. The alkylhydroperoxide reductase activity taken together with the unique lipid‐binding properties of HMP suggests that this flavoHb may be involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress.

Cyanide Binding to Lucina pectinata Hemoglobin I and to Sperm Whale Myoglobin: An X-Ray Crystallographic Study

The x-ray crystal structures of the cyanide derivative of Lucina pectinata monomeric hemoglobin I (L. pectinata HbI) and sperm whale (Physeter catodon) myoglobin (Mb), generally taken as reference models for monomeric hemoproteins carrying hydrogen sulfide and oxygen, respectively, have been determined at 1.9 A (R-factor = 0. 184), and 1.8 A (R-factor = 0.181) resolution, respectively, at room temperature (lambda = 1.542 A). Moreover, the x-ray crystal structure of the L. pectinata HbI:cyanide derivative has been studied at 1.4-A resolution (R-factor = 0.118) and 100 K (on a synchrotron source lambda = 0.998 A). At room temperature, the cyanide ligand is roughly parallel to the heme plane of L. pectinata HbI, being located approximately 2.5 A from the iron atom. On the other hand, the crystal structure of the L. pectinata HbI:cyanide derivative at 100 K shows that the diatomic ligand is coordinated to the iron atom in an orientation almost perpendicular to the heme (the Fe-C distance being 1.95 A), adopting a coordination geometry strictly reminescent of that observed in sperm whale Mb, at room temperature. The unusual cyanide distal site orientation observed in L. pectinata HbI, at room temperature, may reflect reduction of the heme Fe(III) atom induced by free radical species during x-ray data collection using Cu Kalpha radiation.

Structural Basis of Enzymatic S-Norcoclaurine Biosynthesis.

The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1Å resolution. NCS shares no common features with the functionally correlated “Pictet-Spenglerases” that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-Å-long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.

Antibody–drug conjugates: targeting melanoma with cisplatin encapsulated in protein-cage nanoparticles based on human ferritin

A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4(+) melanoma cell line, but not to a CSPG4(-) breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt-Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt-Pt-Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt-Pt-Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt-Pt-Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.

The Heck Reaction of Allylic Alcohols Catalyzed by Palladium Nanoparticles in Water: Chemoenzymatic Synthesis of (R)‐(−)‐Rhododendrol

The use of phosphine‐free perfluoro‐tagged palladium nanoparticles immobilized on fluorous silica gel (FSG), either through fluorous–fluorous interactions or covalent bonding, in the Heck reaction of aryl iodides with allylic alcohols under aerobic conditions in water is described. 4‐(4‐Methoxyphenyl)‐butan‐2‐one, an important fine chemical, is readily accessed by this procedure. A two‐step one‐pot process, involving a Heck reaction followed by an enantioselective enzyme‐catalyzed reduction, to form chiral alcohols is applied to the synthesis of (R)‐(−)‐rhododendrol. The palladium catalysts can be recycled several times, both in the Heck reaction and in the one‐pot chemoenzymatic process.

A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca

The gene coding for a hemoglobin‐like protein (Tf‐trHb) has been identified in the thermophilic actinobacterium Thermobifida fusca and cloned in Escherichia coli for overexpression. The crystal structure of the ferric, acetate‐bound derivative, was obtained at 2.48 Å resolution. The three‐dimensional structure of Tf‐trHb is similar to structures reported for the truncated hemoglobins from Mycobacterium tuberculosis and Bacillus subtilis in its central domain. The complete lack of diffraction patterns relative to the N‐ and C‐terminal segments indicates that these are unstructured polypeptides chains, consistent with their facile cleavage in solution. The absence of internal cavities and the presence of two water molecules between the bound acetate ion and the protein surface suggest that the mode of ligand entry is similar to that of typical hemoglobins. The protein is characterized by higher thermostability than the similar mesophilic truncated hemoglobin from B. subtilis, as demonstrated by far‐UV CD melting experiments on the cyano‐met derivatives. The ligand‐binding properties of Tf‐trHb, analyzed in stopped flow experiments, demonstrate that Tf‐trHb is capable of efficient O2 binding and release between 55 and 60 °C, the optimal growth temperature for Thermobifida fusca.

Sulfide Binding Properties of Truncated Hemoglobins

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high affinity for hydrogen sulfide (K = 5.0 x 10(6) and 2.8 x 10(6) M(-1) for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though lower with respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 x 10(8) M(-1)). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH(-)-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH(-) whereas the YCD1 and YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm(-1) is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen sulfide is thought to have a possible physiological significance as H(2)S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis.

Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

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