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A unique defense mechanisms by which Mycobacterium tuberculosis protects itself from nitrosative stress is based on the O2-dependent NO-dioxygenase ( NOD) activity of truncated hemoglobin 2/2HbN ( Mt2/2HbN). The NOD activity largely depends on the efficiency of ligand migration to the heme cavity through a two-tunnel (long and short) system; recently, it was also correlated with the presence at the Mt2/2HbN N-terminus of a short pre-A region, not conserved in most 2/2HbNs, whose deletion results in a drastic reduction of NO scavenging. In the present study, we report the crystal structure of Mt2/2HbN-ΔpreA, lacking the pre-A region, at a resolution of 1.53 Å. We show that removal of the pre-A region results in long range effects on the protein C-terminus, promoting the assembly of a stable dimer, both in the crystals and in solution. In the Mt2/2HbN-ΔpreA dimer, access of heme ligands to the short tunnel is hindered. Molecular dynamics simulations show that the long tunnel branch is the only accessible pathway for O2-ligand migration to/from the heme, and that the gating residue Phe(62)E15 partly restricts the diameter of the tunnel. Accordingly, kinetic measurements indicate that the kon value for peroxynitrite isomerization by Mt2/2HbN-ΔpreA-Fe( III) is four-fold lower relative to the full-length protein, and that NO scavenging by Mt2/2HbN-ΔpreA-Fe( II)-O2 is reduced by 35-fold. Therefore, we speculate that Mt2/2HbN evolved to host the pre-A region as a mechanism for preventing dimerization, thus reinforcing the survival of the microorganism against the reactive nitrosative stress in macrophages. Database Coordinates and structure factors have been deposited in the Protein Data Bank under accession number . [ABSTRACT FROM AUTHOR]

Two domino Diels-Alder adducts were obtained from 3,7-bis(cyclopenta-2,4-dien-1-ylidene)- cis-bicyclo[3.3.0]octane and dimethyl acetylenedicarboxylate or N-methylmaleimide under microwave irradiation. From the first adduct, a C20H24 diene with C2 v symmetry was obtained by Zn/AcOH reduction, hydrolysis, oxidative decarboxylation, and selective hydrogenation. Photochemical [2+2] cycloaddition of this diene gave a thermally unstable cyclobutane derivative, which reverts to the diene. However, both the diene and the cyclobutane derivatives could be identified by X-ray diffraction analysis upon irradiation of the diene crystal. New six-membered rings are formed upon the transannular addition of bromine or iodine to the diene. The N-type selectivity of the addition was examined by theoretical calculations, which revealed the distinct susceptibility of the doubly bonded carbon atoms to the bromine attack. [ABSTRACT FROM AUTHOR]

Nonsymbiotic hemoglobins AHb1 and AHb2 discovered in Arabidopsis thaliana are likely to carry out distinct physiological roles, in consideration of their differences in sequence, structure, expression pattern, and tissue localization. Despite a relatively fast autoxidation in the presence of O, we were able to collect O-binding curves for AHb2 in the presence of a reduction enzymatic system. AHb2 binds O noncooperatively with a p50 of 0.021 ± 0.003 Torr, a value consistent with a recently proposed role in O transport. The analysis of the internal cavities derived from the structures sampled in molecular dynamics simulations confirms strong differences with AHb1, proposed to work as a NO deoxygenase in vivo. Overall, our results are consistent with a role for AHb2 as an oxygen carrier, as recently proposed on the basis of experiments on AHb2-overexpressing mutants of A. thaliana. © 2011 IUBMB IUBMB Life, 63(5): 363-362, 2011. [ABSTRACT FROM AUTHOR]