Ligation Tunes Protein Reactivity in an Ancient Haemoglobin: Kinetic Evidence for an Allosteric Mechanism in Methanosarcina acetivorans Protoglobin

Protoglobin from Methanosarcina acetivorans (MaPgb) is a dimeric globin with peculiar structural properties such as a completely buried haem and two orthogonal tunnels connecting the distal cavity to the solvent. CO binding to and dissociation from MaPgb occur through a biphasic kinetics. We show that the heterogenous kinetics arises from binding to (and dissociation from) two tertiary conformations in ligation-dependent equilibrium. Ligation favours the species with high binding rate (and low dissociation rate). The equilibrium is shifted towards the species with low binding (and high dissociation) rates for the unliganded molecules. A quantitative model is proposed to describe the observed carbonylation kinetics

Sulfide-Binding Hemoglobins: Effects of Mutations on Active-Site Flexibility

The dynamics of Hemoglobin I (HbI) from the clam Lucina pectinata, from wild-type sperm whale (SW) myoglobin, and from the L29F/H64Q/V68F triple mutant of SW, both unligated and bound to hydrogen sulfide (H(2)S), have been studied in molecular dynamics simulations. Features that account for differences in H(2)S affinity among the three have been examined. Our results verify the existence of an unusual heme rocking motion in unligated HbI that can promote the entrance of large ligands such as H(2)S. The FQF-mutant partially reproduces the amplitude and relative orientation of the motion of HbI's heme group. Therefore, besides introducing favorable electrostatic interactions with H(2)S, the three mutations in the distal pocket change the dynamic properties of the heme group. The active-site residues Gln-64(E7), Phe-43(CD1), and His-93(F8) are also shown to be more flexible in unligated HbI than in FQF-mutant and SW. Further contributions to H(2)S affinity come from differences in hydrogen bonding between the heme propionate groups and nearby amino acid residues

Structural flexibility of the heme cavity in the cold-adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125

Truncated hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two‐on‐two α‐helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group‐II truncated hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph‐2/2HbO), a cold‐adapted Antarctic marine bacterium hosting one flavohemoglobin and three distinct truncated hemoglobins. The Ph‐2/2HbO aquo‐met crystal structure (at 2.21 Å resolution) shows typical features of group‐II truncated hemoglobins, namely the two‐on‐two α‐helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal‐site hydrogen‐bonded network that includes water molecules and several distal‐site residues, including His(58)CD1. Analysis of Ph‐2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph‐2/2HbO can access diverse heme ligation states. Among these, detection of a low‐spin heme hexa‐coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme‐Fe ligand. Altogether, the results show that Ph‐2/2HbO maintains the general structural features of group‐II truncated hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment.Fil: Giordano, Daniela. Consiglio Nazionale delle Ricerche; ItaliaFil: Pesce, Alessandra. Università degli Studi di Genova; ItaliaFil: Boechi, Leonardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bustamante, Juan Pablo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caldelli, Elena. Universita Degli Studi Di Firenze; ItaliaFil: Howes, Barry D.. Universita Degli Studi Di Firenze; ItaliaFil: Riccio, Alessia. Consiglio Nazionale delle Ricerche; ItaliaFil: di Prisco, Guido. Consiglio Nazionale delle Ricerche; ItaliaFil: Nardini, Marco. Università degli Studi di Milano; ItaliaFil: Estrin, Dario Ariel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Smulevich, Giulietta. Universita Degli Studi Di Firenze; ItaliaFil: Bolognesi, Martino. Università degli Studi di Milano; ItaliaFil: Verde, Cinzia. Consiglio Nazionale delle Ricerche; Italia. Universita Di Roma; Itali