title: The reactivity with CO of AHb1 and AHb2 from Arabidopsis thaliana is controlled by the distal HisE7 and internal hydrophobic cavities.
abstract: The nonsymbiotic hemoglobins, AHb1 and AHb2, have recently been isolated from Arabidopsis thaliana. Using steady-state and time-resolved spectroscopic methods, we show that Fe2+ AHb1 contains a mixture of penta- and hexacoordinated heme, while Fe2+ AHb2 is fully hexacoordinated. In the CO complexes, polar interactions and H-bonds with the ligand are stronger for AHb1 than for AHb2. The ligand binding kinetics are substantially different, reflecting the distribution between the penta- and hexacoordinated species, and indicate that protein dynamics and ligand migration pathways are very specific for each of the two proteins. In particular, a very small, non-exponential geminate rebinding observed in AHb1 suggests that the distal heme cavity is connected with the exterior by a relatively open channel. The large, temperature-dependent geminate rebinding observed for AHb2 implies a major role of protein dynamics in the ligand migration from the distal cavity to the solvent. The structures of AHb1 and AHb2, modeled on the basis of the homologous rice hemoglobin, exhibit a different cavity system that is fully compatible with the observed ligand binding kinetics. Overall, these kinetic and structural data are consistent with the putative NO-dioxygenase activity previously attributed to AHb1, whereas the role of AHb2 remains elusive.
authors.affiliation: Dipartimento di Biochimica e Biologia Molecolare, UniversitA degli Studi di Parma, Parma, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruno
authors.forename: Serena
authors.initials: S
authors.lastname: Faggiano
authors.forename: Francesca
authors.initials: F
authors.lastname: Spyrakis
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
authors.forename: Stefania
authors.initials: S
authors.lastname: Abbruzzetti
authors.forename: Elena
authors.initials: E
authors.lastname: Grandi
authors.forename: Cristiano
authors.initials: C
authors.lastname: Viappiani
authors.forename: Alessandro
authors.initials: A
authors.lastname: Feis
authors.forename: Stephan
authors.initials: S
authors.lastname: Mackowiak
authors.forename: Giulietta
authors.initials: G
authors.lastname: Smulevich
authors.forename: Elena
authors.initials: E
authors.lastname: Cacciatori
authors.forename: Paola
authors.initials: P
authors.lastname: Dominici
mesh_terms: D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry*; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry*; D006639:Histidine; D057927:Hydrophobic and Hydrophilic Interactions; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: C470347:AHB1 protein, Arabidopsis; C109042:AHB2 protein, Arabidopsis; D029681:Arabidopsis Proteins; D006454:Hemoglobins; D008024:Ligands; D006639:Histidine; D002248:Carbon Monoxide; D007501:Iron