title: Structural plasticity and functional implications of internal cavities in distal mutants of type 1 non-symbiotic hemoglobin AHb1 from Arabidopsis thaliana.
abstract: The increasing number of nonsymbiotic plant hemoglobins discovered in genomic studies in the past decade raises intriguing questions about their physiological role. Among them, the nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana deserves particular attention, as it combines an extremely high oxygen affinity with an internal hexacoordination of the distal histidine HisE7 to the heme iron in the absence of exogenous ligands. In order to gain insight into the structure-function relationships of the protein, the ligand binding properties of mutants of two conserved residues of the distal cavity, HisE7 --> Leu and PheB10 --> Leu, were investigated by experimental and computational studies and compared to results determined for the wild type (wt) protein. The Fe(2+)-deoxy HisE7 --> Leu mutant exists, as expected, in the pentacoordinated form, while a mixture of penta- and hexacoordinated forms is found for the PheB10 --> Leu mutant, with an equilibrium shifted toward the pentacoordinated form with respect to the wt protein. Spectroscopic studies of the complexes of CO and CN(-) with AHb1 and its mutants show a subtle interplay of steric and electrostatic effects by distal residues on the ligand binding to the heme. Moreover, stopped-flow and flash photolysis experiments reveal substantial kinetic differences triggered by those mutations, which are particularly manifested in the enhanced geminate rebinding and bimolecular association rate. These findings are discussed in light of the drastic alterations found by molecular dynamics simulations in the nature and distribution of internal cavities in the protein matrix of the mutants, revealing an extremely large sensitivity of the protein structure to changes in distal HisE7 and PheB10 residues. Overall, data are consistent with the putative NO-dioxygenase activity attributed to AHb1.
authors: Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Parma, Italy.
authors: Serena
authors: S
authors: Faggiano
authors: Stefania
authors: S
authors: Abbruzzetti
authors: Francesca
authors: F
authors: Spyrakis
authors: Elena
authors: E
authors: Grandi
authors: Cristiano
authors: C
authors: Viappiani
authors: Stefano
authors: S
authors: Bruno
authors: Andrea
authors: A
authors: Mozzarelli
authors: Pietro
authors: P
authors: Cozzini
authors: Alessandra
authors: A
authors: Astegno
authors: Paola
authors: P
authors: Dominici
authors: Silvia
authors: S
authors: Brogioni
authors: Alessandro
authors: A
authors: Feis
authors: Giulietta
authors: G
authors: Smulevich
authors: Oliver
authors: O
authors: Carrillo
authors: Peter
authors: P
authors: Schmidtke
authors: Axel
authors: A
authors: Bidon-Chanal
authors: F Javier
authors: FJ
authors: Luque
mesh_terms: D000042:Absorption; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D007834:Lasers; D008024:Ligands; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010649:Phenylalanine; D010782:Photolysis; D011487:Protein Conformation; D055550:Protein Stability; D013059:Spectrum Analysis, Raman; D013997:Time Factors
chemical_list: C470347:AHB1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D006454:Hemoglobins; D008024:Ligands; D050505:Mutant Proteins; D010649:Phenylalanine; D006639:Histidine; D002248:Carbon Monoxide