Ligand migration and binding in nonsymbiotic hemoglobins of Arabidopsis thaliana.
We have studied carbon monoxide (CO) migration and binding in the nonsymbiotic hemoglobins AHb1 and AHb2 of Arabidopsis thaliana using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures. Both proteins have similar amino acid sequences but display pronounced differences in ligand binding properties, at both physiological and cryogenic temperatures. Near neutral pH, the distal HisE7 side chain is close to the heme-bound ligand in the majority of AHb1-CO molecules, as indicated by a low CO stretching frequency at 1921 cm(-1). In this fraction, two CO docking sites can be populated, the primary site B and the secondary site C. When the pH is lowered, a high-frequency stretching band at approximately 1964 cm(-1) grows at the expense of the low-frequency band, indicating that HisE7 protonates and, concomitantly, moves away from the bound ligand. Geminate rebinding barriers are markedly different for the two conformations, and docking site C is not accessible in the low-pH conformation. Rebinding of NO ligands was observed only from site B of AHb1, regardless of conformation. In AHb2, the HisE7 side chain is removed from the bound ligand; rebinding barriers are low, and CO molecules can populate only primary docking site B. These results are interpreted in terms of differences in the active site structures and physiological functions.
Nienhaus
Karin
K
Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology, 76128 Karlsruhe, Germany.
Dominici
Paola
P
Astegno
Alessandra
A
Abbruzzetti
Stefania
S
Viappiani
Cristiano
C
Nienhaus
G Ulrich
GU
D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation
C470347:AHB1 protein, Arabidopsis; C109042:AHB2 protein, Arabidopsis; D029681:Arabidopsis Proteins; D006454:Hemoglobins; D008024:Ligands; D009569:Nitric Oxide; D002248:Carbon Monoxide