Determinants of substrate internalization in the distal pocket of dehaloperoxidase hemoglobin of Amphitrite ornata.
Dehaloperoxidase (DHP) is a small heme protein in the coelom of the terebellid polychaete Amphitrite ornata. It can act both as an oxygen storage protein (hemoglobin function) and as a dehaloperoxidase (peroxidase function). The X-ray structure of the ferric form shows that the phenolic substrate can bind inside the protein, which is not the case for a typical peroxidase. In the present study, we have used CO-ligated DHP to mimic the distal pocket of the peroxidase DHP and to probe under which conditions both a halophenol and a diatomic ligand can be accommodated in the distal pocket. To vary the structure of the distal pocket, we have compared wild-type DHP and mutants H55V and H55R at different pH values, using flash photolysis in the visible and FTIR spectroscopy in the CO stretching bands. The latter technique is extremely sensitive to even small structural changes in the CO environment and thus can report substrate binding in the distal pocket. Our results on wild-type DHP and its variants indicate that halophenols and a diatomic ligand can indeed simultaneously be present in the distal pocket if the distal histidine is in the low-pH conformation, in which its side chain is swung out of the distal pocket. The markedly different pH dependencies of enzyme activity and substrate binding are not consistent with the hypothesis that substrate dehalogenation occurs within the interior of DHP.
Nienhaus
Karin
K
Institute of Biophysics, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Nickel
Elena
E
Davis
Michael F
MF
Franzen
Stefan
S
Nienhaus
G Ulrich
GU
D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D011077:Polychaeta / Q000201:enzymology*; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity
D006454:Hemoglobins; D008024:Ligands; C098661:DHP I dehaloperoxidase; D010544:Peroxidases